Method of enriching for mammalian stem cells

Mammalian stem cells are obtained and maintained in vitro whose genome has at least one nucleic acid construct encoding an antibiotic resistance gene operatively linked to a promoter specific for mammalian stem cells. The preferential expression of the antibiotic resistance gene in the stem cells results in the preferential survival of the stem cells in the presence of antibiotic.

SUMMARY OF THE INVENTION 
This invention relates to methods of isolating and/or enriching and/or 
selectively propagating animal stem cells, genetically modified animal 
cells and animals for use in said method, transgenic animals providing a 
source of such cells and selectable marker constructs for producing 
genetically modified cells and transgenic animals. 
Stem cells are progenitor cells which have the capacity both to self-renew 
and to differentiate into mature somatic tissues. 
Embryonic stem cells are the archetypal stem cell, being capable 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 
capable of differentiating into many cell types. Other types of stem 
cells, for example bone marrow stem cells and epidermal stem cells, 
persist in the adult animal. These stem cells have a more restricted 
capacity for differentiation. 
In general, when required for research purposes or for medical 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, 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 (31/2 day) 
embryonic cells from murine strain 129 and Black 6, or hybrids thereof. 
There has developed a pressing need to isolate and maintain in vitro 
embryonic stem cells from other murine strains and more especially from 
other species including other laboratory animals (e.g. rats, rabbits and 
guinea pigs), domesticated animals (e.g. sheep, goats, horses, cattle, 
pigs, birds, fish, etc.) and primates. Similarly, numerous medical 
applications for other pluripotential cells such as haematopoictic stem 
cells also demand their isolation and culture in vitro. 
However hitherto the problems associated with producing cultures of stem 
cells including the problem of producing cell populations of a 
satisfactorily low degree of heterogeneity and the problem of overgrowth 
in culture of non-pluripotent cells have not been solved. A particular 
problem associated with the continuing presence of certain differentiated 
cell types is that these can cause elimination of stem cells from the 
culture by inducing their differentiation or programmed cell death. 
We have now developed a technique by which the aforementioned problems can 
be overcome. 
According to one aspect of the invention there is provided a method of 
isolating and/or enriching and/or selectively propagating animal stem 
cells, which comprises maintaining a source of said cells under culture 
conditions conducive to cell survival, characterised in that the source of 
cells includes cells containing a selectable marker which is capable of 
differential expression in (a) stem cells and (b) cells other than the 
desired stem cells, whereby differential expression of said selectable 
marker results in preferential isolation and/or survival and/or division 
of the desired stem cells. In the context of this invention, the term 
"animal cell" is intended to embrace all animal cells, especially of 
mammalian species, including human cells. 
Examples of stem cells include both unipotential and pluripotential stem 
cells, embryonic stem cells, gonadal stem cells, somatic stem/progenitor 
cells, haematopoietic stem cells, epidermal stem cells and neuronal stem 
cells. 
In carrying out the method of the invention, the source of cells may 
include pluripotential cells having a positive selectable marker and 
expression of the said marker is used to permit isolation and maintenance 
of the plunrpotential cells. Alternatively, the source of cells may 
include a negative selectable marker which is expressed in cells other 
than the desired pluripotential cells and is used selectively to deplete 
the source of cells of cells other than the desired pluripotential cells. 
The selectable marker may, for example, be a foreign gene, a cellular gene 
or an antibiotic resistance gene such as for example the bacterial 
neomycin resistance gene. 
Alternatively the selectable marker may be a growth stimulating gene, for 
example an immortalising gene, an oncogene or a gene coding for the 
polyoma or SV40 T antigens or derivatives thereof, or the selectable 
marker may be a gene coding for a growth factor or a growth factor 
receptor or a signal transducing molecule or a molecule that blocks cell 
death. 
In one particular embodiment the isolation and/or enrichment and/or 
selective propagation of the desired pluripotential cells is dependent on 
the presence of cells other than the desired pluripotential cells and the 
simultaneous maintenance of both cell types is dependent on expression of 
a selectable marker, in one or the other cell population, which is capable 
of rescuing cells that do not express the marker but which neighbour cells 
which do themselves express the marker. In this instance, the selectable 
marker may, for example, be the hypoxanthinc phosphoribosyl tranferase 
(HPRT) gene. 
In another embodiment the selectable marker may be a gene encoding a 
product which is toxic per se, or a toxic gene product which is 
conditionally active in combination with a suicide substrate. An example 
of such a gene product is a herpes simplex virus thymidine kinase (HSV-TK) 
in combination with ganciclovir. 
Expression of the selectable marker may be achieved by operatively 
inserting the selectable marker into an expression construct prior to 
introduction to the cell source, in which case expression of the 
selectable marker can result from the introduction of either a stable or 
transiently integrated construct. Alternatively, expression of the 
selectable marker results from operatively inserting the selectable marker 
into an endogenous gene of the cell source. 
Various means of introducing the selectable marker may be employed, 
including introduction into the cells by transfection, lipofection, 
injection, ballistic missile, viral vector or by electroporation. 
The source of the cells may be a single cell such as a fertilized oocyte, 
or it may comprise a mixture of cells, such as cells derived from an 
embryo, blood or somatic tissue of a normally bred or transgenic animal or 
cell line. In the latter case the selectable marker may be incorporated 
into the transgenic animal's genome. 
Most preferably, in carrying out the method of the invention a gene or gene 
fragment operatively linked to and regulating expression of the selectable 
marker is/are associated with a pluripotential stage of cellular 
development. Such a gene or gene fragment may be active in pluripotential 
cells of the developing embryo, especially in the inner cell mass and/or 
primitive ectoderm, or may be active in adult stem cells. 
In preparing a source of cells for use in accordance with the invention one 
of the following protocols may advantageously be adopted: 
introducing into a source of cells containing stem cells, a selectable 
marker construct, wherein said selectable marker construct is adapted to 
operatively link to an endogenous gene which provides said differential 
expression, or 
introducing into a source of cells containing stem cells, a selectable 
marker construct, wherein said selectable marker construct has been 
previously linked to one or more gene fragments which provide said 
differential expression. 
The genetic marker preferably comprises a selectable marker operatively 
linked to a promoter which is differentially active in the desired 
pluripotent cells (e.g. primitive ectoderm). By "selectable marker" is 
meant a selectable gene which may be a foreign gene or a cellular gene 
which is not naturally expressed, or such a gene which is naturally 
expressed, but at an inappropriate level, in the target cell populations. 
This gene in use acts as a selection marker by adapting the phenotype of 
the target cell population in such a way that cells with the so-adapted 
phenotype may be enriched or depleted under particular culture conditions. 
In the case where stem cells are embryonic cells it is preferred that the 
selectable marker is operatively linked to a promoter which is 
differentially active in stem (e.g. primitive ectoderm, primordial germ 
cells) and non-stem cells. Promoter and other cis-regulatory elements may 
be included in the expression construct prior to introduction into the 
cells or by targeting promoter-less constructs into specific genes by site 
specific recombination. 
A wide variety of gene products may be relied upon for selective isolation 
and propagation of the desired stem cells, including markers which are 
designed to protect the desired cells from the effects of an inhibiting 
factor present in the culture medium. In this instance, the inhibiting 
factor can, for example, be an antibiotic substance which inhibits growth 
or reproduction of cultured cells, not expressing the gene (i.e. cells 
other than the desired cells). The selectable marker (e.g. HPRT) may also 
provide protection both for the desired cells in which it is expressed as 
well as other closely associated cells by means of metabolic rescue. 
Alternatively the selectable marker may selectively permit the growth of 
stem cells. In this instance the marker may encode a growth factor, a 
growth factor receptor, a transcription factor, an immortalising or an 
oncogenic product (e.g. temperature sensitive simian virus 40 T antigen). 
Alternatively, the selectable marker may be a cell surface antigen or other 
gene product which allows purification or depletion of expressing cells 
for example by panning or fluorescence-activated cell sorting (FACS). The 
invention thus enables stem cell populations to be obtained/maintained 
having a satisfactory degree of homology. 
Alternatively the selectable marker may be a conditionally toxic gene for 
instance herpes simplex virus thymidine kinase [HSV-TK]. In this instance 
expression of the selectable marker is directed to cells other than the 
desired cells and not to stem cells. Cells other than the desired 
phenotype may be selectively depleted by addition of a lethal substrate 
(e.g. ganciclovir). 
The genetic marker may be introduced into the source of cells by a variety 
of means, including injection, transfection, lipofection, electroporation 
or by infection with a viral vector. 
Further, the source of cells may be produced by transfection 
extemporaneously, or the source of cells may be derived from a transgenic 
animal, e.g., the founder transgenic animal or an animal at least one 
ancestor of which has had the aforementioned genetic marker introduced 
into its genetic complement. In such transgenic animals it is possible for 
the marker to pass down the germ line and eventually results in the 
production of progeny, from the tissues of which (especially from the 
embryonic tissue) the required source of cells can be derived. 
Thus according to further aspects of the invention, there is provided an 
animal cell capable of being cultured under appropriate selective culture 
conditions so as to enable isolation and/or enrichment and/or selective 
propagation of stem cells, characterised in that said cell contains a 
selectable marker wherein differential expression of the selectable marker 
in (a) the desired stem cells and (b) cells other than the desired stem 
cells enables selective growth of the desired stem cell to occur. 
The invention further provides an animal cell capable of being cultured 
under selective culture conditions so as to grow as stem cells, 
characterised in that said cells contain stem cells containing a genetic 
marker, whereby a gene product associated with the genetic marker is 
produced and which under said culture conditions causes selective survival 
and/or division of the desired stem cells to occur. 
The animal cells according to this aspect of invention are preferably 
characterised by possessing the preferred characteristics described above. 
The invention further provides according to another aspect thereof, a 
transgenic animal having genetic characteristics such that it or its 
progeny, during embryonic development or later life, constitute a source 
of animal pluripotential cells as defined above. 
Such transgenic animal may be produced according to the invention by 
introducing a genetic marker into a fertilised oocyte or an embryonic 
cell, or an embryonic stem cell in vitro, the genetic marker having the 
characteristics defined above, and utilising the resulting transformed 
oocyte or embryonic cell as a progenitor cell for the desired transgenic 
animal. 
Vectors for use in producing an animal cell defined above form a further 
aspect of the invention. 
Thus the invention further provides vectors for use in genetically 
modifying animal cells so as to produce transformed cells suitable for use 
as the source of cells for the method referred to above, said vector 
comprising a first genetic component corresponding to said selectable 
marker and a second genetic component which in the genetically modified 
animal cells (1) results in the said differential expression of the 
selectable marker from either a transiently or stably integrated construct 
or (2) enables site-directed integration of the selectable marker into a 
specific gene so as to provide operative coupling of the selectable marker 
with targeted endogenous gene regulatory elements. 
Such vectors may be in the form of expression vectors in which said second 
genetic component includes control sequences which are differentially 
activated (a) in stem cells and (b) in cells other than the desired stem 
cells. 
The invention covers vectors which when used to transform animal cells 
become integrated into the animal genome as well as vectors which do not 
become so integrated. 
The expression vectors referred to above may comprise a DNA sequence coding 
for the afore-mentioned selectable marker operatively linked to a genetic 
control element, or sequence enabling targeting of a promoterless marker 
to an endogenous gene which is expressed differentially in the said stem 
cells and in cells other than the desired stem cells. 
For the generation of pluripotential embryonic stem cells the expression 
constructs preferably comprise a DNA sequence coding for said selectable 
marker operatively linked or targeting to a genetic control element(s) 
which is associated with a stage of embryonic development prior to 
differentiation of pluripotential embryonic cells. Most preferably the 
genetic control elements derive from a gene specifically active in the 
inner cell mass of the mouse blastocyst, in primitive ectoderm, and in 
primordial germ cells of the early embryo. 
In more detail, the present invention has resulted in the development of 
expression constructs which direct specific expression of selectable 
markers in stem cells and not in differentiated cell types. Having 
introduced an expression construct by transfection or via the generation 
of transgenic animals, stem cells present within mixed cell populations 
can be isolated by culturing in the presence of the selection agent in 
vitro, or by otherwise manipulating the culture conditions. 
One example of a gene which displays a suitably restricted stem cell 
expression pattern and therefore may provide suitable "stem cell specific" 
regulatory elements for the expression of a selectable marker in 
accordance with the invention is the Oct4 gene. 
Octamer binding transcription factor 4 is a member of the POU family of 
transcription factors (reviewed by Scholer, 1991). Oct4 transcription is 
activated between the 4- and 8-cell stage in the developing mouse embryo 
and it is highly expressed in the expanding blastocyst and then in the 
pluripotent cells of the egg cylinder. Transcription is down-regulated as 
the primitive ectoderm differentiates to form mesoderm (Scholer et al., 
1990) and by 8.5 d.p.c. (days post coitum) is restricted to migrating 
primordial germ cells. High level Oct4 gene expression is also observed in 
pluripotent embryo carcinoma and embryonic stem cell lines, and is 
down-regulated when these cells are induced to differentiate (Schmoler et 
al., 1989; Okamoto et al., 1990). 
Selectable marker genes under the control of the Oct4 promoter may, 
according to the in vention, be applied to the isolation of embryonic stem 
cell lineages. Furthermore, reports describing low level Oct4 expression 
in some adult tissues (Takeda et al., 1992) may extend the utility of 
these expression constructs beyond embryonic stem cells to include other 
stem cells essential to tissue homeostasis and repair in other systems 
including the haematopoietic system. In the event that Oct4 is not 
expressed in somatic stem cells, other transcriptional regulatory 
elements, such as those associated with the haematopoietic stem cell 
specific antigen CD34, may be utilised in a similar manner. 
Two specific approaches are provided according to the invention for 
generating the desired spatial and temporal restrictions in transgenic 
expression. The first approach is through the generation of transgenic 
animals in which a partially characterised Oct4 gene promoter fragment 
(Okazawa et al., 1991) is employed to drive stem cell specific 
transcription of the selectable marker. An appropriate selectable marker 
is the neomycin phosphotransferase gene which confers resistance to the 
antibiotic G418. An alternative is to utilise a selectable marker which is 
associated with the production of a gene product which can counteract a 
deficiency in a metabolite, e.g. the hypoxanthine-guanine phosphoribosyl 
transferase (HPRT) gene in HPRT-deficient cells (Hooper et al., 1987). 
This approach may be advantageous in situations where stem cells require 
continuous support from closely associated differentiated cells. In this 
instance direct cell contact will permit metabolic rescue of the 
neighboring support cells by the stem cells despite the lack of selectable 
marker gene expression in the support cells. 
The second approach utilise the endogenous Oct4 gene locus, and therefore 
the associated Oct4 gene regulatory elements, to link resistance marker 
gene expression as closely as possible with the endogenous Oct4 gene 
expression profile. This may be accomplished by high efficiency gene trap 
targeted mutagenesis of the Oct4 gene in embryonic stem cells. This 
approach provides more tightly regulated control of selectable marker gene 
expression by avoiding random integration site effects which often result 
in unpredictable expression patterns of randomly integrated constructs.

DETAILED DESCRIPTION 
The invention will now be described in more detail in the following 
Example, with particular reference to the accompanying drawings of which 
FIG. 1 illustrates the structure of plasmid Oct-4-Neo-.beta.S, FIG. 2 
illustrates the structure of plasmid Oct-4-Neo-.beta.fos and FIG. 3 
illustrates the structure of the plasmid Oct4-tgtvec. 
EXAMPLE 1 
1. Isolation of OCT4 Promoter Sequences: 
We screened a strain 129 mouse genomic lambda library with a 330 bp 5'Oct4 
cDNA fragment. Several clones were isolated and screened by restriction 
analysis and Southern blot hybridization. A 1.4 kb Bam HI-Hind III 
fragment containing the Oct4 promoter element (Okazawa et al., 1991) was 
isolated from clone 1 and ligated into pBluescript II KS(-) (Stratagene) 
to generate pOct4 (5' genomic). 
2. Construction of Plasmids: 
To generate the Oct4-Neo promoter constructs an engineered Neomycin 
resistance gene (neo), designed to provide an Nco I restriction site at 
the translation initiation codon, was isolated from pLZIN (Ghattas et al., 
1991) as a 1.1 kb Xba I-Sph I fragment encompassing encephalomyocarditus 
virus internal ribosome entry site sequence (EMCV-IRES, Ghattas et al., 
1991) and 5'-Neo coding sequences and cloned into pSP72 (Promega Biotech). 
The Kpn I-Nco I EMCV-IRES sequence was replaced with a 1.3 kb Oct4 
promoter fragment isolated from pOct4 (5' genomic) by Kpn I and subsequent 
partial Nco I restriction digest. Neo3'-coding, rabbit .beta.-globin gene 
(intron) and SV40 polyadenylation sequences were isolated as a 1.7 kb Sph 
I fragment from 6P-IRESNeo-.beta.S and ligated into the Sph I site to 
generate Oct4-Neo-.beta.S (FIG. 1). To generate the Oct4-Neo-.beta.fos 
construct (FIG. 2), an Oct4-Neo-.beta.S Bam HI fragment incorporating the 
Oct4 promoter, neo gene and the rabbit .beta.-globin intron was inserted 
5' to a human c-fos genomic sequence. This 1.7 kb genomic sequence (Bal 
I-Sph I) encodes human c-fos mRNA 3' coding and non-coding sequences 
previously associated with mRNA destabilization (Wilson and Triesman, 
1988), and, the c-fos polyadenylation sequence. 
The Oct4-neo construct (Oct4-tgtvec) is designed for targetted integration 
into the Oct4 gene (FIG. 3). The Oct4 targetting construct contains 1.7 kb 
of 5'Oct4 gene sequence and 4.2 kb of 3'Oct4 gene sequence. Following 
homologous recombination this construct incorporates a lacZ-neomycin 
fusion gene (.beta.geo, encoding a bifunctional protein, Freidrich and 
Soriano, 1991) into the first intron of the Oct4 gene. Splicing from the 
splice donor sequence of the first exon-intron boundary to the integrated 
IRES-.beta.geo sequence is facilitated by the inclusion of a murine 
engrailed-2 splice acceptor sequence (Skarnes et al., 1992) immediately 5' 
to the IRES-.beta.geo sequence. Translation of the .beta.geo cistron of 
the Oct4-.beta.geo fusion transcript is facilitated by the inclusion of 
the EMCV-IRES immediately 5' to the .beta.geo coding sequence. 
3. ES Cell Transfection and Colony Selection: 
Mouse 129 ES cells (line CGR-8) were prepared and maintained in the 
presence of Differentiation Inhibiting Activity (DIA) or Leukemia 
Inhibitory Factor (LIF) as described by Smith (1991). Plasmid DNA for 
transfection was linearised by Sal I digest, ethanol precipitated and 
resuspended at 10-14 mg/ml in PBS. Following 10 hours culture in fresh 
medium, near confluent ES cells were dispersed by trypsinisation, washed 
sequentially in culture medium and PBS, and resuspended at 
1.4.times.10.sup.8 /ml in PBS for immediate transfection. Routinely, 0.7 
ml of cell suspension was mixed with 0.1 ml DNA containing solution and 
electroporated at 0.8 kV and 3.0 .mu.FD using a Biorad Gene Pulser and 0.4 
cm cuvettes. Transfections were plated on gelatinised tissue culture 
dishes at 5-8.times.10.sup.4 /cm.sup.2 in growth medium for 16 hours prior 
to the addition of selection medium containing 200 .mu.g/ml (active) G418 
(Sigma). Single colonies were picked 8-10 days post-transfection and 
transferred in duplicate into 24 well tissue culture plates for further 
expansion in growth medium containing 200 .mu.g/ml G418. 
Clonal cell lines bearing the Oct4-Neo-.beta.S and Oct4-Neo-.beta.fos 
constructs (referred to as Oct4-Neo cell lines) were grown for two days, 
washed twice with PBS and the medium replaced with fresh G418 medium with 
or without DIA. Cell lines which grew normally in the presence of DIA but 
did not survive in the absence of DIA were selected for expansion and 
further analysis. 
Clonal cell lines bearing the Oct4-tgtvec targetting construct (referred to 
as Oct4-targetted cell lines) were expanded in duplicated 24 well plates. 
Once confluent, one series of cells were frozen for storage while the 
reminder were analysed by Southern analysis. 
4. Further Characterisation of Oct4-Neo and Oct4-targetted Cell Lines: 
Selected Oct4-Neo cell lines were assayed for ES cell growth and 
differentiation in DIA supplemented or non-supplemented medium at various 
G418 concentrations. Cells were plated at 1.times.10.sup.4 /cm.sup.2 in 12 
well tissue culture plates in the various media preparations and cultured 
for 6 days. Fresh medium was applied every 2 days until day 6 when cells 
were fixed and stained as previously described (Smith, 1991.) 
Oct4-targetted cell lines positive by genomic Southern analysis were 
analysed by lacZ staining and growth and differentiation in DIA 
supplemented or non-supplemented medium in 200 .mu.g/ml G418. 
5. Production of Embryoid Bodies from Oct4-Neo Cell Lines: 
Embryoid bodies were generated by the hanging drop method (Hole and Smith, 
in press) and maintained in suspension culture for 2, 4, 6 or 8 days in 
the presence or absence of G418. Control embryoid bodies were generated 
from the parental cell line CGR-8 in the absence of G418. Embryoid bodies 
were then collected and transferred to gelatinised tissue culture dishes 
to enable adherence and expansion of the aggregates for analysis of 
contributing cell types. All embryoid bodies were maintained for 4 days in 
the absence of DIA and G418 prior to inspection. 
6. Production of Chimeras from Oct4-Neo and Oct-4 Targetted Cell Lines: 
Selected Oct4-Neo cell lines were cultured in the absence of G418 for 7 
days prior to embryo injection as previously described (Nichols et al., 
1990). Briefly, blastocysts for injection were collected 4 d.p.c. from 
C57BL/6 donors, injected with 10-20 cells and allowed to re-expand in 
culture prior to transfer to the uteri of pseudopregnant recipients. 
Chimeras were identified by the presence of patches of sandy coat colour 
on the C57BL/6 background. Male chimeras were test bred for transmission 
of the Oct4-Neo transgene. Transgenic mice were then crossed onto 
different genetic backgrounds. 
7. Results 
The Oct4-Neo-.beta.S construct generated approximately 50 colonies/10.sup.6 
cells transfected while the Oct4-Neo-.beta.fos construct generated 
approximately 10 fold fewer colonies. Three clones were selected on the 
basis of their differential survival in medium containing G418 (200 
.mu.g/ml) in the presence or absence of DIA. All three cell lines 
displayed apparently normal growth rates in DIA-supplemented G418 
containing media and died when cultured in the absence of DLA in G418 
medium. Cultures maintained in DIA supplemented G418 medium grew as 
essentially pure ES cells while cultures maintained in DIA supplemented 
medium in the absence of G418 grew as mixed cultures of ES cells and 
differentiated progeny closely resembling those of the parental CGR-8 
line. Thus G418 selection eliminates differentiated cell types and allows 
propagation of pure stem cell populations. The three cell lines selected 
were designated Oct4-Neo-.beta.S18, Oct4-Neo-.beta.S21 and 
Oct4-Neo-.beta.fos11. These cell lines have been introduced into host 
blastocysts and resulting chimaeras may be test bred. Similar results were 
obtained with ES clones targetted with the Oct4-tgtvec construct. In 
addition, histochemical staining of these cultures for 
.beta.-galactosidase activity confirmed that expression of .beta.geo was 
restricted to undifferentiated stem cells (Mountford et al, 1994). 
Embryoid bodies were generated from the Oct4-Neo cell line 
Oct4-Neo-.beta.fos11 to examine the effect of G418 selection on mixed cell 
aggregates and to test the utility of the selection system in isolating ES 
cells from mixed cell populations. Embryoid bodies generated with both the 
experimental cell line (Oct4-Neo-.beta.fos11) and the parental cell line 
(CGR-8) and cultured in the absence of G418 were composed almost entirely 
of differentiated cells with few if any ES like cells. In contrast, visual 
analysis of the expanded colonies revealed that the Oct4-Neo-.beta.fos11 
embryoid bodies cultured in the presence of G418 contained high 
proportions of ES cells. The feasibility of isolating stem cells from 
differentiating systems is thus confirmed. 
8. Summary 
ES cells capable of germ line transmission have previously been established 
from only 2 inbred strains of mice, 129 and C57BL/6. Combining the 
Oct4-neomycin selection scheme with established of ES cell isolation and 
propagation procedures (Evans and Kaufman, 1981; Martin, 1981; Nichols et 
al., 1990; Yoshida et al, 1994) should enable ES cell line derivation from 
previously non-productive mouse strains and other mammalian species in 
which Oct4 is differentially expressed. 
Selection against non-stem cell phenotypes in mixed cell populations may be 
advantageous for several reasons. Firstly, selection against 
differentiated cells in mixed populations provides a method for extensive 
stem cell enrichment. Secondly, selective removal of differentiated cells 
prevents their overgrowth in the cultures. Thirdly, elimination of 
differentiated cells may enhance stem cell self-renewal due to the loss of 
differentiation inducing activity associated with differentiated cells. 
EXAMPLE 2 
RESCUE AND RECOVERY OF PLURIPOTENTIAL STEM CELLS FROM ES CELL EMBRYOID 
BODIES 
Methods 
1. Cell Culture 
ES cells were routinely maintained in medium supplemented with 
Differentiation Inhibiting Activity (DIA) as described by Smith (1991). 
Embryoid bodies were formed by aggregation of ES cells in the absence of 
DIA. The aggregates were produced by plating dissociated ES cells in 10 
.mu.l or 30 .mu.l drops of medium at a density of 100 cells/drop. Arrays 
of drops were plated on the lid of a 10 cm tissue culture dishes using a 
multipipettor. This was then inverted over the base of the dish, which 
contained 10 ml of water in order to maintain humidity, and the hanging 
drops were cultured at 37.degree. C. in a 7% CO.sub.2 atmosphere. 
2. Histology and .beta.-Galactosidase Staining 
Embryoid bodies were fixed in Bouin's solution and embedded in agar. 
Paraffin sections were then prepared by standard procedures and stained 
with haematoxylin and cosin. Alkaline phophatase staining of embryoid body 
outgrowths was carried out using Sigma Kit 86-R. Histochemical staining 
for .beta.-galactosidase was performed with Xgal as described (Beddington 
et al, 1986). 
Results 
3. Cell Lines and Selection Systems 
Fos11 is a derivative of the ES cell line CGR8 which has been transfected 
with the Oct4neofos construct. Fos11 cells express low levels of G418 
resistance under control of the Oct4 proximal promoter element, but 
differentiated progeny show no expression of the transgene and are 
therefore sensitive to G418. OKO160 and OKO8 are derivatives of the ES 
cell lines CGR8 and E14TG2a respectively in which an IRES-.beta.geopA 
cassette has been introduced into one allele of the Oct4 gene by 
homologous recombination as described. OKO cell lines express high levels 
of .beta.geo in the undifferentiated state and therefore stain strongly 
with Xgal and are G418-resistant. Differentiated progeny lose expression 
of .beta.geo and become negative for Xgal staining and sensitive to G418. 
In monolayer cultures, Fos11 and OKO cells are maintained as pure ES cell 
populations by culture in the presence of DIA and selection in G418. Under 
conditions which favour differentiation, however, such as low density and 
absence of DIA (Smith, 1991), G418 selection results in the complete 
elimination of these cultures over 3-5 days. Rb40 cells are a derivative 
of CGR8 which are constitutively resistant to G418 due to expression of 
neoR directed by the human .beta.-actin promoter. 
4. Formation of Embryoid Bodies in the Presence and Absence of Selection 
against Differentiated Cells 
Production of embryoid bodies by the conventional procedure (Doetschman et 
al, 1986) of detachment of clumps of cells followed by aggregation in bulk 
suspension culture results in a mixed population of aggregates, 
heterogeneous in both size and differentiation status. In order to obtain 
more uniform and consistent development, embryoid bodies in the present 
study were formed by aggregation of defined numbers of cells in individual 
drops of culture medium (see Methods). After 48 hours in hanging drop 
culture, the aggregates were transferred en masse into suspension culture 
in the presence or absence of G418. 
Under G418 selection against differentiated progeny aggregates still formed 
from both Fos11 cells and the OKO clones. Although some dead cells 
appeared around the periphery of the aggregates, the bodies themselves 
increased in size during the culture period. Samples were harvested 
periodically from t he bulk cultures and processed for histological 
examination. After several days enbryoid bodies formed in the absence of 
selection were mostly cystic and contained a variety of morphologically 
differentiated cell types. Undifferentiated cells were rarely apparent. By 
contrast, aggregates maintained under selection showed no indications of 
cellular specialisation and the bodies appeared to consist of solid balls 
of undifferentiated cells. The great majority of cells in these 
undifferentiated aggregates appeared healthy and viable and there was no 
evidence of necrosis, although occasional pyknotic nuclei, suggestive of 
apoptosis, were seen. Embryoid bodies formed in G418 were noticeably 
smaller than their counterparts formed in the absence of selection, 
however. This can be attributed to a combination of the lack of cyst 
development and the removal of differentiated cells. 
5. Persistence of Pluripotential Stem Cells in Embryoid Bodies Formed Under 
Selection Against Differentiated Cells. 
The absence of any undifferentiated aggregates in control cultures implied 
that it was unlikely that the effect of G418 was due to selection of a 
subpopulation of non-differentiating aggregates. In order to exclude 
definitively this possibility, however, and also to facilitate 
quantitative determination of the effects of G418 selection, a modified 
protocol was used which allows assessment of the behaviour of individual 
aggregates. Cultures were initiated in 30 .mu.l hanging drops in the 
presence or absence of G418 and maintained in drop culture for 7-8 days. 
Embryoid bodies were then transferred individually to gelatin-coated 
96-well tissue culture plates and the media diluted 6-fold with media 
lacking G418. Th e stem cell maintenance factor DIA was added at this 
stage to allow expansion of any undifferentiated ES cells which were 
present. The cultures were allowed to attach and outgrow -for 48 hours 
then fixed and stained for alkaline phosphatase or for 
.beta.-galactosidase as appropriate. 
The data summarized in Table 1 show that in the absence of any selection 
undifferentiated stem cells are almost completely eliminated from embryoid 
bodies within 7 days of suspension culture. Outgrowths contained a variety 
of morphologically differentiated cell types, but areas of cells with ES 
cell morphology were not observed. In the OKO cells expression of 
.beta.-galactosidase is coupled to the stem cell-specific transcription 
factor Oct4 (Mountford et al, 1994) and therefore serves as a marker of 
undifferentiated cells. Isolated Xgal-staining cells were occasionally 
seen in OKO outgrowths, but clusters of staining cells were not detected 
under these conditions (but see Discussion). 
The efficiency of embryoid body formation in G418 was identical to that in 
non-selected cultures, essentially 100%. In marked contrast to the 
untreated embryoid bodies, however, embryoid bodies established under 
continuous G418 selection gave rise to outgrowths consisting largely of ES 
cells. The undifferentiated nature of these cells was indicated by the 
characteristic morphology of ES cell colonies a nd by staining with 
alkaline phosphatase and was confirmed by Xgal staining of the OKO 
outgrowths. 
Several outgrowths from embryoid bodies formed under selection were picked 
and transferred to 2 cm wells. All of the colonies picked were readily 
expanded into mass cultures of undifferentiated cells. These cultures 
remained dependent on DIA and differentiated in similar fashion to 
parental ES cells when plated in non-supplemented media. Furthermore, 
these derivatives differentiated efficiently into multiple cell types on 
aggregation, confirming their pluripotency. 
These findings demonstrate that the selective elimination of differentiated 
progeny results in the persistence of pluripotential stem cells in ES cell 
aggregates. 
6. Stem Cell Extinction in Mixed Aggregates 
The implication that differentiated progeny may be directly responsible for 
stem cell extinction in embryoid bodies was addressed further. The 
behaviour of OKO cells was assessed following formation of mixed 
aggregates with Rb40 ES cells which can differentiate in the presence of 
G418. Rb40 cells express neomycin phosphotransferase constitutively and 
G418 selection has no discernible effect on their differentiation, either 
in monolayer culture or in aggregates. Hanging drop cultures were 
established, using a 3:1 ratio of OKO cells to Rb40 cells. Paraffin 
sections of mixed embryoid bodies revealed that they underwent extensive 
differentiation in both the absence and the presence of G418. The 
effective elimination of undifferentiated stem cells under both conditions 
was confirmed by Xgal-staining of outgrowths (Table 1). 
This result provides direct evidence that the presence of differentiated 
progeny induces the elimination of pluripotential stem cells. This implies 
that certain differentiated stem cell progeny are a source of inductive 
signals which either instruct further differentiation of remaining stem 
cells or possibly induce them to enter apoptosis. 
Conclusion 
Aggregation induces ES cells to develop into differentiated structures 
known as embryoid bodies. Pluripotential stem cells rapidly become extinct 
in these embryoid bodies due to the efficient induction of differentiation 
and possibly also to selective cell death. However, if differentiated 
progeny are specifically eliminated from the aggregates using methods 
according to the invention, the stem cells persist and can be propagated. 
The findings detailed above constitute a clear demonstration that through 
the use of a stem cell-specific selection system according to the 
invention it is possible to recover stem cells from conditions which would 
normally force their elimination by either differentiation or death. 
TABLE 1 
______________________________________ 
Disappearance or Persistence of Oct-4 Expressing 
ES Cells in Embryoid Bodies. 
No. No. Xgal 
% +ve 
Culture G418* No. Drops Outgrowths +ve Drops 
______________________________________ 
OKO8 - 25 25 0 0 
OKO8 + 25 24 24 96 
OKO160 - 30 30 0 0 
OKO160 + 30 30 30 100 
OKO160:Rb40 - 30 29 0 0 
OKO160:Rb40 + 30 30 0 0 
______________________________________ 
*500 .mu.g/ml 
EXAMPLE 3 
PROCEDURES FOR ESTABLISHING EMBRYONIC STEM CELL CULTURES FROM MOUSE EMBRYOS 
Lines of transgenic mice were established in which the neomycin 
phosphotransferase gene conferring resistance to G418 is expressed with 
the specificity of the Oct4 gene. The .beta.S21 line harbour the 
Oct4neo.beta.S transgene whilst in the OKO line the neo gene has been 
incorporated into the endogenous Oct gene via gene targeting with the 
Oct4-tgtvec construct. These mice were outcrossed for two generations with 
MF1 outbred albino mice and with inbred CBA mice. Neither of these mouse 
strains produce ES cells using standard procedures. 
Four preferred procedures for isolating stem cells are described. In all 
cases the embryos are cultured in standard ES cell culture medium 
supplemented with either Differentiation Inhibiting Activity (Smith, 1991) 
or interleukin-6 plus soluble interleukin-6 receptor (Yoshida et al, 
1994). G418 is added at concentrations of 200 .mu.g/ml-1 mg/ml. 
Procedure 1 
Blastocysts are flushed on the fourth day of pregnancy. Groups of 4-10 
blastocysts are cultured in 1 cm tissue culture wells under G418 
selection. Outgrowths are individually detached and dissociated with 
trypsin as described nichols et al, 1990) after 4-6 days in culture and 
replated in single wells. G418 selection is maintained. Colonies with the 
characteristic morphology of ES cells which appear in the cultures over 
the next 14 days are picked and expanded under continuous selection. 
Procedure 2 
As Procedure 1, except that blastocysts are put into implantation delay 
before harvesting by ovariectomy of the dams on the third day of 
pregnancy. Blastocysts are flushed 4 days after the ovariectomy. 
Procedure 3 
Post-implantation embryos between 5.5 and 7.5 days post-coitum are isolated 
and the primitive ectoderm separated by microdissection and/or protease 
digestion. The primitive ectoderm is gently dissociated into clumps of 
20-50 cells which are then cultured as in Procedure 1. 
Procedure 4 
Embryos prepared as for Procedures 1, 2 or 3 are cultured in hanging drops 
under G418 selection for a period of 5-7 days before transfer to tissue 
culture wells and subsequent manipulation as in Procedure 1.