Note: Descriptions are shown in the official language in which they were submitted.
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PROCESS FOR OBTAINING STEM CELLS
This invention relates to nuclear reprogramming of somatic cells, to a method
of obtaining stem cells from nuclear transplanted embryos, and relates in ,
particular to a method of obtaining embryonic stem (ES) cells. It also.
relates
to pluripotent cells and to assays for reprogramming of somatic cell nuclei
into
pluripotent cell nuclei, and to assays for factors capable of reprogramming a
somatic cell nucleus.
ES cells are pluripotent stem cells and can be used to generate a large range
of somatic tissues such as haematopoietic stem cells, neural precursor cells
and skin cells. The therapeutic value of being able to generate human
embryonic stem cells in vitro would be considerable. Dominko et a! have
suggested bovine oocyte cytoplasm can successfully reprogram somatic nuclei
from different mammalian species and initiate early embryonic development.
It is thus hoped that ES cells will be obtainable from a wide range of
mammalian embryos and will show great value in the development of
transgenic animals. Many have claimed obtaining ES cells, but it has hitherto
not been possible to prove that ES cells have in fact been obtained other than
from the mouse, because they have until now been isolated on the basis of
morphology. This is an unreliable test as embryonic structures derived from
non-ES cells look virtually identical to embryonic structures that do contain
ES
cells.
Transplantation in mammals is presently hampered by the problems associated
with rejection. When foreign tissues are introduced into a host its immune
response is to attack what is perceived as invading tissue. This severe and
life
threatening response can be prevented by the use of immunosuppressant
drugs, however, this leaves the host open to opportunistic infection. The
immune response is not elicited when the host's own tissues are implanted.
It would therefore be of advantage in transplantation if tissues that were
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genetically identical to the host's could be generated.
Embryonic development in mammals commences with a series of cleavage
divisions that generates equivalent totipotent cells. Maternal factors
deposited
in the oocyte dictate a characteristic number and timing of cleavages for each
species. Activation of the zygotic genome occurs during the cleavage process.
This is followed by the divergence of two distinct cell lineages, an outer
epithelial cell layer, the trophectoderm, and an internal cluster of cells,
the inner
cell mass (ICM). The trophectoderm subsequently contributes the embryo-
derived component of the placenta, whereas the ICM is the founder of the
embryo proper. The ICM is pluripotent in that it gives rise to all foetal cell
types, including the germ cells, and also to some extraembryvnic tissues.
Pluripotential cell lines, embryonic stem (ES) cells, can be derived by direct
culture of ICM or its immediate pluripotent successor epiblast (Brook and
Gardner, 1997; Evans and Kaufman, 1981; Martin, 1981; Thomson et al.,
1996).
Once isolated the pluripotent stem cells can be cultured in the presence of
particular growth factors and other signalling molecules that induce their
differentiation into particular cell types. Particular tissue types are needed
in
different medical diseases and conditions. Haematopoietic stem cells could be
used to treat individuals who are suffering from leukaemia and have been
subject to loss of their own haematopoietic cells due to chemotherapy. Neural
progenitor cells could be used to treat individuals suffering from
neurodegenerative diseases such as Alzheimer's disease or Parkinsonism. Skin
cells could be generated that are suitable for grafts in cases where an
individual
has suffered severe burns or scarring. Thus there is great therapeutic
potential
in being able to generate pluripotent stem cells that are genetically
identical to
a patient.
It is desirable to devise a further method by which human pluripotent stem
cells
can be made. It is in addition desirable to be able to make pluripotent stem
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cells from a given individual. !t is also desirable to be able easily to
identify
which cells in the early embryo are undifferentiated pluripotent stem cells.
Furthermore, it would be advantageous to be able to isolate these pluripotent
stem cells and then induce them to differentiate into a particular cell type.
Finally, it is desirable to have an assay which is able to identify those
factors
that are necessary to maintain an undifferentiated state in the ES cell.
Nuclear replacement in oocytes is used to produce embryos and live offspring
from somatic cells. This cloning procedure enables the production of
genetically identical tissues or individuals, but the efficiency of current
procedures is very low and only a small percentage of nuclear transfer embryos
give viable offspring. It is likely that this is due in part to incomplete
reprogramming of the donor cell nucleus. International patent publications WO-
97/07668 and WO-97/07669 describe a method of transferring the nucleus of
a somatic cell to a recipient cell, which could be ~an oocyte. Both WO-
97/07fi68 and WO-97/07669 teach that the donor cell nucleus must be in a
quiescent state for successful embryogenesis and subsequent implantation to
occur. It would be advantageous to be able to identify which of the embryos
produced by nuclear transplantation contain pluripotent cells prior to further
culturing or implantation into a host mother.
The present invention aims to provide stem cells and an improved method for
obtaining stem cells. It also aims to provide an improved method for obtaining
human ES cells including transgenic human ES cells. The present invention
further aims to provide an assay for factors capable of reprogramming somatic
cell nuclei to a totipotent state, and an assay for identification of embryos
or
embryonic structures containing ES cells.
The present invention provides a combination of transplantation of a nucleus
from a somatic cell into an enucleated oocyte with identification of daughter
cells of the oocyte that express a gene characteristic of a desired stem cell.
fn preferred embodiments of the invention, cells expressing a gene
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characteristic of embryonic stem cells are identified.
Accordingly, a first aspect of the invention provides a method of obtaining a
stem cell of a predetermined species, comprising:-
obtaining a somatic cell of the predetermined species, which cell
contains a gene coding for a marker which is differentially expressed in
(a) desired stem cells, and (b) cells other than desired stem cells;
obtaining an enucleated oocyte;
transferring the nucleus of the somatic cell into the enucleated oocyte
to form a transgenic oocyte;
culturing the transgenic oocyte in vitro to produce a culture of cells
derived from the transgenic oocyte; and
identifying those cells of the culture that express the marker and,
optionally, selectively propagating them.
It is a particular advantage of the invention that it facilitates the
identification
of embryos, and embryonic structures that will successfully go to term, and
form adults. The high failure rate of prior art methods, in which as low as 1
per cent or less of embryos are viable has hitherto rendered this technology
highly unreliable. The method of the invention may thus further comprise
culturing the transgenic oocyte to form an embryo or embryonic structure, and
determining whether the cells of the embryo or embryonic structure express the
marker gene. Pluripotent cells are known to form the inner cell mass of the
embryonic structure, and the method typically comprises culturing the
transgenic oocyte to form an embryo or embryonic structure comprising an
inner cell mass and trophectoderm cells, and determining whether the cells of
the inner cell mass express the marker gene.
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The somatic cell containing the marker gene may be obtained by introducing
a transgene into a somatic cell, or by isolating a somatic cell containing the
transgene from a transgenic animal. The invention thus encompasses first and
descendant generations of transgenic stem cells. For generation of first
generation transgenic stem cells, it is especially preferred that, starting
with a
somatic cell, a transgene is introduced into the somatic cell before the
subsequent step of nuclear transfer into an enucleated oocyte. The somatic
cell can be obtained via any standard biopsy procedure.
A key component of the present invention technology is the use of markers for
ready identification of desired stem cells. In a specific embodiment of the
invention, cells that are pluripotent stem cells express green fluorescent
protein
(GFP). Using UV fight, visual inspection allows sorting of the cells or
embryos
so that non-viable ones can be discarded.
In one embodiment of the invention, the marker is a selectable marker wherein
cells not expressing the selectable marker can be selectively killed or
ablated
in culture. For example, the selectable marker may be antibiotic resistance,
such as a gene coding for neomycin resistance or 6418 resistance.
The isolation and propagation of pluripotent ES cells is facilitated by
elimination
of differentiated cells. This is suitably achieved by expressing a selectable
marker under control of regulatory sequences of the ICM marker gene, oct4,
or of an Oct-4 responsive gene such as fgf4, or the gene for an Oct-4 co-
factor
such as Sox2, or of any other gene that is expressed selectively in ICM and
epiblast cells. The selectable marker can be integrated into the endogenous
gene by homologous recombination or may be inserted in a transgene
construct. In addition a conditional immortalising gene can be introduced into
a stem cell-specific locus to facilitate expansion of the pluripotent cells. A
key
indicator of functional reprogramming and a prerequisite for foetal
development
should be formation of a pluripotential ICM/epiblast that expresses Oct-4 in
most or ail cells, as it is known that Oct-4 is essential for development of
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pluripotent cells in the mammalian embryo. Such embryos will give a high rate
of post-implantation development and can be used as a source of ES cells.
An alternative is for the marker to be a reporter gene, wherein cells
expressing
the marker can be identified visually in culture. One suitable reporter is one
which codes for a product that fluoresces in a particular light, such as GFP
in
UV light as mentioned or luciferase. Another is /3-galactosidase which gives a
blue colour with Xgal or a fluorescent signal with fluorescein digalactoside
(FDG). It is particularly convenient that all can be used with the light
microscope, so simple techniques are sufficient to identity the desired cells.
The stains are preferably vital stains, in that they do not require fixing -
killing
of the stained tissue.
In use of the method of a specific embodiment of the invention, a transgene is
introduced into a plurality of human somatic cells, the transgene containing a
promoter active exclusively in embryonic stem cells and directing expression
of GFP or of GFP fused to the stem cell specific gene product. The somatic
cell
nuclei are then each transferred into an enucleated pig oocyte. The oocytes
are then cultured to form embryonic structures and these are subjected to
illumination by UV and inspection under a microscope. Those few that show
fluorescence within the ICM can be easily identified and isolated. From that
point onwards the isolated embryonic structures can be further cultured in
vitro
or implanted. The use of the marker hence eases discarding the non-viable
embryos that previously would have had to be identified on the basis of
inability to implant, inability to go to term or subsequent morphological
change
indicating they are not in fact viable embryos.
A further alternative is for the transgene to code for an immortalising gene.
This aids maintenance of a Tong term culture of embryonic stem cells obtained
by the method.
In use of the method of a further specific embodiment of the invention, a
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transgene is introduced into a plurality of human somatic cells, the transgene
containing a promoter active exclusively in embryonic stem cells and directing
expression of resistance to neomycin. The somatic cell nuclei are then each
transferred into an enucleated cow oocyte. The oocytes are then cultured to
form embryos from which cells which appear to be pluripotent stem cells are
isolated. These cells are cultured in the presence of 6418 and thus only those
that are undifferentiated ES cells are able to survive.
In use of the method of a specific embodiment of the invention, a transgene is
70 introduced into a plurality of human somatic cells, the transgene
containing a
promoter active exclusively in neuronal stem cells and directing expression of
a selectable marker. The somatic cell nuclei are then each transferred into an
enucleated recipient cell. The recipients are then cultured and these are
subjected to selection. Those few that survive can be easily identified and
isolated. From this point onwards the isolated neuronal stem cells can be
further cultured in vitro. The use of the marker hence eases discarding the
non-neuronal stem cells. In embodiments of the invention in which the aim is
to obtain target stem cells other than ES cells, such as in the above
embodiment for obtaining neuronal stem cells, the recipient is either an
oocyte
or preferably a target stem cell of a different individual. Thus an enucleated
neuronal stem cell is used as the recipient for a transplanted somatic
nucleus.
Thus, to obtain neuronal stem cells, a plurality of somatic cells is first
genetically manipulated to introduce a selectionireporter gene such as ~3geo
under control of the regulatory elements of sox1, sox2 or sox3, genes that are
expressed uniformly in neuroepithelial precursor cells. For example, a
selectionireporter gene, ~3geo, is integrated by homologous recombination into
the sox2 gene, which is expressed uniformly in precursor cells in the neural
plate and neural tube. A transgene containing this construct is introduced
into
somatic cells, and application of 6418 after nuclear transplantation results
in
the efficient isolation of viable,Qgeo-positive cells. These cells express
markers
of neuroepithelial precursor cells. They can be propagated and will
differentiate
efficiently into networks of neuron-like cells that express a variety of
neuronal
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markers. Thus, an in vitro system for genetic and molecular dissection of
mammalian neural differentiation is provided by the invention, and also a
route
for the production of pure populations of normal or genetically modified
neural
precursors and neurons for functional studies including transplantation. The
CD34, CD44 and SCL genes are suitable for obtaining haematopoietic
progenitors, and the Nkx2.5 or GATA-4 gene for cardiac progenitors. For
generating myogenic progenitors, MyoD or myf-5 are suitable.
It is an additional option that the method of the invention further comprises
introducing into the somatic cell a selectable marker and a reporter marker,
both expressed in the desired stem cells. This enables the visual
identification
and isolation of viable embryos, which can then be cultured under selection
for
the subsequent isolation of ES cells. The resultant culture contains stem
cells
that are genetically identical to the original donor somatic cell, except for
the
presence of the transgenes and of recipient cytoplast mitochondria. Use of
excision sites flanking the transgenes are suitable to enable removal of the
transgenes, such as by site specific recombinases.
The technology of the present invention is of application to all mammalian
cell
types, and to obtaining embryonic stem cells of a species for which there is a
low for no) availability of recipient oocytes. It is preferred that the
somatic cell
is a human cell, and surprisingly the invention provides for creation of human
embryonic stem cells from a non-human oocyte - pig or cow oocytes are used
in the recited examples below, as they are readily available.
Once embryonic stem cells are isolated, they can be used as a resource for
deriving progenitor cells of a selected lineage. As described and claimed in
co-
pending GB patent application 9807935.3 filed on 14th April 1998, neuronal,
haematopoietic progenitors, cardiac and other progenitors are obtainable using
a further transgene expressed substantially only in the desired progenitors,
and
also cells of a selected differentiated non-stem cell lineage are similarly
obtainable.
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A second aspect of the invention provides a method of obtaining stem cells
comprising:-
obtaining a somatic cell from an individual which cell comprises a
transgene which is differentially expressed in (a) desired stem cells, and
(b) cells other than desired stem cells;
transferring the nucleus of the somatic cell into an enucleated oocyte;
culturing the oocyte to farm an embryo or an embryonic structure; and
determining whether cells in the resultant embryo or embryonic structure
are expressing the transgene.
The method is preferably for obtaining mammalian embryonic stem cells,
wherein the transgene is differentially expressed in (a) cells of the inner
cell
mass, and (b) cells other than cells of the .inner cell mass, and the method
comprises culturing the oocyte to form an embryo or embryonic structure and
determining whether the cells of the inner cell mass express the transgene. It
is particularly preferred that the transgene is expressed substantially only
in
cells of the inner cell mass. It is an option that blastocysts are developed
in
vivo, e.g. in ligated oviducts.
The transgene can code for a selectable marker, wherein cells not expressing
the selectable marker can be selectively killed or ablated in culture; or the
transgene can code for a reporter marker which fluoresces when illuminated
with light of a certain wavelength, and wherein cells expressing the reporter
marker can be identified by their fluorescence in the presence of the light.
Other preferred and alternative features of the first aspect of the invention
apply equally to the second aspect, and further aspects, of the invention.
A third aspect of the invention provides an assay for the effect of a factor
on
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a nucleus of a somatic cell, comprising:-
introducing the nucleus into an enucleated cell, wherein the nucleus
comprises a marker that is differentially expressed in (a) a desired stem
cell, and (b) a cell other than the desired stem cell;
introducing the factor into the cell;
maintaining the cell in in vitro culture; and
determining whether the cell or any of its daughter cells are expressing
the marker, whereby expression of the marker indicates the somatic cell
nucleus has been reprogrammed to be a nucleus of the desired stem cell.
15' By "reprogrammed", and derived terms, we mean that a nucleus from a cell
with restricted developmental potential acquires greater developmental
potential, e.g. that a unipotent nucleus from a differentiated somatic cell is
thereby able to be directed to develop into a pluripotent nucleus of a stem
cell,
or that a multipotent nucleus from a somatic stem cell is thereby able to be
directed to develop into a pluripotent stem cell nucleus. Preferably, the
somatic
cell nucleus is able to be reprogrammed such that when it is implanted into
the
enucleated oocyte it can facilitate the development of a normal embryo, which
can then be implanted and go to term to form an adult animal or which can
then be used for derivation of embryonic stem cells and, subsequently,
differentiated cells if so desired.
An advantage of the assay is that it assists screening for a factor or factors
that induce reprogramming of nuclei. Factors so identified can then be used
as agents further to increase the reliability and efficiency of the technique.
The
assay is suitably performed using enucleated oocytes. The assay is also
suitably performed using enucleated cells other than oocytes, and this has the
advantage that the effect of the factor is assayed without interference from
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any nucleus reprogramming factor that might be already coincidentally present,
as might be the case if an oocyte is used. Fibroblasts are one suitable cell
type.
For identification of factors to reprogram nuclei into p[uripotential nuclei,
the
marker used in the assay is differentially expressed in la) pluripotential
stem
cells, and (b) cells other than pluripotential stem cells.
The assay is suitable for assay of a library of factors, such as a library of
factors obtainable by expression of a cDNA library obtained from mRNA
extracted from the cytoplasm of an oocyte.
As an alternative to the above recited procedure, it is an option for the
assay
to be carried out without nuclear transfer, but nevertheless providing for
identification of a factor that reprograms the nucleus of a somatic cell.
Specifically, the assay may comprise:-
maintaining a culture of a cell, which cell comprises a marker
differentially expressed in (a) a desired stem call and Ib) a cell other than
a desired stem cell;
introducing into the cell a factor to be assayed;
determining whether the cell or any of its daughter cells are expressing
the marker, whereby expression of the marker indicates that somatic cell
nucleus has been reprogrammed to be a nucleus of a desired stem cell.
This embodiment of the inventiori can conveniently be used for assay of an
oocyte library to identify a factor that activates OCT 4 in, say, a fibroblast
cell.
It is also desirable to assay for embryos that have the potential to go to
term,
and for embryonic structures that contain pluripotent cells. Accordingly, a
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fourth aspect of the invention provides an assay to determine whether an
embryo or embryonic structure contains embryonic stem cells, comprising:-
introducing into an enucleated oocyte the nucleus of a somatic cell
comprising a marker differentially expressed in (a) embryonic stem cells,
and (b) cells other than embryonic stem cells;
obtaining therefrom an embryo or embryonic structure; and
determining whether cells of the embryo or embryonic structure express
the marker.
In an embodiment of the invention, the assay comprises introducing into the
somatic cell a transgene containing a sequence coding for the marker under
control of a promoter activated substantially only in epablast cells. One
specific
example is the oct4 promoter, or a promoter activated in cells expressing the
oct4 gene.
After introduction of the transgene, there will be some somatic cells that are
stably transfected, but many that are not. In a preferred embodiment of the
invention the transgene also contains or is cotransfected with a sequence
coding for a second marker under control of a promoter activated in
substantially all cells, and the assay comprises determining whether the
transgene has been stably integrated into the nucleus by determining whether
the somatic cell expresses the second marker.The beta-actin promoter is one
example. In this way, stable transfectants can be identified before
determination of those that express the first marker, and optionally before
the
step of nuclear transplantation. Preferably, the second marker is a selectable
marker, enabling selection of stable transfectants. The second marker may be
flanked with excision sites to enable its removal prior to further
manipulation.
In fifth to twelfth aspects of the invention there is provided the cells
obtainable
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by nuclear transfer according to the invention, namely:-
a human embryonic stem cell derived from a human oocyte;
a human embryonic stem cell derived from a non-human oocyte;
a human embryonic stem cell derived from a non-human mammalian
oocyte;
a human embryonic stem cell derived from a pig or cow oocyte;
a human stem cell derived from a human oocyte;
a human stem cell derived from a non-human oocyte;
a human stem cell derived from a non-human mammalian oocyte; and
a human stem cell derived from a pig or cow oocyte;
The invention still further provides a stem cell for transplantation into a
first
human and being immunologically compatible with the first human, derived
from an oocyte of a different human; and an embryonic stem cell of a first
mammalian species, derived from an oocyte of the same species or of a species
other than the first species, and comprising a transgene coding for a
selectable
marker which is substantially only expressed in the embryonic stem cell; and
a multipotential cell that expresses a transgene which identifies its
undifferentiated state, which cell is genetically identical, apart from the
presence of the transgene, to an adult animal.
The invention yet further provides a method of obtaining a human embryonic
stem cell, suitable for derivation of human stem cells or human somatic cells
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for transplantation into a first human individual, comprising introducing into
an
oocyte other than of that first individual the nucleus from a somatic cell of
that
first individual. The human embryonic stem cell preferably contains a
transgene coding for a selectable marker which is substantially only expressed
in the human embryonic stem cell.
The invention again further provides a method of reprogramming a somatic
nucleus into a pluripotent nucleus, comprising:-
identifying a factor that induces reprogramming of the nucleus, by
carrying out the assay according to the third aspect of the invention;
obtaining a somatic cell nucleus;
obtaining an enucleated cell;
transferring the somatic cell nucleus into the enucleated cell to form a
recombinant cell;
introducing the factor into the recombinant cell.
The invention yet again further provides a method of reprogramming a somatic
nucleus into a pluripotent nucleus, comprising:-
identifying a factor that induces reprogramming of the nucleus, by
carrying out the assay according to the third aspect of the invention;
obtaining a somatic cell, and introducing the factor;
obtaining an enucleated cell;
transferring the nucleus of the somatic cell into the enucieated cell to
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form a recombinant cell;
culturing the recombinant cell in vitro.
In these above two methods, it is optional to introduce into the somatic cell
a
gene coding for a marker which is differentially expressed in (a) desired
totipotent cells, and (b) cells other than desired totipotent cells; and
determine
whether cells of the embryo are expressing the marker. Even though a
reprogramming factor has been identified by the assay, using these optional
steps facilitates isolation of reprogrammed nuclei, which nuclei can then be
propagated if desired and can be introduced into enucleated oocytes.
In use of a typical method of the invention a pluripotent cell specific
selectable
marker and/or reporter and/or immortalising gene is introduced into a somatic
cell culture, for example a culture of foetal or adult fibroblasts, and a
nucleus
from a cell harbouring said markers) is transferred into an enucleated oocyte
or other suitable cytoplast. Visualisation of, or selection for, expression of
the
stem cell specific marker then allows detection of, or selection for,
successful
reprogramming. Pluripotential stem cell cultures are isolated and propagated
under selection for marker gene expression and /or influence of the
immortalising gene.
In a specific embodiment of the invention, described in an example below, the
selectable marker/reporter is introduced under control of the regulatory
elements of the gene encoding the essential pluripotent cell-specific
transcription factor Oct-4 (Mountford et al., 1994). In another embodiment,
the selectable marker is placed under regulatory control of an Oct-4
responsive
sequence such as the fgf4 gene or of a stem cell restricted co-factor of Oct-4
such as Sox2, or of another ICM/epiblast specific gene. The selectable marker
preferably encodes both a reporter gene function, such as ~3-galactosidase or
GFP, and a drug resistance function. This dual function can be achieved in the
form of a fusion gene, such as the iacZlneo fusion ~3geo, or of a dicistronic
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construct incorporating an internal ribosome entry site (IRES), for example
gfpl RESpac. In an alternative embodiment the transgene construct may
incorporate an immortalising gene to facilitate subsequent growth of the
pluripotent stem cells. This may be included instead of, or in addition to,
the
selectable rnarker/reporter. One such immortalising gene is that encoding SV40
large T protein. The immortalising gene is rendered conditional, for example
by
using a temperature or hormone-regulated variant and/or by incorporation of
target sequences for site-specific recombination or endonuclease degradation.
A combination of reporter, selectable marker and immortalising gene can be
introduced either in a single construct using gene fusions and/or IRES
elements,
or by introduction of separate transgenic constructs.
The POU transcription factor Oct-4 is expressed in all pluripotent cells in
the
ICM and epiblast and in germ cells. Oct-4 is absent from mature
trophectoderm and all other differentiated cell types. The inventors have
discovered that Oct-4 is essential for the establishment of the pluripotential
identity of the ICM. In blastocyst stage embryos in which the oct-4 gene has
been inactivated, internal cells fail to develop ICM character and are
diverted
into the trophectoderm lineage. Oct-4 function is also continuously required
to maintain pluripotency of ES cells. Inactivation of the oct-4 gene in
established ES cell cultures results in a failure of self-renewal and terminal
differentiation. Oct-4 controls the expression of multiple target genes. Genes
that are positively regulated by Oct-4, such as the gene for fibroblast growth
factor-4 (FGF-4), exhibit a similar restricted pattern of expression in ICM
and
epiblast. Oct-4 functions in collaboration with co-factors such as the HMG-box
transcription factor Sox2 and the E 1 A-like activity whose expression may be
similarly restricted in the early embryo.
The selectable marker is preferably placed under transcriptional control of
Oct-4
regulatory sequences either by homologous integration into the oct-4 gene or
random integration of a transgene construct. In the example the selectable
marker is integrated into the 3'UTR of the oct4 gene and is preceded by an
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EMCV IRES sequence inserted immediately 3' to the oct4 termination codon.
Alternatively, the selectable marker is integrated either directly at the ATG
start
codon, or in-frame within the Oct-4 coding sequence, or in any frame within
the Oct-4 coding sequence in which case it should be preceded by a
translational termination codon in the oct-4 reading frame and an IRES
element.
Optionally, the transgene may be flanked by recognition sequences for a site
specific recombinase such as Cre or Flp to enable subsequent deletion, or may
contain a site for a rare restriction site endonuclease such as I-scel. For
introduction into.somatic cells in culture the transgene construct will
normally
contain or be co-transfected with a construct containing an independent
selectable marker to enable isolation of stable transfectants. This selectable
marker should be distinct from that inserted into the oct-4 gene and must be
under the transcriptional control of a promoter active in the target somatic
cell,
for example the promoter of a ubiquitously expressed gene such as ~3-actin.
The independent selectable marker may be flanked bar recognition sequences
for a site specific recombinase to allow subsequent excision. These sites
should be for a different recombinase than sites flanking the stem cell-
specific
marker transgene to avoid excision of the latter.
The transgene construct may be introduced via pronuclear injection to generate
a transgenic animal from which somatic cells can be isolated or is introduced
directly into somatic cells in vivo or in culture by microinjection, viral
transduction, lipofection, electroporation, calcium phosphate co-precipitation
or other method. Integrity of the transgene and integration by legitimate or
illegitimate recombination are determined by genomic DNA analysis. Where
appropriate transient introduction of the appropriate site-specific
recombinase
is used to delete the independent selectable marker.
Nuclei from cells harbouring the transgene are transferred into enucleated
oocytes or other suitable cytoplasts using established procedures ICampbeil et
al., 1996; Wilmut et al., 1997) or technical modifications thereof such as the
use of microinjection rather than fusion. Recipient oocytes may be of the same
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species as the donor cell or of a different species. Thus bovine or other
animal
oocytes may be used for reprogramming human somatic cell nuclei. Following
nuclear transfer, activated reconstituted oocytes may be transferred to the
reproductive tract of a primed female for development in vivo with subsequent
recovery and examination at the blastocyst stage. In vivo development of
nuclear transfer embryos could also be achieved by encapsulation and
placement at a favourable ectopic site, e.g. in the reproductive tract or
subcutaneously, in an animal of the same or different species. Alternatively
embryos may be developed in vitro by culture intact to the blastocyst stage.
Expression of the reporter may be determined by histochemical or
immunohistochemical analysis of fixed specimens, but more normally is
examined in live embryos by visualisation of vital fluorescent staining,
either
directly for GFP or using a fluorescent substrate for ~i-galactosidase.
Embryos showing specific and' uniform staining in the I~CM/epiblast are
selected
for transfer to recipients to produce embryos or live offspring. For
derivation
of stem cell lines, embryos may be explanted directly into culture.
Alternatively
the stem cell compartment may first be expanded in vivo, either by allowing
implantation and a brief period of post-implantation development, and/or by
transplantation of cleavage stage embryos, blastocysts or egg cylinders to an
ectopic site such as the kidney capsule or testis of a syngeneic or
immunocompromised host for teratoma development (Solter et al., 19701. The
embryo and recipient are not required to be of the same species for teratoma
formation. Induction of human teratomas can be achieved in nude or SCID
mice, for example. Cultures initiated from embryonic material or teratomas are
maintained in the presence of the selection drug until continuously growing ES
cells are established. Pluripotent EG cell cultures (Matsui et al., 1992) may
also be initiated from primordial germ cells following uterine transfer and
implantation. Again, the cultures are carried out in the presence of selection
agent to eliminate non-stem cells.
There now follows description of specific embodiments of the invention
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illustrated by drawings in which:-
Fig. 1 shows an Oct4-/3geo expression construct according to the
invention;
Fig. 2 shows an egg cylinder stage embryo, 6.5 dpc (upper panel) and
blastocysts from a backcross mating (lower panel), illustrating
expression of ~3-galactosidase in ICM and epiblast of transgenic embryos;
Fig. 3 shows cultured epiblast of a transgenic embryo, illustrating
expression of ~3-galactosidase is maintained in culture;
Fig. 4. shows ~3-galactosidase staining in (A) morulae and (B) blastocyst
stage Oct4-bgeo transgenic mouse embryos; and
Fig. 5. shows /3-galactosidase staining in nuclear transfer mouse
embryos following transfer of Oct4-bgeo somatic cell nuclei into non-
transgenic recipient cytoplasts.
Example 1
Mouse genomic clones spanning the Oct-4 locus were isolated from a strain
129 genomic library and mapped by restriction analysis. A transgene construct
was prepared comprising the distal and proximal 5' enhancer elements, the
entire structural gene, a IRES~3geo cassette inserted at the translation
termination codon, the 3' untranslated sequence and several kb of 3' sequence
(Figure 1 ).
Transgenic rats harbouring this transgene were generated by pronuclear
injection. In these rats expression of the transgene faithfully reproduced
expression of Oct-4. Thus Q-galactosidase activity was present in the inner
cell
mass of blastocysts and the epiblast of early post-implantation embryos but
was absent from extraembryonic lineages and from differentiating embryonic
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germ layers (Figure 2). Post-gastrulation expression of ~3-galactosidase was
confined to germ cells. Significantly also, ~3-galactosidase activity ie
specific
expression of the selectable marker was maintained in culture (Figure 3).
Having confirmed that the transgene contained appropriate regulatory
sequences to dictate pluripotential cell-specific expression, it was modified
for
transfection into cultured somatic cells. As Oct-4 is not expressed in these
cells, this required introduction of an independently expressed selection
marker.
A hygromycin resistance-thymidine kinase fusion gene (hph/tk) under
transcriptional control of the mouse pgk-7 promoter was introduced into the 3'
end of the transgene, downstream of the untranslated region. The pgk-hph/tk
cassette was flanked by /oxPsites and the entire transgene fragment was
flanked by frt sites.
The linearized construct was introduced into primary rr~ouse embryo
fibroblasts
by electroporation and clonal isolates selected in the presence of hygromycin.
Clones were assayed for ectopic activity of the Oct-4 transgene by plating
aliquots in the presence of 200,ug/ml 6418. Only those that failed to grow in
6418 were studied further. Three such clones (I, II and III) with single copy
integrations determined by Southern analysis were transiently transfected with
Cre recombinase and selected in the presence of gancyclovir. Gancyclovir
resistant colonies were screened by PCR to confirm Cre-mediated excision of
the pkg-hph/tk cassette. Deletion of this cassette removes the potential for
interference with activity of the Oct-4 transgene.
Resultant clones carrying the Oct-4,~geo transgene only were used for nuclear
transfer into mouse, ovine and bovine enucieated oocytes. The oocytes were
quiesced by serum-deprivation and nuclear transfer was carried out as
described by Campbell et al (1996). In some cases a serial round of nuclear
transfer was performed. After nuclear transfer the activated oocytes were
cultured in embryo culture medium and monitored for cleavage, morula
formation and blastocyst development. Embryos were fixed and stained for ~3-
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galactosidase activity using Xgal. No staining was observed after nuclear
transfer of clone II derivatives, presumably due to a non-permissive
integration
site. Derivatives of clones ! and III in contrast gave ICM-specific staining
in 30-
50% of embryos that developed to the blastocyst stage, regardless of oocyte
origin. Of these approximately half showed mosaic staining and half showed
uniform staining in all ICM cells. Thus only around 25% of morphologically
developed blastocyts show normal expression of the Oct-4 transgene.
The developmental capacity of Oct4/3geo expressing and non-expressing
embryos generated after nuclear transfer into mouse oocytes was then
compared. A vital fluorescent substrate for Q-galactosidase (Molecular Probes)
was used to visualise transgene expression in live embryos. Blastocysts were
categorised as non-staining, mosaic ICM staining or uniform ICM staining and
divided accordingly into three groups. Each group was transferred into the
uteri of separate pseudopregnant mouse recipients. Mice were sacrificed 5
days later for analysis of implantation and development. The first two groups
gave fewer than 50% implantations. High numbers of resorption sites and
abnormal embryos were found in both cases. Morphologically normal embryo
development was not observed after transfer of class I embryos and only in 1
in 10 implantations generated by class II embryos. In contrast, class Ill
embryos gave greater than 50% implantation rates and the majority of embryos
were normal. The analysis was then repeated and pregnancies allowed to go
the term. Liveborn offspring were obtained from class III embryos only.
In order to isolate stem cell lines, nuclear transfer embryos were developed
in
vivo to the late blastocyst or early implantation stage. Ep~blasts were
microdissected (Brock and Gardner, 1997) and placed in culture in ES cell
medium in the presence of 6418. After several days the epiblasts were
dissociated and replated. Expanding populations of undifferentiated stem cells
were generated. Stem cell lines were also derived form embryos developed in
vitro by immunosurgical isolation of the ICM at the blastocyst stage followed
by microsurgicaf removal of the primitive endoderm and culture in ES cell
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medium plus 6418.
Fibroblast clones 1 and III in which the transgene was competent for
reprogramming were also used to assay for factors that mediate
reprogramming. Messenger RNA was prepared from oocytes, fractionated on
a sucrose gradient, and microinjected directly into fibroblast nuclei. 6418
selection was then applied to identify RNA pools containing reprogramming
activity. cDNA expression libraries were prepared from positive pools and
transfected into the fibroblasts to enable isolation of sequences encoding
reprogramming factors.
Example 2
Mouse genomic clones spanning the Oct-4 locus were isolated from a strain
129 genomic library and mapped by restriction analysis. A transgene construct
was prepared comprising the distal and proximal 5' enhancer elements, the
entire structural gene, a IRES /3geo cassette inserted at the translation
termination codon, the 3' untranslated sequence and several kb of 3' sequence
(Figure 1 ).
Transgenic mice harbouring this transgene were generated by pronuclear
injection. /3-galactosidase activity was monitored by Xgal staining of fixed
embryos and tissue and is evident in morulae and blastocysts stage pre-
implantation mouse embryos (Figure 4). As expected, Q-galactosidase activity
was not detected in somatic cells including cumulus cell and foetal fibroblast
nuclear donors.
Nuclei from somatic cells of transgenic mice were used for nuclear transfer.
Enucleated and non-enucleated wild-type oocytes were used as recipient
cytoplasts. In some cases a serial round of nuclear transfer was performed.
After nuclear transfer the activated oocytes were cultured in embryo culture
medium and monitored for cleavage, morula formation and blastocyst
development. Embryos were fixed and stained for ~3-galactosidase activity
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using Xgal. Staining corresponding to Oct4 expression was observed in
approximately 50% of the nuclear transfer embryos at the eight-cell stage of
development (Figure 5) confirming successful nuclear reprogramming. Embryos
which did not show /3-galactosidase activity failed to reprogram the Oct4-bgeo
transgenic nucleus, or to functionally incorporate the transgenic nucleus and
were therefore scored as compromised in developmental capacity.
Nuclear transfer embryos can be stained with the vital fluorescent substrate
for
~3-galactosidase (Molecular Probes) to visualise transgene expression in live
embryos. Embryos categorised as non-staining, mosaic or uniform staining can
be divided according to nuclear status and associated developmental capacity
based on the transgene expression profile.
References
Brook, F. A., and Gardner, R. L. 11997). The origin and efficient derivation
of
embryonic stem cells in the mouse. PNAS 94, 5709-5712.
Campbell, K. H. S., McWhir, J., Ritchie, W. A., and Wilmut, I. (1996). Sheep
cloned by nuclear transfer from a cultured cell line. Nature 380, 64-66.
Dominko T, Mitalipova M, Haley B, Beyhan Z, Memili E and First N,
Theriogenology, Jan 1 st 1998, Vol. 49, No. 1, page 385.
Evans, M. J., and Kaufman, M. (1981). Establishment in culture of
pluripotential cells from mouse embryos. Nature 292, 154-156.
Martin, G. R. (1981 ). Isolation of a pluripotent cell line from early mouse
embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc.
Natl. Acad. Scl. USA 78, 7634-7638.
Matsui, Y., Zsebo, K., and Hogan, B. L. M. (1992). Derivation of
pluripotential
embryonic stem cells from murine primordial germ cells in culture. CeII70, 841-
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847.
McWhir, J., Schnieke, A. E., Ansell, R., Wallace, H., Colman, A., Scott, A.
R.,
and Kind, A. J. (1996). Selective ablation of differentiated cells permits
isolation of embryonic stem cell lines from murine embryos with a non-
permissive genetic background. Nature Genetics 14, 223-226.
Mountford, P., Zevnik, B., Duwel, A., Nichols, J., Li, M., Dani, C.,
Robertson,
M., Chambers, I., and Smith, A. ( 1994). Dicistronic targeting constructs:
reporters and modifiers of mammalian gene expression. Proc. Natl. Acad. Sci.
USA 91, 4303-4307.
Solter, D., Skreb, N., and Damjanov, I. (1970). Extrauterine growth of mouse
egg cylinders results in malignant teratoma. Nature 227, 503-504.
Thomson et al, Proc. Natl. Acad. Sci., 1995 or 1996, Isolation of a Primate ES
cell (details to be confirmed).
Thomson et al, Science 282, 1998, pp1 145-1 147, Embroynic stem cell lines
derived from human blastocysts.
Wilmut, I., Schnieke, A. E., McWhir, J., Kind, A. J., and Campbell, K. H. S.
(1997). Viable offspring derived from fetal and adult mammalian cells. Nature
385, 810-813.
This invention thus enables increased efficiency of cloned and transgenic
animal production by nuclear transfer, provides an assay for factors that
mediate or suppress reprogramming, provides a generic method of ES cell
derivation for mammals, and provides a method of establishing pluripotent stem
cell cultures from human individuals of any age.
