Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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CYTOKINE EXPRESSED BY DIA/LIF-DE~:TCIENT EMBR~ONIC STEM CELLS FOR THE INHIBI-
T~ON OF DIFFERENTIATION
Field of the Invention
This invention reiates to substances having the capacity to inhibit
differentiation of embryonic stem (ES) cells. The invention further provides
methods of deriving and propagating ES cells, ES cells per se and cell lines useful
in deriv;ng and assaying novel factors.
Backqround to the Invention
Embryonic stem cells are the archetypal stem cell, being capable both of
unlimited self-renewal and of differentiating to form the whole gamut of cell types
found in the adult animal. Such stem cells are described as pluripotential as they
are capabie of differentiating into many cell types. These cells will participate fully
in normal embryogenesis following reintroduction into blastocysts and can
contribute functional differentiated progeny to all somatic tissues and to the germ
line. ES cells can also be induced to differentiate into a wide variety of cell types
in culture, recapitulating in vitro processes responsible for tissue diversification in
the developing embryo. ES cells therefore provide a unique system for the analysis
of factors that control early embryonic growth and differentiation.
ES cells have the additional property of being able to participate fully in
normal embryogenesis following reintroduction into host embryos and contribute
functional differentiate progeny to all somatic tissues and to the germ line. Their
~ ability to form gametes allows ES cells to be used as a means of transmitting
genetic modifications into animals. This is exploited for gene discovery and
mutation though gene trapping strategies and most importantly for precise gene
alteration via gene targeting.
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In general, when required for research purposes or for medicai use, stem
cells have to be isolated from tissue samples by various fractionation procedures,
but even after careful segregation of cell types, these stem cell preparations consist
of mixed cell types and while enriched for stem cells, include high proportions of
differentiated cells which are not categorised as stem cells.
Furthermore, most stem cells cannot be grown readily in culture and when
attempts are made to culture stem cells, and more particularly to maintaining
established lines of ES cells, the cells being cultured ~which ordinarily contain a
mixed population of cell types) grow at different rates and stem cells rapidly
become overgrown by non-stem cell types. An exception is that embryonic stem
cells from two specific strains of mice (129 and Black 6) can be cultured in vitro.
Thus established lines of embryonic stem cells can be obtained by culturing early
(3 1/2 day) embryonic cells from murine strain 129 and Black 6, or hybrids thereof.
Proven embryonic stem cells with the capacity for germ line transmission
have to date only been established from specific inbred strains of mice (notablystrains 129 and C57BL/~i). They are derived by culturing early embryonic cells in
the presence of a feeder layer of embryonic fibroblasts and/or of cytokine
leukaemia inhibitory factor (LIF~ or related cytokines which act through the signal
transducer glycoprotein 130 (gp130) (Yoshida eta/., 1994). In the absence of a
source of LIF the stem cells differentiate and cannot be propagated. E~y contrast,
ES cells cultures in the presence of DIA/LIF or of a feeder layer of DIA/LIF-
producing cells maintain their proliferative capacity, retain characteristic stem cell
morphology, and express stem cell markers such as alkaline phosphatase, stage-
specific embryonic antigen-1 and the stem cell-specific transcription factor Oct-3/4.
ES cells expanded in the presence of LIF remain pluripotential and competent to
produce germline chimaeras when reintroduced into mouse blastocysts
(Nichols etal., 1990; 1994).
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It is known that cytokines play an important role in the maintenance of the
pluripotentiai phenotype of ES ceils in vitro. Thus the investigation of factorswhich regulate proiiferation and differentiation of ES cells led to the purification of
a glycoprotein of apparent molecular weight 45 kDa named " Differentiation
inhibiting activity" or DIA. This is identical to the cytokine "Myeloid leukaemia
inhibitory factor" or LIF. DIA/LIF acts to sustain the self-renewal of undifferentiated
ES cells and thereby allows their propagation in vitro. ES cells maintained in the
presence of this factor retain their full development potential. Moreover ES cells
can be established de novo by direct cutture of blastocysts in medium
supplemented with DIAILIF. (The term "cytokine" as used herein refers to any
substance which acts from outside a cell and is capable of affecting cell survival
and/or growth andlor differentiation. Primarily the term denotes a proteinaceousfactor capable of inhibiting differentiation of ES ceils).
In the absence of a source of DIAILIF, the stem cells differentiate and cannot
be propagated. By contrast, ES cells cultured in the presence of DIA/LIF or of afeeder layer of DlA/LlF-producing cells maintain their proliferative capacity, retain
characteristic stem cell morphology and express stem cell marker proteins such as
alkaline phosphatase and stage-specific embryonic antigen-1 (SSEA-1 l (Williams et
al., 1988; Smith et al., 1988). Most significantly, ES cells expanded in the
presence of DIAILIF remain pluripotential and competent to produce chimeras whenreintroduced into mouse blastocysts (Nichols et al., 1990; Pease et al., 1990).
Further study has revealed that undifferentiated ES cells express low levels
of mRNA which gives rise to the matrix-localised form of DIAILIF, whereas
differentiated cells express relatively high levels of both soluble and matrix-
associated DIAILIF. It has been proposed that the enhanced production of DIAILIFby newly differentiated cells may provide a mechanism for regulating the balancebetween differentiation and self-renewal in stem cell populations. The physiological
importance of DIAILIF has been established by the finding that homozygous
DlAlLlF-deficient female mice are sterile due to an incapacity to support embryoimplantation. However, DIA/LIF -/- embryos are viable. This observation suggests
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that other factors may compensate for the absence of DIA/LIF expression during
early development. It has recently been shown that a number of cytokines,
including DlA/LlF, interleukin-6 (IL-6), ciliary neurotrophic factor (CNTF) and
oncostatin M (OSM), share the same effector molecule gpl 30. This presumably
underl;es the considerable overlap in biological activities reported for these factors.
Activation of gp130 signalling processes by any of these cytokines is sufficient to
support ES cell self-renewal. However, it is not yet clear which, if any, of these
molecules plays a role in the maintenance of stem cell renewal early during
embryogenesis or whether other factor~s) are involved in this process.
There hag developed a pressing need to isoiate and maintain in vitro
embryonic stem cells from other murine strains and more especially from other
species including other laboratory animals (eg. rats, rabbits and guinea pigs),
domesticated animals (eg. sheep, goats, horses, cattle and pigs~ and primates
including human. The demand for this is twofold. Firstly to extend gene targeting
and other sophisticated genome manipulation technologies such as chromosome
modification (Smith A.J.H. et al., 1995) into other species, notably the rat, which
is the experimental model of choice for the pharmaceutical industry, and the pig,
which requires genetic adaptation for use in xenotransplantation. The second need
is for the development of human ES cells as a universal source of donor cells for
transpiantation. However, no known techniques exist for generating and
maintaining established lines of true embryonic stem cells other than from the
mouse strains noted above and from hybrids thereof. Also, the only known
method of maintaining ES cells used either DIA/LIF or related cytokines as
described above or feeder layers as a source of such cytokines. A further
disadvantage of the known ES derivation methods is that expression of gp130 by
donor embryo cells is essential.
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Statement of Prior Art
A paper entitled "Pluripotent Embryonic Stem Cells from the Rat Are Capable
of Producing Chimeras", lannaccone, P.M. eta/., Dev. E;iol. 163, 288-292 (1994~
describes a cell line designated RESC-01 which is stated in the paper to consist of
"diploid rat embryonic stem cells". Further the RESC-01 cell line is stated to be
capable of being "used to produce chimeras by injection into rat blastocysts". The
aforementioned paper does not characterise the RESC-01 cell line by reference toits karyotype and in the absence of such characterisation, the data presented isinsufficient to support the allegation that the RESC-01 celi line consists of rat ES
cells. It is understood from subsequent disclosed information (personal
communication, not published) that the RESC-01 cell line has a mouse karyotype,
not rat. It is apparent therefore that lannaccone et al's stated aim "The ability to
establish a stem cell population from the rat and make chimeras with them is thefirst step toward providing an important addition to the repertoire of genetic
manipulation techniques in mammals" has hitherto been unfulfilled.
Further a paper entitled "Isolation of a primate embryonic stem cell line ",
Thomson J.A. etal., Proc. Natl. Acad. Sci. U.S.A. describes a cell line designated
R278.5 which is stated to be an ES cell line. However although R278.5 possess
a number of the characteristics of ES cells, it has not been shown to be a true
ES cell line by at least one of the following criteria:
(i~ extensive contribution to chimeras without tumour formation,
(ii) reconstitution of host tissues,
(iii) contribution of functional gametes to chimeras and generation
of offspring.
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Summarv of the invention
In order to define the contribution of DIA/LIF in vitro and establish an assay
system for detecting factors which can regulate ES self-renewal, we have
generated ES cells in which both copies of DIA/LIF gene have been deleted. We
report here that different;ated DlA/LlF-deficient ES cells synthesize a novel soluble
factor which inhibits ES cell differentiation. This factor is distinct from previously
characterised ES cell maintenance factors and most significantly acts independently
of direct activation of gpl30. ES cells have aiso been generated which lack LIF
receptor. The LlF-negative and LIF receptor-negative cells (LIF(-) and rLlF~-)) are
additionally useful in assay procedures which form further aspects of the invention.
According to one aspect of the present invention, there is provided a novel
cytokine designated ESRF (ES cell Renewal Factor) and characterised by the
capacity to inhibit differentiation of ES cells in the absence of DIAILIF and without
direct interaction with gpl30.
More specifically, there is provided a cytokine designated ESRF and
characterised by the capacity to inhibit differentiation of ES cells (i) in the absence
of DIAILIF and ~ii) in the absence of cytokines which act through gp130 and ~ inthe absence of interaction with gp t 30. The capacity to inhibit differentiation of ES
cells (iv) in the absence of interaction with LlF-receptor may further be used to
characterise the cytokines of the invention.
The cytokine of the invention may be further characterised by features
selected from the following:
- being distinguishable from DIAILIF
- being distinguishable from IL-61slL-6R
- being distingufshable from CNTF
- being distinguishable from oncostatin M
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- being distinguishable from cardiotrophin-1
taken individually or in combination. ~"IL-6/slL-6R" is an abbreviation of
"interleukin-6/soluble interleukin-6 receptor").
The cytokine designated ESRF herein is intended to include ESRF derived
directly or indirectly from any mammalian species, including laboratory animals (i.e.
rodents~, domestic and farmyard animals (e.g. dogs, cats, sheep, horses, cows,
pigs etc.) as well as primates including humans. The factor may be obtained by
isolation of endogenously produced factor in an appropriate cell line, or by
recombinant expression.
As indicated, the cytokine of the invention is believed to inhibit
differentiation by a mechanism which is distinct from gp ~ 3(~ and this feature may
be used as a further characterising feature thereof.
Additionally its capacity to inhibit differentiation of ES cells cannot be
eliminated by neutralising anti-DlA/LlF antiserum, nor by neutralising anti-lL-6soluble receptor antiserum, nor by neutraiising anti-CNTF antiserum.
The novel cytokine of the invention was discovered by generating cell lines
that were DIA/LIF deficient and identifying in supernatants from such ceil lines an
activity that inhibited differentiation of ES cells. The supernatants from these cells
in crude or partially purified form also form part of the present invention. Thus the
present invention further provides an at least partially purified composition
obtainable from supernatants of DIA/LIF deficient cells and comprising at least one
polypeptide having the following characteristics
- the capacity to inhibit differentiation of ES cells (i~ in the absence
of DIA/LIF and (ii) in the absence of cytokines which act through
gp130 and (iii) in the absence of interaction with gp130.
The capacity to inhibit differentiation of ES cells (iv) in the absence of
interaction with LlF-receptor may further be used to characterise the partially
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purified compositions. These may optionally further be characterised by featuresselected from the following:
- being distinguishable from DIA/LIF
- being distinguishable from IL-6/slL-6R
- being distinguishable from CNTF
- being distinguishable from oncostatin M
- being distinguishable from cardiotrophin-1
taken individually or in combination.
At least partially purified polypeptide components of said compositions also
fall within the present invention.
The invention may alternatively be defined as a cytokine designated ESRF
which is obtainable by culturing cell line D7A3-PE and is characterised by
- the capacity to inhibit differentiation of ES cells
- being distinguishable from l~IA/LIF
- being distinguishable from IE-6/slL-6R
- being distinguishable from CNTF
- being distinguishable from oncostatin M
- being distinguishable from cardiotrophin-1
The invention further provides a method of producing the novel cytokine
ESRF which comprises culturing a ESRF-producing cell line and isolating ESRF from
the supernatant therefrom. Suitably the ESRF-producing cell line comprises the cell
line D7A3-PE or a cell derived therefrom. The deposited cell line D7A3-PE
constitutes a further aspect of the present invention.
The invention further provides culture procedures which utilise ESRF. One
is a method of propagating ES cells which comprises propagating the cells in thepresence of the cytokine designated ESRF. Another is a method of establishing EScells which comprises culturing early embryos in the presence of the cy~okine
designated ESRF. These procedures can utilise ESRF as the sole ES cell-
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propagation enhancing agent, or others may be included in the culture medium, for
example DIA/LIF or other cytokines including ones which act through gp130.
Propagation and/or establishment of ES cells in accordance with the invention may
be monitored by culturing cells which include a selectable marker such as Oct-
4Rgeo. Further the method of the invention may be utilised to promote the
propagation of somatic stem cells such as haematopoietic stem cells.
In a particular embodiment of the invention, the cytokine of the invention,
in combination with a second cytokine known to act via gpl 30, is used to derive,
propagate and/or maintain a culture of embryonic stem celis in an undifferentiated
state. For exarnple, a combination of the cytokine of the invention with LIF is
suitable for deriving, propagating and/or maintaining embryonic stem cells of rats
or other species.
In this procedure for generating ES cells it is advantageous to culture
embryos, which preferably are zona-free, on a feeder layer of ceils which express
matrix-associated LIF.
Suitable feeder layer cells are derived from fibroblasts. Examples include
mouse embryo fibroblasts that have been transfected so as to express matrix-
associated LIF. A suitable stably transfected cell line is designated DIA-M and has
been deposited at the European Collection of Animal Cell Cultures (ECACC~ on
31 May 1996 under Accession No. 9~;053101.
DIA-M was produced as a clonal derivative of C3HlOT1/2 mouse embryo
fibroblasts stably transfected with an expression vector containing a cDNA for the
matrix-associated form of mouse LIF (Rathjen etal., 1990) and an IRES-linked neoselectable marker. (See also WO 94/24301 for details of the expression system).
Thus in its more specific aspects there is provided a new methodology for
isolating and propagating embryonic stem cells from other species. The approach
is based on a novel combination of soluble LIF plus the cytokine Embryonic Stem
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Cell Renewal Factor (ESRF) plus a feeder layer of genetically modified fibroblasts
which overexpress the matrix-associated isoform of LIF ~Rathjen ef a/., 1 g90J.
Using this protocoi rat embryonic stem cells can be propagated indefinitely
whilst retaining a euploid rat karyotype and characteristic stem cell morphology and
marker expression. Furthermore a novel method for freezing and thawing the ES
cells is described and procedures are given for chimaera production.
According to a further aspect of the invention there is provided a method for
assaying and/or detecting growth factors that affect differentiation by a meohanism
that is distinct from mechanisms involving DIA/LIF and/or gpl30 interactions,
which comprises culturing ES cells in the presence of a sample to be assayed, and
detecting variation in growth or diffe~entiation compared to cells cultured in the
absence of the sample, characterised in that the ES cells have an LlF-negative
(LIF(-)) and/or LIF receptor-negative (rLlF(-)) phenotype.
A specific embodiment of the invention is a cytokine having the following
physicochemical properties:-
~ apparent MW equal or greater than 100,000 Daltons in salineor 4M urea
~ isoelectric point = 4.25-4.5
~ stable above pH3, but inactivated below pH3
~ inactivated by 0.5M NaOH
~ sensitive to reduction by 10mM dithiothreitol, 30min, 37~C.
~ inactivated by incubation with trypsin
inactivated by incubation at 50~C
~ stable to proionged (6 months) storage at 4~C
~ stable to freeze/thaw
~ stable to exposure to 4M urea
~ stable in 1% CHAPS or CHAPSO
~ inactivated by 0.01% SDS
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The invention further provides an established line of embryonic stem cells
characterised by possessing at least five and preferably at least seven of the
following features:
(i) the characteristic morphology of stem cells, including
- growth in clumps as small tightly packed cells with a
high nuclear to cytoplasmic ratio,
(ii) expression of one or more specific markers selected
from (a) alkaline phosphatase, (b) stage-specific
embryonic antigen-1, (c) Oct-3/4,
(iii~ non-expression of differentiation markers, for example H19 RNA,
(iv) sub'stantial or uniimited propagation potentiai,
(v) stabiiity to freezing and thawing,
(vi) stable euploid karyotype,
(vii) propagation dependent on cytokines,
(viii~ in vitro differentiation inducible by withdrawal of
cytokine(s), aggregation or chemical differentiation
inducers,
(ix) ability to form teratocarcinomas comprising derivatives
of endoderm, mesoderm and ectoderm,
(x) ability to colonise and/or reconstitute host tissues through the
production of somatic stem cells and functionally differentiated
progeny,
(xi) ability to colonise host embryos with contribution of
functional differentiated progeny to chimeras,
(xii) ability to produce functional gametes in chimeras and
generation of viable offspring,
(xiiil ability to integrate exogenous DNA,
and further characterised in that said cell line has a karyotype other than mouse.
Such cells preferably possess at least five and preferably at least seven of thespecified features (i) to (viii), and most preferably are further characterised by
possessing at least one of the specified features (ix) to (xiii).
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It will be appreciated that established lines of embryonic cells provided
according to the invention need not necessarily exhibit each and every one of the
features ~i) to (xiii) listed above. Thus for certain species, one or more
characteristics may be absent. For example in feature ~ rodent embryonic stem
cells may display all three of the specific markers mentionad above, i.e. (a) alkaline
phosphatase, (b) stage-specific embryonic antigen-1 and (c~ Oct-3/4,. However,
stage-specific embryonic antigen-1 (feature (ii~(b~ may be absent or undetectable
in embryonic stem cells of other species, especially large mammals such as
primates .
Further, in respectof mammals having long gestation periods and/or reaching
sexual maturity after a long period of time, it may not be practical to demonstrate
the existence of features tx) and ~xi~. Also moral and legal constraints may make
it impossible to demonstrate features ~x) and (xi~ in certain species such as human.
Description of Figures
The invention will now be described in more detail by way of example with
particular reference to the accompanying drawings of which:
Figure 1 illustrates the response of lif-r-/- ES cells to cytokines
Figure 2 shows a phase contrast photomicrograph of rat ES cells
Figure 3 shows a metaphase chromosome spread from rat ES cells
Figures 4A show marker expression in rat ES cells of A. Alkaline
& 4B phosphatase and B. Oct-4 immunostaining
Figures 5A, show differentiation of rat ES cells into A. Trophoblast,
5B & 5C B. Parietal endoderm and C. Bipolar cells
The production, isolation and characterisation of ESRF in accordance with
the invention will now be described in more detail by way of example.
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EXAMPLE 1
1.1 Generation of DlA/LlF-deficient ES cell lines
In order to abolish DIA/LIF function in ES cells, both alleles of the gene were
deleted via two rounds of homologous recombination. ES cells in which one copy
of the gene is inactivated were generated using a replacement vector in which the
entire coding sequence of the DIA/LIF gene is replaced by a selectable HPRT mini-
gene. After transfection into HPRT-deficient E1 4TG2a cells, 1 5AT-resistant ES cell
clones which had undergone the recombination event were identified by DNA
hybridisation analysis using a probe external to the 3' arm of homology. To verify
the expected replacement event, DNAs of positive clones were restricted with theappropriate restriction enzyme and hybridized to a 5' external probe. In total, 11
correctly targeted clones were identified from a primary screen of 89 colonies.
One germline competent clone, D6, was used for the deletion of the second
allele. A hygromycin resistance cassette was substituted for the HPRT marker in
the targeting vector. In parental cells this construct gave rise to homologous
replacement events at a comparable frequency (10%) to that obtained with the
HPRT vector. This second construct was electroporated into the D6 clone and
transfectants were isolated by selection in hygromycin B. Three classes of
homologous recombinant were identified amongst 162 colonies screened :
recombination into the previously targeted allele had occurred in 30% of clones;integration into the 5' or 3' homology region of the wild-type allele was detected
in 4 cases; and only three ctones had undergone replacement of the wiid-type
allele. Deletion of DIA/LIF coding sequences was confirmed for these three ciones
by the absence of hybridisation with a full-length cDNA probe whereas clones
which had undergone recombination into only one homology arm retained the gene.
The loss of DIA/LIF mRNAs was confirmed using a highly sensitive ribonuclease
protection assay. E14TG2a ES cells express relatively high levei of both matrix-associated and diffusible forms of DIA/LIF mRNAs after induction of differentiation,
as previously described for other ES cell lines. In contrast, no protected fragment
could be detected in RNA preparations from undifferentiated or differentiated
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doubie knock-out ES cells. These experiments confirm unambiguously that the
DIA/LIF deletion is a null mutation. The similar phenotype of two independent
clones, named D1 C2 and D7A3, which were isolated from separate plates during
the drug selection is reported in the following experiments.
1.2 Capacity of homozygous DlAlLlF-negative ES cells for differentiation
in vivo
~ IA/LIF-deficient ES cell clones were used to generate chimaeras. DIC2 andD7A3 ES cells were injected into C57~L/6 blastocysts and chimaeric offspring were
produced. Chimaeras showed high ES cell contribution as judged by the sandy
coat colour. THis was confirmed by the determination of the degree of ES cell
contribution to blood and tail of five chimaeric mice by glucose phosphate
isomerase isozyme analysis. Both clones contributed to gametogenesis as
indicated by the production of viable germ-line offspring. These results establish
that the two rounds of selection applied to generate double knock-out ES cell lines
have not abolished their normal developmental potential.
1.3 Stem cell renewal can occur in the absence of DIA/LIF
The differentiation of ES cells in high density monolayer culture does not
result in the complete elimination of stem cells. We have previously proposed a
model of feedback regulation of stem cell renewal in which the synthesis of DIA/LIF
by newly differentiated ES cells contributes to the rescue of residual
undifferentiated stem cells. The validity of this hypothesis was examined by
investigation of the capacity of DlA/LlF-deficient ES ce~ls for stem cell rescuefollowing induction of differentiation. Wild-type, heterozygous and homozygous
mutant ES cells were induced to differentiate at high cell density by exposure to
3-methoxybenzamide ~ME3A~ for three days. After a further four days is basal
medium, the number of undifferentiated ES colonies was deterrnined by both
morphological inspection and alkaline phosphatase staining. A similar number of
undifferentiated ES colonies were recovered from the wild-type and two
heterozygous ES cell cultures. In contrast, the number of ES colonies was around3-fold lower for the two DlA/LlF-negative ES clones. This decrease confirms that
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the autocrine and paracr;ne production of DIA/LIF is a major component in the feed
back regulation of stem cell renewal in differentiating ES cell cultures.
Significantly, however, stem cell renewal was not completely abolished in the
absence of DIA/LIF, suggesting that another regulatory factor is operative in this
system.
1.4 Inhibition of ES cel~ differentiation by a soluble factor (ESRF3
In order to preclude the possibility that the culture conditions imposed a
selection for rare differentiation-defective variants, the stem cell rescue capacity
of DlA/LlF-deficient ES cells was further investigated in co-culture assays.
Convenient indicator cell lines were generated by integration of a 13-galactosidase
reporter gene into the Oct-4 locus of DlA/LlF-negative ES cells by homologous
recombination. The expression of l~-galactosidase in such targeted clones is
restricted to undifferentiated stem cells. The indicator cells were plated on layers
of MBA-induced differentiated wild-type or mutant clones. After 4 days, the
number of undifferentiated ES cells colonies derived from the indicator population
was determined by staining for 13-galactosidase activity. Once again, the results
indicate that the capacity of DlA/LlF-deficient differentiated cells to inhibit ES cell
differentiation is reduced relative to the parental cell line, but is not abolished.
Similar results were obtained using retinoid-induced differentiated cells as thefeeder layer.
To determine whether the effect was due to a diffusible factor, a second
kind of co-culture experiment was carried out in which the indicator cells were
plated on a microporous insert above the layer of differentiated cells. The insert
membrane prevents cell-cell contact between the two cell populations but allows
the access of diffusible factors. Both parental and Oct-4-tagged ES cells were used
as indicators, staining for alkaline phosphatase and f3-galactosidase respectively.
Similar results were obtained in both cases. The greater activity in wild-type
cultures is significantly reduced in the presence of neutralizing DIA/LIF antiserum,
but is not eliminated. This indicates that wild-type cells also synthesize active
factors other than DIA/LIF. The residual activity in the presence of anti-DlA/LlF
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was similar to that produced by D!A/LlF-negative cells which suggests that
expression of the responsibie factor is not significantly up-regulated ;n these cells.
These data establish that a solubie factor or soluble factors other than
DlA/LiF which is able to prevent ES cell differentiation is synthesized by both wild-
type and DlA/LlF-deficient differentiated cells. However, the decreased numbers
of colonies generated in response to the mutant cells or to wild-type cells in the
presence of neutralising anti-DlA/LlF indicate thatthis factor is produced in limiting
amounts .
1.5 Isoration of a differentiated cell line expressing high leveis of a stem
cell renewal factor ~ESRF)
In order to facilitate characterization of the stem cell renewal activity, we
established differentiated cell lines from embryoid body outgrowths of the DIA/LIF-
negative ES cells. Several of these cell lines produced levels of activity which were
readily detectable in their culture supernatants. One cell line, named D7A3-PE,
could be propagated indefinitely and produced high levels of a soluble factor which
could maintain undifferentiated ES cells. In addition to their characteristic
morphology, ES cells cultured in D7A3-PE conditioned medium continued to
express markers of the undifferentiated state such as alkaline phosphatase and
Rex-1 mRNA and in the case of Oct-4 tagged cells retained 13-galactosidase
activity. This conditioned media was effective on several independent ES cell lines.
ES cells cultured in the presence of conditioned media or partially purified ESRF
formed tight, rounded up colonies, morphologically distinguishable from both
differentiated ceils and ES cells maintained in DIA/LIF. ES cells serially passaged
in the presence of ESRF and retained the capacity for multilineage differentiation.
The active factor could be concentrated at least 20-fold by ultrafiltration and was
destroyed by incubation with trypsin, consistent with a proteinaceous
macromolecule. The factor was inactivated by heating to 50~C or by acidificat;onbelow pH3.
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1.6 Purif;cation and biochemical properties
For purification of ESRF, conditioned medium is prepared from D7A3-PE cells
in the absence of serum. Cells are inoculated into 1 50cm2 tissue culture fiasks in
normal growth media and grown to near confluence. Cultures are then rinsed with
PBS and transferred into defined medium consisting of a 50:50 mix of GMEM and
Ham's F12 basal media supplemented with 1 QO,rlM 2-mercaptoethanol, 1,ug/ml
insulin, 5,ug/mi transferrin and 1 OnM sodium selenite. Cultures are incubated for
4 hours then the medium is discarded and replaced with fresh defined medium
(50ml/flask). This medium is conditioned by incubation with the D7A3-PE cells at37~C for 96 hours. The medium is then harvested and replaced. Three successive
harvests can be obtained from each cuiture. The harvested medium is clarified bycentrifugation and passed through a sterile 0.2~m filter. The ability to inhibit ES
cell differentiation is routinely detectable at a 1 in 10 dilution of the unfractionated
conditioned medium though full activity over a four day assay requires no greater
than 25% dilution. Activity in the conditioned media is stable upon freezing andthawing, but is lost upon incubation at 50~C for 30 min, acidification to pH2 orincubation with trypsin. Activity can be concentrated by uitrafiltration in an
Amicon cell using a membrane with a nominal molecular weight cut-off of 100,000
Daltons. Quantitative recovery of biological activity is obtained, indicating that
native ESRF has a molecular weight in excess of 100,000 Daltons.
ESRF is quantitativeiy recovered from conditioned medium by stepwise
precipitation with saturated ammonium sulphate. ESRF activity is recovered in the
25-35% fraction. This fraction contains 20-25% of total protein. Upon
reconstitution in 1/100 volume of starting medium, a viscous solution is obtained
which partitions into fluid and gel phases upon cold storage. Both phases contain
biologically active ESRF. The gel material is likely to consist of highly glycosylated
extracellular matrix components. ESRF can be extracted from the gel by incubation
with salt buffers or more efficiently with 0.1 % CHAPS. This finding suggests that
ESRF has avidity for ECM components. Consistent with this, significant levels ofESRF activity can be extracted directly from monolayers of D7A3-PE cells by
incubation for 2 hours with basal medium containing 0.1% CHAPS. (Under such
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18
conditions the ceils remain viable with no evidence of significant Iysis). It ispossible that ESRF may exist in isoforms which associate differentially with
extracellular matrix. Alternative protocols for obtaining an ESRF-containing fraction
by ammonium sulphate precipitation are given in Appendix 9.
Ultracentrifugation studies (see appendix 10) suggest that ESRF activity is
not significantly associated with plasma membranes or subcellular organelles butis a soluble protein or protein complex.
The maximum concentration required for biological activity is 1 .5nM, based
on the protein 'content of urea resolubilised ammonium sulphate precipitated
material (see ~ppendix 9) and assuming a molecular weight equal to 100,000
Daltons.
Further fractionation of ESRF may be achieved on lectin columns. ESRF
binds to soya bean lectin and to lentil lectin, but not to wheat germ agglutinin(appendix 1 1~. Bioactivity is not affected by the addition of heparin to the assay
medla.
Upon size exclusion chromatography of the ammonium sulphate fraction
using TSK G3000SWXL HPLC column in 50mM sodium phosphate buffer, pH7.2,
ESRF migrated as a single peak with a retention time shorter than BSA, indicative
of a molecular weight of 100-150,000 Daltons.
ESRF can be further purified by solution phase electrofocussing of the
ammonium sulphate fractionated material. Focussing may be performed in the
presence of 1% CHAPS and 2% ampholytes in a BioRad Rotofor cell. ESRF is
recovered quantitatively in 2-4 fractions of pH 4.25-4.5. ESRF is insoluble in these
fractions, but resolubilizes upon direct addition to culture medium for the ES cell
bioassay or upon adjustment of buffer pH. SDS gel electrophoresis shows that twomajor proteins with apparent molecular weights of 1 15,000 and 180,000 Daltons
are specific to the active fractions.
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Further purification of ESRF may be achieved by heparin agarose affinity
chromatography. The sample is loaded in 20 mM phosphate buffer, p~i3.~,
containing 0.1 % CHAPS. Under these conditions most material does not bind to
the column and a significant purification of ESRF can be obtained. ESRF is not
eluted with 1 OOmM salt, but elutes over a broad range of higher salt
concentrations. The heterogenous elution profile is suggestive of variable
glycosylation. (Similarly heterogenous elution profiles are also obtained on anion
exchange chromatography). Bioactive fractions contain very low levels of protein(undetectai~le at 280nm). Protein is also undetectable by either coomassie blue
staining, conventional silver staining, or silver staining combined with periodic acid
preincubation (for improved detection of glycoproteins~ of SDS PAGE gels loaded
with up to 10-fold higher amounts of material than required for full biological
activity. The low amount of protein present indicates that ESRF is fully active at
nanomolar concentrations.
Biotinylation of pooled fractions of active material followed by SDS PAGE in
the presence of reducing agent and immunoblotting revealed the presence of a
heterogeneous doublet of apparent molecular weight 70-80,000. No other bands
were apparent. This observation confirms that these fractions are highly purified.
It further indicates that native ESRF may be a disulphide-linked dimer.
The concentration of IL-6 in the conditioned medium was determined using
the highly sensitive B9 cell proliferation assay. Conditioned medium did induce
proliferation of B9 cells but the mitogenic response 'equated to a concentration of
less than ~ pg/mi IL-6. That this activity was due to IL-6 was confirmed by the use
of a neutralizing IL-6 soluble receptor antibody, RS 13. The bioactivity was
inhibited totally in the presence of RS13. In contrast, inhibition of ES cell
differentiation by D7A3-PE cell conditioned medium was not modified by the
addition of RS13, indicating that this low level of IL-6 is not responsible for the
effect of this media. This conclusion was further substantiated by the finding that
the conditioned medium was not mitogenic for BAF-mgp130 cells. The latter are
responsive to IL-6 only in the presence of soluble IL-6 receptor, so the negative
~
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result in this assay indicates that the D7A3-PE cell conditioned media does not
çontain any IL-6 soluble receptor.
The potential involvement of CNTF was examined both by specific
immunodepletion of conditioned medium with the 4-68 anti-CNTF mAb, and by the
use of a neutralizing anti-CNTF antiserum. Neither treatment affected the ability
of D7A3-PE cell conditioned medium to inhibit ES cell differentiation. Furthermore,
activity was retained on addition to RS13 anti-lL-6R antiserum to medium
previously depleted with anti-CNTF.
These results establish that neither IL-6/slL-6R nor CNTF are responsible for
the inhibition of ES cell differentiation by differentiated DlA/LlF-deficient ES cells.
1.7 The self-renewai factor is distinct from mouse oncostatin M and from
cardiotrophin- 1
\/louse cardiotrophin-1 has a molecular weight of 22,0Q0 ~altons. Mouse
oncostatin M has a molecular weight of 30-40,000 Daltons. ESRF, in contrast, hasan apparent molecular weight in excess of 100,000 Daltons. It is quantitatively
retained by ultrafiltration membranes with a cut-off of 100,000 Daltons even in the
presence of 4M urea and has a mobility on size exclusion chromatography
corresponding to a molecular weight greater than 100,000 Daltons.
1.8 ESRF is active on LlF-receptor deficient ES cells
LIF and related cytokines act through a heterodimeric receptor consisting of
the low affinity LIF receptor and gp130. ES cells lacking LIF receptor were
generated by two rounds of gene targeting. These cells can be propagated using
the combination of IL-6 and soluble IL-6 receptor which acts via gp 130 homodimers
without involvement of LlF-receptor. LlF-receptor negative ES cells are pluripotent
and contribute to multiple tissues in chimaeras. They show no self-renewal
response to LIF, CNTF, oncostatin M or cardiotrophin-1. As shown in Figure 1,
however, LlF-receptor deficient ES cells remain responsive to ESRF. This findingconfirms that ESRF is distinct from the LIF group of cytokines.
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1.9 The self-renewal facto- (ESRF) does not act through direct interaction
with gp~t30
All the cytokines described to date which are capable of sustaining ES cell
self-renewal act through receptor complexes containing the gp 130 signal
transducer. Monoclonal antibodies have been raised against mouse gp130 which
are capable of blocking gp1 30-mediated signal transduction (Saito et al.,
unpublished data). In the presence of these antibodies, ES cell responses to
DIAILIF, IL-6/slL-6R, CNTF and human OSM are inhibited and the cetls differentiate
(Saito et al, unpublished data). The activity of WEHI-3E~ cell conditioned medium
(a source of mouse cardiotrophin-1~ on ES ceits atso is completely abotished by anti
mouse gp 1 30 .
The effect of two different neutralising anti-mouse gp130 antibodies on the
maintenance of ES cell self-renewal by ESRF was investigated. In the presence ofESRF, neither antibody inhibited the production either of alkaline phosphatase-
positive undifferentiated colonies by wild-type ES cells, or of 13-galactosidase-
positive, G41 8-resistant colonies by Oct-4-targeted cells (Mountford eta/, 1994).
The integrity of the antibodies at the end of the assay was confirmed by retention
of the ability to block mitogenic stimulation of BAF-mgp130 cells by IL-6/slL-6R.
The resutts of these experiments with neutralising antibodies against mouse
gp130 indicate that the effect of ESRF on ES cell maintenance is not mediated via
direct activation of gp 130, in contrast to the actions of ali previously described ES
ceil self-renewal factors.
Addition of cytokines which act via gp130 to ES cells results in activation
of STAT3 and induction of the immediate early gene tisl l. Under the same
conditions, however, ESRF administration causes no apparent increase in STAT3
activity as determined by gel shift analysis and no induction of tis 1 1 transcripts by
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RNase protection assay. This indicates that ESRF action is mediated via a distinct
intracellular signalling pathway(s) from gp130 signal transduction.
The biological response of ES cells to ESRF is also distinguishable from their
response to LIF or reiated cytokines. The ES cells are characteristically more
compact in the presence of ESRF and form very tight, rounded, colonies which
have a tendency to detach from the culture surface. On transfer to LlF-containing
medium these colonies adopt a siightly more flattened and spread appearance,
typical of ES cells cultured in LIF. Undifferentiated colonies can be maintained for
at least 11 days using ESRF alone. However, the colony size does not appear to
increase after the first 4 days, in contrast to the effect of LIF which promotescontinuous stem cell expansion. Additive or synergistic effects on stem cell
propagation are apparent on combination of sub-optimal amounts of LIF and ESRF.
These data are consistent with distinctive effects on the self-renewal process.
1.10 Mouse ES cells maintained using ESRF remain pluripotential
ESRF sustains the undifferentiated phenotype of ES cells in vitro. This effect
persists for at least 11 days. However, proliferation is limited and cultures cease
to expand after about four days. This contrasts with cultures propagated in the
presencè of LIF which undergo a continuous increase in stem cell numbers. To
determine whether ES ceils maintained in ESRF are capable of contributing to
chimaeras, cells were cultured at cional density in the presence of ESRF for four
days. They were then transferred to LlF-containing medium to facilitate expansion
of the stem cells prior to blastocyst injection. This protocol was applied to the ES
cell line Zin40 which carries a constitutively expressed nuclear localised 13-
galactosidase marker.
Zin40 cells plated in control medium underwent complete differentiation and
did not give rise to any ES cell colonies upon replating in the presence of LIF. Cells
plated in ESRF, however, remained undifferentiated as described above and
engendered many alkaline phosphatase positive stem cell colonies on replating.
Cells from replated and expanded cultures contributed extensively to chimaeras as
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23
determined by the widespread presence of 13-galactosidase positive cells in mid-gestation embryos. These chimaeric embryos were morphologically normal. The
levels of Zin40 contribution were comparable to those obtained from parallel
injections of cells maintained in LIF only.
1.11 Uses of ESRF
Regulation of the balance between stem cell renewal and differentiation is
crucial for tissue diversification during embryogenesis and for tissue renewal and
repair in adult mammals. The generation of DlA/LlF-deficient ES cells allowed usto study the role of this cytokine in stem cell renewal and differentiation and to
characterize other factors involved in the regulation of this process. The results
establish that DlA/LlF-deficient differentiating cultures retain some capacity for
maintaining a population of undifferentiated cells. The findings indicate that factors
and signalling pathways other than those characterised to date have the capacityto maintain the self-renewal of pluripotential ES cells. This may prove of
fundamental importance for the isolation and propagation of stem cells from other
species .
The propagation and differentiation of murine pluripotential embryonic stem
(ES) cells is controlled by specific cytokines. The proliferation of ES cells in vitro
is sustained through the activation of intracellular processes associated with the
signal transducer gp130. In an attempt to define the relative contributions of
different cytokines to self-renewal in ES cell cultures we generated ES cells lacking
a functional gene for the cytokine Differentiation Inhibiting Activity ~DIA/LIF).
These cells show a significantly reduced capacity for stem cell renewal when
induced to differentiate, indicating that DIA/LIF is the major regulatory cytokine
present in normal ES cell cultures. However, undifferentiated ES cell colonies are
still produced in the complete absence of DIA/LIF. This is due to the secretion of
a soluble, macromolecular, activity by differentiated ES cell progeny. In addition
to DIA/LIF, the cytokines ciliary neurotrophic factor (CNTF), interleukin-6 in
combination with soluble interleukin-6 receptor ~IL-6/slL-6R~, cardiotrophin-1 and
oncostatin M can each activate gp130 and support ES cell propagation. The
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involvement of either CNTF or IL-~/slL-6R in our system has been precluded
through the use of neutralising antisera against these factors. Most significantly,
the effect of all the aforementioned cytokines on ES cells is abolished in the
presence of neutralising antibodies against mouse gp130, whilst the activity in
D7A3-PE conditioned media is unaffected. These findings establish that ES cell
self-renewal can be sustained via a gp 1 30-independent signalling pathway and that
differentiated ES cell derivatives secrete a factor which activates this pathway.
The cell line designated D7A3-PE has been deposited at the European
Collection of Animal Cell Cultures (ECCAC) on 1 8th November 1994 under
Accession No 941 1 1845.
FXAMPLE 2- Derivation of Non-Mouse ES Cells
There has only been a single report to date of establishment of purported
ES-like cells from the rat (lannacone et~/[19941 Dev. Biol.) despite intense effort
in several laboratories. However, as indicated above, lannacone's cells were later
shown to have a mouse and not a rat karyotype. The potential application of ESRFto derivation of rat ES cells has therefore been investigated.
Z. ~ Derivation and propagation of rat embryonic stem cells in the presence
of ESRF
Rat blastocysts were placed in culture on irradiated feeder layers of
C3H10T1/2 derived feeder cells in standard ES cell culture medium containing LIFplus ESRF. Inner cell mass ciumps were picked, dissociated in trypsin and replated
in identical conditions. Colonies of small, morphologically undifferentiated cells
which arose were picked individually, dissociated and replaced. In this way stemcell cultures have reproducibly been initiated from approximately 50% of rat
blastocysts of Fischer and Sprague Dawley, and at a lower percentage of BDIX andDA strains. The undifferentiated cells can be maintained for prolonged periods and
expanded extensively, by regular passaging. They can be frozen by conventional
procedures and recovered from storage in liquid nitrogen.
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In more detail, the procedure was as follows:
2.2 Protocol for rat ES cell derivation
2.2.1 Female rats are paired with males and checked every morning
for the presence of sperm in the vagina. On the morning of the 5th day of
pregnancy (day on which sperm found = day 1 ) blastocysts are flushed from the
uteri, using medium 1 (Appendix 1 ) . Zonae pellucidae, if present, are removed by
brief exposure to acid Tyrode's medium.
2.2.2 Feeder iayers are prepared from DIA-M fibroblasts. DIA-M cells
are a clonal derivative of C3H1OT1/2 mouse embryo fibroblasts stably transfectedwith an expression vector containing a cDNA for the matrix-associated form of
mouse LIF (Rathjen et71., 1990) and an IRES-linked neo selectable marker. These
celis express high levels of recombinant matrix LIF. Feeder layers are made by
dispensing approximately 75,000 gamma-irradiated DIA-M cells to a 15 mm
diameter well of a 4-well tissue-culture plate (from Nunclon). 0.5 ml medium 2
~Appendix 1 ) is added to each well.
2~2.3. The zona-free embryos are cultured in these wells for 5 days.
After the embryos attach to the feeders (after 2-3 days) the medium-(medium 2)
is changed daily.
2.2.4. After 4 days in culture, the large masses of cells are picked
individually from the feeder layers, roughly broken up by trituration in fine Pasteur
pipettes, and transferred individually to wells containing DIA-M feeder layers
(prepared as above) and 0.5 ml medium 3 (Appendix 1 ) per well.
Dishes are incubated at 37~C, 7%CO2 in air, and the medium is changed
daily. These culture conditions are used in all further steps.
Z.2.5. Small colonies of small, clear, morphologically undifferentiated
cells can be seen in the wells after ~-7 days in culture. When large enough, these
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are picked for trypsinisation or breaking up. (See step 5). If too small for
trypsinisation, colonies are transferred intect to fresh feeders. Alternatively, if the
well does not contain too much differentiating tissue, colonies may be removed
from the monolayer and allowed to reattach.
2.2.6. Colonies to be trypsinised are rinsed briefly in 3 changes of
phosphate-buffered saline and incubated in .05% trypsin with 0.5 mM EDTA and
0.1% chicken serum for 45-60 seconds. They are then transferred to a drop of
medium 3, disaggregated into single cells and small clumps ~y trituration, and
added to a new well (as described in step 3 above3. Alternatively colonies may be
physically broken up into clumps without trypsin treatment by cutting them apartwith glass needles and triturating the fragments with a finely drawn pipette.
2.2.7. Cell lines are maintained by repeated passaging of colonies as
described above. As the number of colonies per well increases, cultures can be
split into several wells and/or transferred to larger wells.
2.2.8. Cells are karyotyped at intervals (every 3-4 passages) to ensure
that euploidy is maintained, according to the protocol detailed in Appendix 2.
Z.2.9. Cells are examined at intervals (every 5-6 passages~ for the
expression of oct-4 protein (Appendix 3), oct-4 mRNA (Appendix 4) alkaline
phosphatase (Appendix 5) and SSEA-1 (Appendix 6).
2.2.10. Cultures are maintained in the presence either of penicillin and
streptomycin, or gentamycin. The type of antibiotic used is changed every 3 to 4weeks to minimise the risk of antibiotic-resistant strains of microorganisms
developing .
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2.Z.11. To make chimaeric animals, cells are disaggregated in trypsin
as described in step 5 above, and introduced into host embryos. Three examples
of protocols for chimaera production are described below:
~ a) Host embryos at the early blastocyst stage are flushed from uteri of
female rats in the morning of the 5th day of pregnancy (early blastocyst
stage) using medium 1. They are cultured for 24 hours in medium 4, and
the next morning disaggregated cells are injected into the blastocyst cavity.
The embryos are allowed to recover in the incubator for two hours, and are
then transferred to the uteri of rats in the 5th day of pseudopregnancy.
b) Host embryos at the early blastocyst stage are collected as in 2.2.1 1 .a.
They are injected immediately with disaggregated cells and transferred
immediately to the uteri of rats in the 4th day of pseudopregnancy.
c) Host embryos are flushed from the oviducts of rats in the 4th day
of pregnancy (8-cell stage) using medium 1. Using a micro-
manipulator, the zonas are slit and several cells are introduced into
the sub-zona space. Embryos are then returned to the uteri of
females in the 5th day of pseudopregnancy.
2.2.12. Cells and host blastocysts are derived from strains with
different coat colours, and chimaeric animals can be identified by colour about a
week after birth. Alternatively, micro satellite markers can be used to distinguish
donor cells and host derived populations.
2.Z.13. Cells may be transfected by the procedure described in
Appendix 7.
2.2.14. Cells may be frozen by the procedure described in Appendix 8.
2.3 Conclusions
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Both LIF and ESRF are essential for maintenance of the undifferentiated cells.
Upon withdrawal of either component the cultures differentiate completely into
trophoblastic cells.
The ES cell status of the undifferentiated cells after extended culture periods
is evidenced by one or more of: ~i) maintenance of overt ES cell morphology; (ii)
induction of overt differentiation by withdrawal of cytokines, ~ expression of the
early embryo and ES cell specific antigen SSEA-1, (iv) expression of the pluripotent
cell specific transcription factor Oct-3/4, (v) expression of alkaline phosphatase;
and (vi) non-expression of H19.
The characteristic stem cell morphology of rat ES cell cultures is shown in
figure 2. these cells are morphologically indistinguishable from mouse ES cells
cultured under the same conditions. The rat chromosome complement of the
cultures is shown in Figure 3. Expression of markers is shown in Figure 4; upperpanel alkaline phosphatase, lower panel Oct-4 immunostaining. Figure 5 shows
examples of rat ES cell differentiation; a. trophoblast, b. parietal endoderm, c.
unidentified bipolar cells.
These-findings provide a direct demonstration that ESRF can collaborate with
LIF (or other cytokines which act via gp130) to enable the establishment of ES
cells.
2.4 Further Characterisation of Rat ES Cells
Using the protocol we give in section (2.2), we can reproducibly derive cell
lines from the rat blastocyst, which are morphologically identical to mouse ES cells
grown in the same conditions. The cells are small, with high nuclear to
cytoplasmic ratio and grow tightly packed together in clumps with a characteristic
rounded, smooth, transparent appearance. These colonies are morphologically
indistinguishable from clumps of mouse ES cells in the same conditions. Althoughthe majority of cells in these cultures maintain an undifferentiated phenotype, some
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29
degree of differentiation occurs. Cells most frequently differentiate as parietal
endoderm and ~iant cells or other trophoblast-like cells, but other cellular
morphologies are occasionally seen.
~ Besides having the characteristic morphology of mouse ES cells, the rat cells
express the same markers. Alkaline phosphatase is expressed by primordial germ
cells in the rat and mouse, and by mouse ES cells. The rat cell lines we have
derived are also strongly positive for alkaline phosphatase, but cease to express
this enzyme when the cells differentiate. The rat cells stain positively with anantibody to SSE~-1 (stage-specific embryonic antigen 1 ) which is also expressedby primordial gel m cells and mouse ES cells. Finally, immunohistochemical staining
has demonstrated that oct-4 (a transcription factor expressed only by totipotentcells, such as primordial germ cells, the inner cell mass of the blastocyst, andmouse ES cells~ is expressed by our rat cells.
We have maintained lines of cells with these morphological characteristics
and which express oct-3/4, SSEA-1 and alkaline phosphatase for more than 25
passages in vitro (over 3 months).
We have confirmed that these are indeed rat cells by examining their
karyotype. They have a modal chromosome number of 42 and possess
metacentric and submetacentric chromosomes characteristic of rats and not found
in normal mouse strains. The cell lines remain euploid at high passage numbers.
If cells are frozen according to the protocol in Appendix 8, colonies may be
stored in liquid nitrogen and subsequently thawed and maintained in culture withno increase in incidence of differentiation, or alteration of ploidy.
The maintenance of an undifferentiated phenotype in these lines is
dependent upon 3 factors, each of which should be present in the system if cell
lines are to remain viable and undifferentiated for more than a few passages. The
medium must contain soluble DIA/LIF and ESRF (see sections 2.1), and the cells
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are preferably grown upon feeder layers of mitotically inactivated DIA-M cells
(section 2.2.2). If either LIF or ESRF is removed from the systems, the cells will
all eventually differentiate (mostly as trophobiast-like cells) or die (Table 1, below).
Once established, rat cells can be propogated on alternative feeder layers or even
on gelatin-coated piastic. Later passage cells may also be grown in the absence
of ESRF.
maximum passage number
with DIA/LIF, ESRF, and DIA-M feeders >2
without DIA/LIF 1-2
without ESRF 4
without DIA/M feeders 4-5
Chimaeras may be produced from these cells by introduction into host
embryos using the procedures given in section 2.2.11.
Genetically modified rats may be produced by transgene integration or by
gene targetting in rat ES cells followed by Chimaera production and germline
transmission.
2.5 Activity of ESRF on ltuman Teratocarcinoma Cells
The effects of conditioned medium and ammonium sulphate fractionated
ESRF on the multipotent human teratocarcinoma cell line GCT 27X1 were
examined. The assay involves growth of cell colonies from single cells in the
absence o~ a feeder layer in serum-containing medium over a 10-14 day period.
Cells are observed on a daily basis using phase contrast microscopy. Under the
conditions of the assay, cell survival and colony formation in control medium islow. ESRF preparations consistently demonstrated a positive effect on stem cell
survival in these assays. Cell viability was enhanced in the early part of the assay
and final colony num~er was increased substantially. Thus ESRF or an activity
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31
associated with it is active in promoting in vitro survival o~ human pluripotent
teratocarcinoma stem cells.
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APPENDIX 1: Media
Medium 1: Flushing med;um. P~31 (see Whittingham, D., 1971. Culture of mouse
ova. J Reprod Fert (Suppl. 14):7-21~ with 10% fetal calf serum and antibiotics.
Medium 2: ESRF medium. 80% GMEM, 20% fetal calf serum, 2000 units humanDlA/ml, and either 50 units/ml penicillin, 50 mg/ml streptomycin, or 50 mg/ml
gentamycin. Semi-purified ESRF is added at a concentration determined by
bioassay of mouse ES cells Isee Appendix 9).
Medium 3: Gardners G1 or G2 (Barnes etal., 1995, 1 luman Reproduction 10:3243-
3247) .
APPENDIX 2: Karyotyping of cells
1. Colonies of cells to be karyotyped are picked from their dishes and cultured
in Colcemid at 0.8 mg/ml for 2 hours in medium 3, in dishes containing
feeder layers of DIA-M cells.
2. Cell clumps are removed and rinsed briefly in changes of PBS (Dulbecco's
phosphate-buffered saline).
3. Clumps are rinsed briefly in hypotonic saline (1% Na citrate, no older than
1 week), transferred to fresh saline and left for 8-10 minutes.
4. Clumps are transferred to a solid watchglass and excess hypotonic saline
removed.
5. The dish is then filled with freshly made up 3:1 fixative (3 parts absolute
ethanol to 1 part glacial acetic acid) and left for 1 hour.
6. Clumps of cells are transferred to a clean microscope slide and watched
under a microscope until the fixative has almost entirely evaporated.
7. 2-4 drops of 60% acetic acid are then added to the cells, causing the tissue
to disaggregate.
8. The drop is kept moving around the slide by blowing until it is completely
evaporated .
9. Slides are stained in 2% Giemsa stain in Giemsa buffer (from Gurr). They
may be mounted, or can be examined unmounted.
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APPENDIX 3: Immunohistochemicai staining for oct-4 protein
1. Medium is removed from wells and cells are washed twice with complete
PBS (Dulbecco's phosphate-buffered saline with 1.5 mM MgCI2 and 1 mM
CaC12~ .
2. Cells are fixed for 10 minutes in 3.7 formalin in complete PBS.
3. Cells are permeabilised for 15 minutes with 0.2~,'~ Triton in complete PBS.
4. Cells are blocked for 20 minutes in goat serum at the dilution suggested in
the protocol of the Vectastain Elite ABC kit, supplied by Vector Laboratories.
5. Primary antibody (affinity-purified anti-oct-4 antiserum from rabbits
Palmieri e't a/., 1995) is diluted 1:5000 in PBS and added to the cells for
30 minutes.
6. Cells are washed in PBS for 10 minutes.
7. The biotinylated secondary antibody (goat anti-rabbit) is prepared according
to the protocol of the Vectastain kit (Vector Laboratories) and added to the
cells for 30 minutes.
8. Cells are washed in PBS for 10 minutes.
9. The "ABC reagent" is prepared according to the protocol of the Vectastain
kit (Vector Laboratories), allowed to stand for 30 minutes, and added to the
ceils for 30 minutes.
10. Cells are washed in PBS for 10 minutes.
11. Peroxidase substrate is prepared from the "VIP kit" supplied by Vector
Laboratories according to instructions.
12. Peroxidase substrate is added to the cells fro 2-10 minutes, until a suitable
intensity of color develops.
13. Cells are washed for 5 minutes in tap water.
14. Cells should be examined and photographed at once.
-
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APPENDIX 4: in situ staining for oct-4 mRNA
Note: ail solutions are made with DEPC-treated water
1. The probe is synthesized from a Stu 1 fragment of a Bluescript piasmid
carrying the POU homeodomain of the oct-4 gene, with 1.75 mM
digoxigenin~ JTP in the synthesis mixture .
2. Cells attached to tissue-culture plates are fixed in 4% paraformaldehyde in
PBS (Duibecco's phosphate-buffered saline) at 4~C overnight.
3. They are rinsed with PBT (PBS + 01.% Tween) and dehydrated in a graded
series of methanol (25%, 50%, 75% and 100%) and stored at-20~C until
needed .
4. Cells are rehydrated through the methanol series, rinsed with PBT, and
washed 3 times in RIPA (1 % NP-40, 0.5% NaDOC, 0.1 % SDS,1 mM EDTA
and 50 mM Tris in 150 mM NaCI).
5. Cells are first washed in 1 :1 hybridisation buffer:PBT (hybridisation
buffer:50% formamide in 10xSSC pH 4.5, with 0.05% of a stock solution
of 100 mg/ml heparin, and 0.1 % Tween 20), and then with 100%
hybridisation buffer.
6. Hybridisation buffer with herring sperm DNA (100,ug/ml) and yeast transfer
RNA (10 mg/ml) is added to the cells, and the dishes are incubated at 70~C
overnight.
7. The probe is denatured, added to the wells at a dilution of 1: 100 - 1 :200,
and the dishes incubated at 70~C overnight.
8. Cells are washed briefly at 65~C with posthybridisation wash buffer (50%
formamide in 2xSSC with 0.1% Tween 20). This wash is followed by 3
further washes at 65~C of 20 minutes each.
9. Cells are allowed to cool to room temperature and washed 3 times with
TBST (0.8% NaCI, 0.02% KCI, Z.5% 1M Tris-HCI pH 7.5).
10. Sheep serum is inactivated by heating to 30 minutes at 70~C with regular
shaking. 10% inactivated sheep serum in TBST is added to the cells and the
plates are left at room temperature for 1 hour.
11. The serum is removed and replaced with anti-digoxigenin alkaline
phosphatase coniugated Fab fragments in 1 % sheep serum diluted in TBST
to a concentration of 1 :2000. Dishes are stored at 4~C overnight.
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- - APPFNDIX 4 ~contd.): in situ staining for oct-4 mRNA
12. Cells are washed briefly 3 times with TBST at room temperature, and then
a further three times for at least 2 hours total time.
13. Cells are washed 3 times in alkaline phosphatase buffer (100 mM NaCI,
50 mM MgCI2, 0.1% Tween 20, 100 mM Tris pH9~.
14. The wash is removed and cells stained in the dark in a solution of 4.5 ul NBT
and 3.5 ul BCIP (x-phosphate) per ml alkaline phosphatase buffer.
15. When a suitable intensity of stain is observed, the reaction is stopped by
rinsing 3 tirnes in PBT with 1 mM EDTA. Cells can be stored for a short time
in this solution at 4~C.
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APPENDiX 5: Alkaline phosphatase staining of cultured cells
1. Cells growing in tissue culture dishes are fixed in cold 80% ethanol for at
least one hour. They may be stored at 20~C in this fix for several weeks.
2. Cells are hydrated by washing for 20 minutes each in solutions of 50%
absolute ethanol, 30% absolute ethanol, and two changes of distilled water.
3. Cells are stained in 0.05% Fast Red TR Salt, 0.01% a-naphthyl phosphate.
0.03% MgCI2, and 0.22% borax for 5-10 minutes.
4. Cells are washed in distilled water and observed immediately. If necessary,
cells can be stored in 50% glycerine in water.
APPENDIX 6: Immunohistochemical staining for SSEA-1
1. Cells growing on feeder iayers attached to glass coverslips are placed in
weiis for ease of handling. They are washed twice in PBS (Dulbecco's
phosphate-buffered saline).
2. Primary antibody ~monoclonal anti-SSEA-1, obtained from J. Ansell) is
diluted 1:1000 in serum-free medium with 0.15% BSA and buffered with
Hepes. This is added to the cells and the dishes incubated at 4~C for 45
minutes.
3. Cells are washed 3 times in cold medium.
4. The secondary antibody (anti-mouse IgM, FITC labelled) is diluted 1 :10 with
medium, added to the cells for 30 minutes at 4~C.
5. Cells are washed 3 times in cold medium.
6. Cells are washed 3 times in cold PBS.
7. Cells are fixed in cold 95% methanol/5% acetic acid for 3 minutes.
8. Cells are rinsed in PBS, the coversiips removed and inverted on drops of
10% PBS:90% giycerin on glass slides.
9. Slides are examined under a fluorescence microscope.
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APPENDIX 7: Transfection of cells
Procedure 1: Calcium phosphate co-precipitation.
1. Cells are trypsinised and transferred to fresh wells containing feeder iayers~ - and medium as usual.
2. Dishes are placed in an incubator at :~7~C with 2.5% C02.
3. Transfection mix (to a final concentration of 50 ul/ml BES, 0.012 M CaCI2
and 1 ug/mi DNA) is added to wells and the cells incubated overnight.
4. The transfection mix is removed next morning and replaced with normal
medium .
5. Transfected cells are selected and/or identified according to the method
determined by the incorporated DNA.
Procedure 2: Lipofection
1. Make up transfection solution, using lipids from the PerFect transfection kit from invitrogen, and Optimem from Gibco.
a. add lipid to Optimem ~12,ul/ml)
b. add DNA to Optimem ~2 ~I/ml)
c. mix lipid/Qptimem with DNA/Optimem
d. Incubate at 37~C for approximately 15 minutes.
2. Wash colonies of cells in three changes of PBS.
3. Transfer colonies to trypsin for 1 minute.
4. Transfer colonies to a drop of Optimem and triturate with a pulled Pasteur
pipette.
5. Remove medium from wells with feeder layers and replace with Optimem
plus lipid and DNA.
6. Add trypsinised cells to the well and incubate for 5 hours.
7. Remove Optimem and replace with cell medium (medium 2~.
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38
Procedure 3: Electroporation
1. Wash colonies 3 times in PBS.
2. Trypsinise 2-3 minutes.
3. Transfer colonies to 50,ul PBS and triturate until cells disaggregate.
4. Transfer PBS with cells to an eiectroporation cuvette containing 650,ul PBS
and 100,ug DNA (in 100,u1 PBS).
5. Leave cuvette on ice for 10 minutes.
6. ~lectroporate at 0.8 kV, 3 ,uF, time constant = 0.1.
7. Leave cuvette on ice for 10 minutes.
8. Divide contents of cuvette into 4 wells (200 ,ul each) and add 300 ,ul medium
2 to each well.
9. Leave cells for 3 hours and change medium (using medium 2).
10. Leave overnight before handling further.
APPENDIX 8: Freezing and thawing of cells
1. Dislodge colonies of cells with a drawn pipette.
2. Transfer colonies quickly to a drop of PBS and then to a drop of 0.25 ml
freezing mix.
3. Transfer the drop of freezing mix quickly to a cryotube containing 0.4 ml of
freezing mix.
4. Transfer to -70~ freezer for overnight freezing, then transfer to liquid N2
freezer.
To thaw:
1. Put ~.5-9.5 ml of Medium into a 25 ml universal.
2. Thaw cryovial in a waterbath then pipette the contents of vial into the
universal .
3. Centrifuge at c 1000 rpm (~00-800 optimal) for 3-~i minutes.
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39
4. Aspirate medium from DIA-M feeders.
5. Aspirate media from universal, leaving celi clumps behind.
6. Add 1 ml of Medium 2 to universal and gently resuspend.
7. Pipette cells onto feeders ~at 0.5 ml per well) and return to culture.
Freezing mixture:
42% FCS
48% Medium 3
1 0% DMSO
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APPENDIX 9: ESRF purification and assay
9.1 Preparation of ESRF-containing ammonium sulphate cut of conditioned
rnedium
1. ESRF-producing adherent cell line (D7A3-P~) is grown to nezr-confluency in
GMEM/10% FCS. Currently, approximately 30-40 large (175 sq.cm.) flasks
are used.
2. Medium is removed, cells rinsed twice with PBS then returned to serum-free
GMEM/Ham's F12 (1:1) supplemented with 10mM HEPES, 3 ,LIM sodium
selenite, 1 ,ug/ml transferrin and 1 ,ug/ml insulin. Cells are gassed and
returned to incubator in this medium for 3-4h minimum, overnight if more
convenient. This is an adaptation and washing step.
3. Medium is removed and replaced with fresh serum-free medium for
conditioning over 3-5 days. 35 ml medium per flask.
4. Conditioned medium is harvested, centrifuged to remove detached cells, and
filter-sterilised. Approximately 1000 ml of medium is collected before
proceeding to step ~.
5. Conditioned medium is broughtto 35% saturation with ammonium sulphate.
Saturated ammonium sulphate is added at 5 ml/min with stirring,in the cold
room and left for a further 60 min. The precipitated protein is pelleted by
centrifugation and constitutes approximately 20% of the total protein in the
conditioned medium (Bradford assay).
6. The precipitate is solubilised in 2M urea. The centrifugation (step 5) is done
in polypropylene bottles and the precipitate ends up as a viscous smear
down the length of the bottle. In view of this, the solubilisation is done on
a rocking roller apparatus for 1 h at room temperature. 15-20 ml in 6 botttes.
7. The solubilised material is finally dialysed overnight against tissue cuiture-
grade PBS ~in cold room) then frozen in aliquots.
9.2 Assay
A number of embryonic stem-cell lines have been used for assaying ESRF activity,most commoniy:-
CP1 for general morphology and alkaline
phosphatase histochemistry
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41
COK018 for general morphology and oct4
expression (detected by lacZ reporter
- - = activity).
The basic protocol is the same in each case.
1. ES cells are seeded on to gelatinised 24-well plates at a density of 5,000
cells per well in 0.5 ml GMEM/10% FCS. Antibiotic is used at an
appropriate concentration (unless the ESRF sample is filter sterilised).
2. After 2-3h test samples are added to the wells and the plates returned to the incubator for 4 days.
3. After 4 days, the cells are stained for alkaline phosphatase activity ~Sigma
Alkaline Phosphatase Leukocyte kit, cat. no. 86-R) or 13-galactosidase
activity (X-gal stain).
4. Cells and colonies are inspected for typical ES-cell phenotype:
- small, round cells
- compact, rounded colonies
- high expression of alkaline phosphatase or expression of oct4 as
reflected by 13-galactosidase activity.
1 unit of ES cell activity is defined as the minimai amount which produces
typical ES-cell phenotype in 0.5 ml assay at the 4d point as described above
(n.b. at this concentration the maiority of cell will not have ES-cell
phenotype~ .
APPENDIX 10: Ultracentrifuga~ion of PE-conditioned medium
1. PE-CM prepared as described (Appendix 9).
2. PE-CM centrifuged at 1 OO,QOO x g for 75 min at 4~C; (S = 106).
3. Supernatant (S1) removed and pellet (P1) solubilised in 0.5 ml 2M urea.
4. Supernatant (S1) re-centrifuged at 265,000 x g for 18 h; (S=3.3).
5. sUPERNATANT (S2) removed and pellet solubilised in 0.5 ml 2M urea (P2).
6. S1, S2, P1 and P2 were desaltediexchanged into PBS on Pharmacia
PD10 (S) or BioGel P6-DG (P) columns.
7. Samples were assayed for ESRF activity.
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Results
S1 activity similar to PE-CM
S2 no activity detected
P1 a very small amount of activity ~less than PE-CM)
P2 very strong activity
APPENDIX 11: Lectin-affinity chromatography of ESRF
1. 25-3~% saturated ammonium sulphate cut of PE-conditioned medium was
sub~ected to fluid-phase isoelectric focussing (Rotofor).
2. precipitated material ~pl =4.25-4.5) was solubilised in 2M urea.
3. solubilised material was exchanged into lectin buffer* on a Pharmacia PD-10
column.
4. 200 ,ul exchanged material was loaded on to columns containing soyabean
lectin, lentil lectin or wheat germ agglutinin (equilibrated with lectin buffer),
left for 30 min, then another 200 111 added and the column left in the cold
overnight.
5. columns were washed through with 5 x column volumes of lectin buffer;
washes were collected.
6. columns were eluted with 5 x column volumes competing sugar** in lectin
buffer and the eluted material collected.
7. wash-through and sugar-eluted material was exchanged into 20mM sodium
phosphate pH 7.3 and assayed for ESRF activity.
*Lectin buffer 20mM sodium phosphate pH 7.3
1 M NaCI
0.1mM CaC12
0.1mM MnCI2
**Sugars soyabean - galactose
- lentil - ~-methyl mannoside
wheat germ - N-acetylglucosamine
Resu~ts
Activity retained and specifically eluted on soyabean and lentil lectin
columns but not on wheat germ agglutinin.
