U.S. patent application number 09/887694 was filed with the patent office on 2003-02-13 for in vitro propagation of embryonic stem cells.
Invention is credited to Gough, Nicholas Martin, Hilton, Douglas James, Williams, Robert Lindsay.
Application Number | 20030032178 09/887694 |
Document ID | / |
Family ID | 46279954 |
Filed Date | 2003-02-13 |
United States Patent
Application |
20030032178 |
Kind Code |
A1 |
Williams, Robert Lindsay ;
et al. |
February 13, 2003 |
In vitro propagation of embryonic stem cells
Abstract
The present invention relates to the use of leukaemia inhibitory
factor (LIF) in the isolation and propagation of embryonic stem
cells in vitro.
Inventors: |
Williams, Robert Lindsay;
(Warrandyte, AU) ; Gough, Nicholas Martin; (North
Balwyn, AU) ; Hilton, Douglas James; (Warrandyte,
AU) |
Correspondence
Address: |
Scully, Scott, Murphy & Presser
400 Garden City Plaza
Garden City
NY
11530
US
|
Family ID: |
46279954 |
Appl. No.: |
09/887694 |
Filed: |
May 2, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09887694 |
May 2, 2001 |
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09584026 |
May 30, 2000 |
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09584026 |
May 30, 2000 |
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09050457 |
Mar 30, 1998 |
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09050457 |
Mar 30, 1998 |
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08278561 |
Jul 21, 1994 |
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08278561 |
Jul 21, 1994 |
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07924809 |
Aug 4, 1992 |
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07924809 |
Aug 4, 1992 |
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07477960 |
May 31, 1990 |
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5166065 |
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Current U.S.
Class: |
435/366 ;
435/325; 435/354 |
Current CPC
Class: |
C12N 5/0606 20130101;
C12N 2501/235 20130101; C12N 2510/00 20130101 |
Class at
Publication: |
435/366 ;
435/354; 435/325 |
International
Class: |
C12N 005/08; C12N
005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 4, 1988 |
AU |
PJ 9644/88 |
Claims
1. A method for the isolation of embryonic stem. (ES) cells from
animal embryos in vitro which method comprises deriving and
maintaining said embryos in culture medium containing an effective
amount of leukaemia inhibitory factor (LIF) for a time and under
conditions sufficient for the development of said ES cells.
2. The method according to claim 1 wherein the culture medium is
free of feeder cells.
3. The method according to claims 1 or 2 wherein the animal embryos
are derived from humans, mice, birds, sheep, pigs, cattle, goats or
fish.
4. The method according to claim 3 wherein the animal embryos are
derived from mice.
5. The method according to claims 1 or 2 wherein the culture medium
is Eagle's medium or modifications thereof or equivalents
thereto.
6. The method according to any one of the preceding claims wherein
the LIF is recombinant LIF.
7. The method according to claim 6 wherein the LIF is recombinant
human or murine LIF.
8. The method according to claim 7 wherein LIF is added to the
culture medium at a concentration of from 10 to 1,000,000
units/ml.
9. The method according to claim 8 wherein the LIF is added to the
culture medium at a concentration of from 100 to 100,000
units/ml.
10. The method according to claim 9 wherein the LIF is added to the
culture medium at a concentration of from 500 to 10,000
units/ml.
11. A method according to any one of the preceding claims wherein
the effective time is from 1 day to 20 weeks.
12. The method according to claim 11 wherein the effective time is
from 1 to 8 weeks.
13. A method for maintaining animal embryonic stem (ES) cells in
vitro while retaining their pluripotential phenotype which process
comprises culturing said cells in a culture medium containing an
effective amount of leukaemia inhibitory factor (LIF) under
conditions sufficient to maintain said cells.
14. The method according to claim 13 wherein the culture medium is
free of feeder cells.
15. The method according to claim 13 or 14 wherein the animal ES
cells are derived from humans, mice, birds, sheep, pigs, cattle,
goats or fish.
16. The method according to claim 15 wherein the animal ES cells
are derived from mice.
17. The method according to claim 13 or 14 wherein the culture
medium comprises Eagle's medium or modifications thereof or
equivalents thereto.
18. The method according to any one of claims 13 to 17 wherein the
LIF is recombinant LIF.
19. The method according to claim 18 wherein the LIF is recombinant
murine or human LIF.
20. The method according to claim 19 wherein the recombinant LIF is
added to the culture medium at a concentration of from 10 to
1,000,000 units/ml.
21. The method according to claim 20 wherein the recombinant LIF is
added to the culture medium at a concentration of from 100 to
100,000 units/ml.
22. The method according to claim 21 wherein LIF is added to the
culture medium at a concentration of from 500 to 10,000
units/ml.
23. Embryonic stem (ES) cells derived from animal embryos in vitro
isolated by deriving and maintaining said embryos in culture
medium, said culture medium containing an effective amount of
leukaemia inhibitory factor (LIF) for a time and under conditions
sufficient for the development of said ES cells.
24. The ES cells according to claim 23 wherein the culture medium
is free of feeder cells.
25. The ES cells according to claim 23 or 24 derived from human,
mouse, bird, sheep, pig, cattle, goat or fish embryos.
26. The ES cells according to claim 25 derived from mouse
embryos.
27. A chimaeric animal or transgenic progeny thereof generated
using ES cells which have been isolated from animal embryos
according to claim 1.
28. A chimaeric animal or transgenic progeny thereof generated
using animal ES cells which have been maintained in vitro according
to the method of claim 13.
29. The chimaeric animal or transgenic progeny thereof according to
claim 27 or 28 wherein said animal is a mouse.
30. The chimaeric animal or transgenic progeny thereof according to
claims 27 or 28 or 29 wherein the ES cells contain additional
genetic material inserted therein.
Description
[0001] This invention relates to the use of a previously discovered
and characterised molecule, leukaemia inhibitory factor (LIF), in
the isolation and propagation of embryonic stem cells in vitro.
[0002] Embryonic stem (ES) cells, the pluripotent outgrowths of
blastocysts, can be cultured and manipulated vitro and then
returned to the embryonic environment to contribute normally to all
tissues including the germline (for review see Robertson, E. J.
(1986) Trends in Genetics 2:9-13). Not only can ES cells propagated
in vitro contribute efficiently to the formation of chimaeras,
including germline chimaeras, but in addition, these cells can be
manipulated in vitro without losing their capacity to generate
germ-line chimaeras (Robertson, E. J. et. al. (1986) Nature
323:445-447).
[0003] ES cells thus provide a route for the generation of
transgenic animals such as transgenic mice, a route which has a
number of important advantages compared with more conventional
techniques, such as zygote injection and viral infection (Wagner
and Stewart (1986) in Experimental Approaches to Embryonic
Development. J. Rossant and A. Pedersen eds. Cambridge: Cambridge
University Press), for introducing new genetic material into such
animals. First, the gene of interest can be introduced and its
integration and expression characterised in vitro. Secondly, the
effect of the introduced gene on the ES cell growth can be studied
in vitro. Thirdly, the characterised ES cells having a novel
introduced gene can be efficiently introduced into embryos by
blastocyst injection or embryo aggregation and the consequences of
the introduced gene on the development of the resulting transgenic
chimaeras monitored during pre- or post-natal life. Fourthly, the
site in the ES cell genome at which the introduced gene integrates
can be manipulated, leaving the way open for gene targeting and
gene replacement (Thomas, K. R. and Capecci, M. R. (1987) Cell
51:503-512).
[0004] However, it is known that ES cells and certain EC (embryonal
carcinoma) cell lines will only retain the stem cell phenotype in
vitro when cultured on a feeder layer of fibroblasts (such as
murine STO cells, e.g. Martin, G. R. and Evans, M. J. (1975) Proc.
Natl. Acad. Sci. USA 72:1441-1445) or when cultured in medium
conditioned by certain cells (e.g. Koopman, P. and Cotton, R. G. H.
(1984) Exp. Cell Res. 154:233-242; Smith, A. G. and Hooper, M. L.
(1987) Devel.Biol. 121:1-91). In the absence of feeder cells or
conditioned medium, the ES cells spontaneously differentiate into a
wide variety of cell types, resembling those found during
embryogenesis and in the adult animal. The factors responsible for
maintaining the pluripotency of ES cells have, however, remained
poorly characterised.
[0005] In work leading to the present invention, it has been found
that LIF has the capacity to substitute for, or be added to, feeder
layers (or conditioned medium) in supporting the maintenance of
pluripotential ES cells in vitro.
[0006] LIF is a protein that has previously been purified, cloned
and produced in large quantities in purified recombinant form from
both Escherichia coli and yeast cells. (International Patent
Application No. PCT/AU88/00093, filed Mar. 31, 1988.) LIF has been
defined as a factor, the properties of which include:
[0007] 1. it has the ability to suppress the proliferation of
myeloid leukaemic cells such as M1 cells, with associated
differentiation of the leukaemic cells; and
[0008] 2. it will compete with a molecule having the defined
sequence of murine LIF or human LIF (defined in International
Patent Application No. PCT/AU88/00093) for binding to specific
cellular receptors on M1 cells or murine or human macrophages. In
addition to the biological properties previously disclosed for
murine and human LIF, LIF has now been found to have the following
properties:
[0009] (a) it allows the derivation and maintenance in the absence
of feeder cells of the pluripotential phenotype in vitro of ES
cells.
[0010] (b) it allows the aforementioned ES cells, after passage in
vitro in the presence of LIF, to contribute to somatic and germline
cell tissues of chimaeric animals such as mice when re-introduced
into the embryonic environment;
[0011] (c) it demonstrates selective binding to high affinity
receptors on murine ES (EKcs-1 (previously known as CS1) and D3)
and EC (PCC3-3A and F9) cells; and
[0012] (d) specific binding of .sup.125I-LIF to high affinity
receptors is not in competition with insulin, IGF-I, IGF-II, acidic
and basic FGF, TGF.beta., TNF.alpha., TNF.beta., NGF, PDGF, EGF,
IL-1, IL-2, IL-4, GM-CSF, G-CSF, Multi-CSF nor erythropoietin, but
is in competition with murine and human LIF.
[0013] Accordingly, a first aspect of the present invention relates
to a method for the isolation of embryonic stem (ES) cells from
animal embryos in vitro which method comprises deriving ES cells
from said embryos in culture medium, said culture medium containing
an effective amount of leukaemia inhibitory factor (LIF), for a
time and under conditions sufficient for the development of said ES
cells. The embryos used may be isolated from animals including, but
not limited to, humans and a number of other animal species such as
birds (eg. chickens), mice, sheep, pigs, cattle, goats and
fish.
[0014] A second aspect of the present invention, contemplates a
process for maintaining animal embryonic stem (ES) cells in vitro
while retaining their pluripotential phenotype, which process
comprises culturing said cell's in a culture medium containing an
effective amount of leukaemia inhibitory factor (LIF) under
conditions sufficient to maintain said cells. The ES cells in
accordance with this aspect of the invention include cells from
humans, mice, birds (eg. chickens), sheep, pigs, cattle, goats and
fish.
[0015] The LIF used in the culture medium is preferably recombinant
LIF produced, by way of example, in accordance with the methods
described in International Patent Application No. PCT/AU88/00093.
In accordance with the present invention, it has been found that
recombinant LIF and in particular recombinant human and murine LIF
are effective substitutes for, or additives to, feeder layers or
conditioned medium in maintaining ES cells in vitro. For the
purposes of the present description recombinant LIF is produced in
E. coli and yeast using the methods described in International
Patent Application No. PCT/AU88/00093, however, it is within the
scope of the present invention to include recombinant LIF produced
in other hosts including mammalian and insect cells and to
synthetic LIF.
[0016] In another aspect, the present invention extends to ES cells
derived from animal embryos by passage in a culture medium
containing LIF, to such ES cells having additional genetic material
inserted therein, and to chimaeric animals such as chimaeric mice
or transgenic progeny of said animals generated by known techniques
using ES cells which have been maintained in vitro in a
LIF-containing culture medium.
[0017] Thus, the invention extends to the generation and
maintenance of ES cells from humans, mice, birds (eg. chickens),
sheep, pigs, cattle, goats and fish and to the generation of
transgenic chimaeric animals and their transgenic progeny using the
ES cells isolated from animal species such as mice, birds (eg.
chickens), sheep, pigs, cattle, goats and fish. This invention also
includes the use of LIF in culture media to modulate the survival
and growth of human and other animal species such as cattle germ
cells and embryonic cells, for example, for use in in vitro
fertilisation and other procedures.
[0018] The present invention may also be described by reference to
the following figures:
[0019] FIG. 1 is a graphical representation showing the effect on
ES cells of different concentrations of LIF.
[0020] FIG. 2 is a pictorial representation showing ES cell
morphology in the presence and absence of LIF.
[0021] FIG. 3 is a graphical (A and C) and pictorial (B)
representation showing the binding of .sup.125I-LIF to ES cells
(EKcs-1) and EC cells (F9 and PCC3-A).
[0022] The present invention is directed to a method for the
isolation and maintenance of embryonic stem (ES) cells from animal
embryos in vitro which method comprises deriving and/or maintaining
said ES cells from said embryos in culture medium containing an
effective amount of leukaemia inhibitory factor (LIF), for a time
and under conditions sufficient for the derivation and/or
maintenance of said ES cells. The animal embryos may be isolated
from a number of animal species'such as humans, mice, birds (eg.
chickens), sheep, pigs, cattle, goats and fish. By reference herein
to "animal embryos" includes reference to "animal blastocysts".
Furthermore, the present invention is exemplified using human LIF
with murine ES cells (heterologous system) and murine LIF with
murine ES cells (homologous system). This is done with the
understanding that the present invention contemplates LIF from any
animal species in heterologous or homologous systems with animal
embryos from animal species such as humans, mice, birds (e.g.
chickens), sheep, pigs, cattle, goats and fish. Although in certain
circumstances, a heterologous system will work effectively, it may
be preferable to use homologous systems. Given the teachings
herein, it will be routine for the skilled technician to ascertain
whether a homologous or heterologous system is required in order to
isolate or maintain particular animal ES cells.
[0023] By "culture medium" is meant a suitable medium capable of
supporting growth of ES cells. Examples of suitable culture media
useful in practicing the present invention are Eagles medium or
modifications or equivalents thereof such as Dulbecco's or Glasgows
modified Eagle's medium with supplements such as 5% -30% (v/v)
foetal calf serum and where necessary 0.01 to 1.0 mm
.beta.-mercaptoethanol but preferably about 0.1 mm
.beta.-mercaptoethanol. The culture medium may or may not contain
feeder cells and LIF may be used to substitute for, or add to, said
feeder cells. When required, LIF, or more particularly synthetic or
recombinant LIF, is added to the medium at a concentration of about
100-1,000,000 units/ml and preferably about 100-100,000 units/ml
and even more preferably 500-10,000 units/ml where 50 units are
defined as the amount of LIF which in one millilitre induces a 50%
reduction in clone formation by murine M1 myeloid cells. By
"recombinant LIF" is meant the LIF prepared by genetic engineering
means such as, for example, according to International Patent
Application No. PCT/AU88/00093 where a number of hosts such as
bacteria (eg. E. coli) or yeast cells may be employed. In
accordance with the present invention, the effective derivation
time is from 1 day to 20 weeks and particularly from 1 to 8
weeks.
[0024] Another aspect of the present invention contemplates a
process for maintaining animal ES cells in vitro while retaining
their pluripotential phenotype which process comprises culturing
said cells in a culture medium containing an effective amount of
LIF under conditions sufficient to maintain said cells. The ES
cells in accordance with this aspect of the invention include cells
derived from humans, mice, birds (eg. chickens), sheep, pigs,
cattle, goats and fish. As with the isolation of ES cells from
animal embryos, the LIF used in the aformentioned process is
preferably recombinant LIF. The culture medium may or may not
contain feeder cells.
[0025] In accordance with the present invention, "pluripotential
cells" and "embryonic stem cells" are those which retain the
developmental potential to differentiate into all somatic and germ
cell lineages.
[0026] The ability of recombinant LIF to maintain the stem cell
phenotype of ES cells is demonstrated by transferring ES cells D3
and HD5 into normal cell culture medium in the presence of varying
concentrations of purified yeast-derived recombinant human LIF
(rY-HLIF) or E. coli--derived recombinant mouse LIF (rE-MLIF). At
concentrations of 1000-5000 units/ml of rY-HLIF or rE-MLIF more
than 90% of the D3 and HD5 ES cells retained their stem cell
phenotype. In contrast, the ES cells maintained in normal culture
medium differentiated over a period of 3-6 days. The proportion of
colonies having the stem cell phenotype was related to the
concentration of LIF in the culture medium. In addition to
maintaining established ES cell lines, six new ES cell lines
(MBL-1,2,3,4,5 & 6) were isolated from blastocysts in the
absence of feeder cells when the media was supplemented with 1000
units/ml rE-HLIF. Long term maintenance of the ES cell lines D3,
HD5 and MBL-1 to 6 in LIF for up to 22 passages (approximately 100
cell generations or 10 weeks) did not noticeably alter the growth
characteristics of these ES cells or their dose dependency on LIF.
The ability of these ES cells to differentiate into all somatic and
germ cell linages was confirmed by reintroduction of D3 and MBL-1
cells into blastocysts. Approximately 50% of the progeny analysed
contained tissues derived from the injected ES cells with levels of
overt chimaerism as high as 90% in individual mice. To test for
germline transmission of ES derived cells male chimaeras were mated
to C57BL/6J mice. Three D3 and two MBL-1 C57BL/6J chimaeras gave
rise to agouti progeny confirming that these ES cells can
contribute to the formation of germ cells.
[0027] The present invention also relates to chimaeric animals
generated by known techniques using the ES cells contemplated
herein. These ES cells may be isolated from animal embryos and/or
maintained in vitro according to the subject invention.
Furthermore, genetically manipulated ES cells may be passaged in
LIF and used to make chimaeric animals. For example, genetically
manipulated ES cells containing a retrovirus vector (N-TK527;
derived from pXT1; C. A. Boulter and E. F. Wagner, (1987) Nucl.
Acids Res. 15:7194) encoding genes for neomycin resistance and
c-src.sup.527 were propagated in the presence of LIF but in the
absence of feeder cells for over 20 passages. These cells still
retained the ability to differentiate as judged by the formation of
normal chimaeras following introduction of these cells into
preimplantation embryos by blastocyst injection.
[0028] Further details of the use of LIF in accordance with the
present invention will be apparent from the following Examples.
EXAMPLE 1
[0029] This example sets out the steps used to maintain ES cells In
vitro in LIF, and to generate chimaeric mice using ES cells so
passaged.
[0030] Step 1: Progation in vitro:
[0031] The ES cells used were the D3 (Doetschman, T. C. et. al.
(1985) J.Embryol.Exp.Morphol. 87:27-45) the EKcs-1 (previously
known as CS1) (Wagner, E. F. et.al. (1985) Cold Spring Harbor
Symp.Quant.Biol. 50:691-700) and the HDS (C. Stewart, unpublished)
ES cell lines isolated from 129 SV He blastocysts and the CBL63 (R.
Kemler, unpublished) ES cells isolated from C57BL/6J blastocysts.
Prior to culture in LIF, the D3 and CBL63 cells were maintained in
Dulbecco modified Eagles medium with 15% (v/v) foetal calf serum on
a feeder layer of primary embryo fibroblasts, and the EKcs-1 and
HD5 ES cells were maintained in Eagle's medium with 15% (v/v)
foetal calf serumand 0.1 mM .beta.-mecraptoethanol, in the presence
of medium conditioned by the bladder carcinoma cell line 5637 (ATCC
No.HTB9).
[0032] The ability of recombinant LIF to maintain the stem cell
phenotype of ES cells was demonstrated by transferring ES cells of
the lines D3 and HD5 into normal cell culture medium in the
presence of varying concentrations of purified yeast-derived
recombinant human LIF (hereafter referred to as rY-HLIF), or E.
coli derived recombinant mouse LIF (rE-MLIF) (previously disclosed
in International Patent Application No. PCT/AU85/00093). The
results are shown in FIGS. 1 and 2. In FIG. 1A, HDS cells
previously maintained in 80% 5637 conditioned medium for eight
passages were transferred to culture media containing 0-5,000 units
ml.sup.-1 of purified, recombinant yeast-derived human LIF (H-LIF;
see below) (.box-solid.-.box-solid.) or purified, recombinant E.
coli-derived mouse LIF (M-LIF; see below)
(.smallcircle.-.smallcircle.). HD5 cells maintained in medium
containig 1,000 units ml.sup.-1 H-LIF for a further 13 passages
were then transferred to 0-1,000 units ml.sup.-1 M-LIF
(.circle-solid.-.circle-solid.). In FIG. 1B, D3 cells maintined on
mouse embryo fibroblasts for 10 passages were transferred to media
containing 1,000-5,000 units ml.sup.-1 H-LIF and after a further 7
or 15 passages the cells were transferred into media containing
0-5,000 units ml.sup.-1 of H-LIF (.box-solid.-.box-solid.) or
0-1,000 units ml.sup.-1 M-LIF (.circle-solid.-.circle-solid.)
respectively. FIG. 2 shows ES cell morphology in the presence of
recombinant LIF. HD5 ES cells cultured in the presence of 80% 5637
conditioned medium were assayed for the ability of purified
recombinant LIF to maintain the stem-cell phenotype by transfer to
media containing 1,000 units ml.sup.-1 M-LIF (A), or to normal
culture media (B). After seven days, the colonies were stained with
Giemsa. Compact stem-cell colonies could be distinguished from
diffuse differentiated colonies. D3 cells maintained in H-LIF for
15 passages were assayed for the ability to differentiate by
transfer into media containing 1,000 units ml.sup.-1 M-LIF (C) or
normal culture media (D). Immunofluorescence of the cells in the
two D3 colony types was carried out using the ECMA-7 monoclonal
antibody which recognizes a stem cell-specific cell-surface
antigen. Cell-surface-specific immunofluorescence was detected on
over 90% of the cells maintained in media containing 1,000 units
ml.sup.-1 recombinant LIF (E) but on less than 1% of the cells
maintained in normal culture media (F). The field of view shown in
(F) contains 21 cells.
[0033] FIGS. 1 and 2 indicate that over 90% of the ES cells
maintained in 1000-5000 units/ml rY-HLIF or rE-MLIF retained their
stem cell phenotype. In contrast, ES cells maintained in normal
culture medium differentiated over a period of 3-6 days. The
different concentrations of rY-HLIF or rE-MLIF used did not result
in any noticeable change in cell number after 6 days in culture,
indicating that there is no selection for a specific subpopulation
able to grow in LIF. Similar results have been obtained using
yeast-derived rMLIF also disclosed in International Patent
Application No. PCT/AU88/00093. The data in FIG. I indicate that
human LIF acts on mouse ES cells, as previously described for the
action of human LIF on M1 myeloid leukaemic cells (Gough, N. M.
et.al. (1988) Proc.Natl.Acad.Sci.USA 85: 2623-2627). The data in
FIG. I also indicate that the action of LIF on ES cells is
independent of glycosylation, as previously described for the
action of LIF on Ml myeloid leukaemic cells.
[0034] Four ES cell lines, D3, EKcs-l, CBL63 and HD5, were
maintained in medium containing 1000-5000 u/ml rY-HLIF for up to 22
passages (10 weeks or approximately 100 generations). Long-term
maintenance of the ES cells in rY-HLIF did not noticeably alter the
growth characteristics of the cells. Furthermore, reduction or
removal of the LIF from the culture medium resulted in the
differentiation of the ES cells with similar kinetics to those
explanted directly from bladder carcinoma 5637 conditioned medium
or a feeder layer of mouse fibroblasts (for example, see FIGS. 1
and 2). The stem cell phenotype of ES cells cultured for multiple
passages in the presence of LIF was confirmed by immunofluorescence
with the ECMA-7 antibody which recognises a cell-surface
stem-cell-specific antigen (Kemler, R. in Progress in Developmental
Biology Band 26 Sauer, H. W. ed page 175; Fisher, Stuttgart, 1980);
ES cells cultured in the presence of LIF expressed the stem cell
marker, whereas in the absence of LIF less than 1% did so (FIG.
2).
[0035] Step 2: Isolation of ES cell lines:
[0036] Murine blastocysts were isolated from 129 Sv He mice at day
4 of development (day 1-day of plug) into either Dulbecco's or
Glasgows modified Eagle's medium with 15% (v/v) foetal calf serum,
0.1 mM .beta.-mercaptoethanol and 1000 units/ml of purified
rE-HLIF. ES cell lines were then isolated by two different
methodologies.
[0037] In the first method the blastocysts were allowed to attach
to the culture dish and approximately 7 days later the outgrowing
inner cell mass picked, trypsinised and transfered to another
culture dish in the same culture media. ES cell colonies appeared
2-3 weeks later with between 5-7 individual colonies arising from
each explanted inner cell mass. The ES cell lines were then
expanded for further analysis. The second method for isolation of
ES cell lines used the immunosurgery technique (described in
Martin, G. R. (1981) Proc. Natl.
[0038] Acad. Sci. USA 78:7634-7638) where the trophectoderm cells
are destroyed using anti-mouse antibodies prior to explanting the
inner cell mass. The efficiency of ES cell lines isolation is shown
in Table 1.
[0039] Step 3: Generation of Chimaeric Mice:
[0040] All the ES cell lines cultured in the absence of feeder
cells but in the presence of LIF (referred to in step 1) or
directly isolated with the aid of culture medium containing LIF
(referred to in step 2) retained the ability to differentiate into
multiple cell types following the removal of LIF indicating that
these cells have retained their pluripotential phenotype. To
confirm their developmental potential, D3 ES cells maintained in
LIF for 7-22 passages and MBL-1 ES cells maintained in LIF for
14-17 passages were reintroduced into the embryonic environment by
blastocyst injection (as described in Williams et al., (1988) Cell
52:121-131). Blastocysts were isolated from the outbred ICR mouse
strain or inbred C57BL/6J mice. The expanded blastocysts were
maintained in oil-drop cultures at 4.degree. C. for 10 min prior to
culture. The ES cells were prepared by picking individual colonies,
which were then incubated in phosphate-buffered saline, 0.5 mM EGTA
for 5 min; a single cell suspension was prepared by incubation in a
trypsin-EDTA solution containing 1% (v/v) chick serum for a further
5 min at 4.degree. C. Five to twenty ES cells (in Dulbecco's
modified Eagle's Medium with 10% (v/v) foetal calf serum and 3,000
units/ml DNAase 1 buffered in 20 mM HEPES [pH 8]) were injected
into each blastocyst. Blastocysts were transferred into
pseudopregnant recipients and allowed to develop normally.
Chimaeric mice were identified by coat markers (Hogan et al.,
(1986) Manipulating the Mouse Embryo, Cold Spring Harbor, N.Y.).
Analysis of the subsequent chimaeric mice revealed that up to
approximately 50% of the progeny contained tissues derived from the
injected cells (Table 2), with levels of overt chimaerism as high
as 90% in individual mice. Furthermore analysis of the organs of
four D3-chimaeras confirmed that the ES cells maintained in LIF
could contribute extensively to the development of all of the
somatic tissues (Table 3).
[0041] The male chimaeras were tested for germline transmission of
ES derived cells by mating to ICR or C57BL/6J females. Three out of
four of the D3-C57BL/6J chimaeras and two out of six of the
MBL-1-C57BL/6J chimaeras gave rise to agouti offspring derived from
the ES cells cultured in LIF (Table 4).
[0042] To test whether genetically altered ES cells could be
maintained in culture medium containing LIF, D3 ES cells were
infected with a retrovirus vector (N-TK527) expressing the neomycin
resistance gene and a c-src gene mutant (c-src.sup.527) (protocol
for infection is described in Williams et al., (1988) Cell 52:
121-131). The ES cell clones isolated were maintained in culture
medium containing LIF for over 20 passages. These genetically
modified ES cells retained the ability to form chimaeric mice
following reintroduction into the embryonic environment by
blastocyst injection (Table 2).
1TABLE 1 Isolation of 129 Sv He ES cell lines in media containing
rE-HLIF ICM Number of ES cell Methodology Blastocyst outgrowing
lines derived Explanted 9 9 4 Immunosurgery 11 3 0 Immunosurgery 7
5 2
[0043] Murine blastocysts were isolated from 129 Sv He mice at day
4 of development (day 1=day of plug) into either Dulbecco's or
Glasgows modified Eagle's medium with 15% (v/v) foetal calf serum,
0.1 mM .beta.-mercaptoethanol and 1000 units/ml of purified
rE-HLIF. The blastocysts were then explanted into the same media
and left to attach to the culture dish and the inner cell mass
picked dissociated in phosphate-buffered saline, 0.5 mM EGTA for 5
min; a single cell suspension was prepared by incubation in a
trypsin-EDTA solution containing 1% (v/v) chick serum and the cells
replated in the cell culture medium described above. The
characteristic ES cell colonies appeared within 1-3 weeks.
[0044] Other blastocysts were treated by immunosurgery (as
described in Martin, G. R. (1981) Proc. Natl. Acad. Sci. USA
78:7634-7638). The blastocysts were allowed to hatch from the zona
pelucida, and then treated with anti-mouse antibodies and destroyed
by the addition of complement. The exposed inner cell mass was then
left to attach to a tissue culture dish and again treated with
anti-mouse antibodies and complement. Within a few days
pluripotential stem cell colonies appeared and were dissociated and
trypsinised as described above.
2TABLE 2 Chimaeric mice derived from ES cells cultured in LIF ES
Blastocysts Pups cells transferred born Chimaeras D3 142 60(42%)
33(55%) MBL-1 51 33(65%) 16(48%) D3 N-TK527 42 22(52%) 12(54%)
[0045]
3TABLE 3 Percentage tissue contributions in individual D3 chimaeric
mice Chimaera Necropsy age C Bl Sp P Li T H D3-1 13 d 35 0 35 20 10
20 40 D3-2 14 d 40 15 35 30 45 30 50 D3-3 11 d 90 50 50 35 50 40 60
D3-4 11 d 50 50 50 30 40 40 50 Chimaera Necropsy age Lu G K M B Sa
D3-1 13 d 30 10 35 30 35 20 D3-2 14 d 35 20 30 50 50 25 D3-3 11 d
45 50 50 70 50 55 D3-4 11 d 50 35 50 50 20 30
[0046]
4TABLE 4 Chimaeric demonstrating germline transmission of ES
derived cells. Passage no. of D3 cells Offspring Mice Chimaerism on
feeders in LIF 129 Sv He C57 775-3 75% 10 16 9 24 778-1 70% 10 22 5
33 778-2 50% 10 22 2 36 778-3 55% 10 22 0 0
[0047] The following relates to Tables 2, 3 and 4:
[0048] D3 and MB1-1 ES cells are derived from 129 Sv He mice
(inbred, agouti, homozygous for the glucose phosphate isomerase
1.sup.a allele). The D3 ES cells were originally cultured on
primary embryo fibroblasts for 10 passages and then transferred to
1,000-5,000 units/ml recombinant LIF for 7-22 passages. The MB1-1
ES cells were isolated in the absence of feeder cells but in the
presence of rE-HLIF these cells were cultured for 14-17 passages.
The ES cells were then injected into ICR (outbred, albino) or
C57BL/6J (inbred, black) blastocysts which were then transfered
into pseudo-pregnant foster mothers. Both the ICR and C57BL/6J mice
are homozygous for the glucose phosphate isomerase 1.sup.b allele.
Chimaeric pups were identified by coat pigmentation (only foster
mothers which became pregnant were counted in estimating the number
of progeny). Tissue chimaerism was estimated using glucose
phosphate isomerase strain differences. The extent of tissue
chimaerism was determined in two D3-ICR (numbers 1 and 2) and two
D3-C57BL/6J chimaeras (numbers 3 and 4). Tissues analysed: C, coat;
B1, blood; Sp, spleen; P, pancrease; Li, liver; T, thymus; H,
heart; Lu, lungs; G, gonads; K, kidneys; M, muscle; B, brain; Sa,
salivary gland. Male chimaeras were mated to ICR or C57BL/6J mice
and offspring identified by coat pigmentation.
EXAMPLE 2
[0049] This example sets out the steps used to document specific
high affinity receptors on ES and EC cells. Accompanying FIG. 3
shows binding of .sup.125I-LIF to ES cells EKcs-1 and EC cells F9
and PCC3-A (Jakob, J. et.al. (1973) Ann.Microbiol.Inst.Pasteur,
124B: 269-282). In relation to FIG. 3, (A), Scatchard analysis of
.sup.125I-labelled LIF binding to F9 (.quadrature.), EKcs-1
(.circle-solid.), PCC3A-1 (.box-solid.) and M1 (.smallcircle.)
cells. Saturation curves for binding were analysed by the method of
Scatchard by plotting the amount of LIF specifically bound (defined
as the difference between binding observed in the absence and
presence of excess unlabelled LIF) versus the ratio of bound to
free LIF. Free LIF values were adjusted for the percent of
.sup.125I-labelled LIF capable of binding specifically to LIF
receptors, in this experiment determined to be 75%. The apparent
dissociation constant for the interaction of LIF with its receptor
was determined from the slopes of the curves and the receptor
number from their intercepts with the ordinate. Results in (A) were
standardized to 5.times.10.sup.6 cells per point and the mean of
duplicate points are shown and curves were fitted using the Ligand
program. (B), Autoradiography of F9 EC cells labelled with
.sup.125I-labelled LIF. (C), Quantitation of silver grains on F9 EC
cells after binding of .sup.125I-labelled LIF.
[0050] Purified recombinant (yeast-derived) human LIF (rY-HLIF) was
radioactively labelled on tyrosine residues as described previously
(Hilton, D. J. et.al.(1988) Proc.Natl.Acad.Sci. USA, 85:5971-5975)
producing .sup.125I-LIF with a specific radioactivity of
approximately 1.2.times.10.sup.7 cpm/pmole. .sup.125I-LIF
(2.times.10.sup.3-5.times.10.- sup.5 cpm) was incubated with
1-4.times.10.sup.6 target cells with or without at least 100-fold
molar excess of unlabelled LIF, in a total volume of 100 .mu.l for
4 hours on ice. Cell-associated and free .sup.125I-LIF were
separated by centrifugation through foetal calf serum (Nicola, N.
A. and Metcalf, (1986) D. J.Cell Physiol. 128:160-188). Specific
cell-associated .sup.125I-LIF was determined by cold
competition.
[0051] FIG. 3 illustrates the specific saturable and high affinity
binding of .sup.125I-LIF to the ES cells EKcs-1 and the EC cells
PCC3-A and F9. The number of LIF receptors per cell derived from
these Scatchard plots were 295, 190 and 330, respectively, with
apparent dissociation constants at 4.degree. C. of approximately 90
pM. This compares with the M1 cell line, a LIF-responsive monocytic
leukaemia, which displays 50-200 LIF receptors/cell with an
apparent dissociation constant of 50-150 pM. All other ES and EC
cells tested--D3, NG2, PC13 and P19--bound similar levels of LIF
(data not shown).
[0052] The binding of .sup.125I-LIF to M1 cells, EKcs-1 and PCC3-A
was also found to be in competition with unlabelled recombinant and
native murine and human LIF, but not with the range of other
hormones and factors, (including several which act on embryonic
cells): insulin, IGF-I, IGF-II, acidic and basic FGF, TGFD, TNFA,
TNFD, NGF, PDGF, EGF, IL-1, IL-4, GM-CSF, G-CSF, Multi-CSF and
erythropoietin.
* * * * *