Note: Descriptions are shown in the official language in which they were submitted.
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PLURIPOTENTIAL CELLS-2
The invention herein described relates to isolated pluripotential cells,
comprising at least part of the cytoplasm derived from an embryonic stem
cell/embryonic germ cell and a nucleus of a somatic cell; methods to prepare
such cells; therapeutic compositions of said cells; and uses thereof.
Animal embryonic development is a highly regulated development process
that combines cell proliferation and cell/tissue differentiation to produce an
intact organism. The co-ordination of cell proliferation and differentiation
is,
and has been, the subject of intense research and the information derived from
this has contributed to our understanding of cell function and disease. For
example and not by way of limitation, regulation of gene expression, cell
differentiation, oncology, teratology.
Mammalian embryonic development is remarkably conserved during the early
stages. Post fertilisation the early embryo completes four rounds of cleavage
to form a morula of 16 cells. These cells complete several more rounds of
division and develope into a blastocyst in which the cells can be divided into
two distinct regions; the inner cell mass, which will form the embryo, and the
trophectoderm, which will form extra embryonic tissue, (eg placenta).
Those cells that form part of the embryo up until the formation of the
blastocyst are said to be totipotent (e.g. each cell has the developmental
potential to form a complete embryo and all the cells required to support the
growth and development of said embryo).
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During the formation of the blastocyst, the cells that comprise the inner cell
mass are said to be pluripotential (e.g. each cell has the developmental
potential to form a variety of tissues).
Embryonic stem cells may be principally derived from two embryonic
sources. Pluripotential cells isolated from the inner cell mass are termed
embryonic stem cells (ES cells). An alternate source of pluripotential cells
is
derived from primordial germ cells isolated from the mesenteries or genital
ridges of days 8.5-12.5 post coitum embryos which would ultimately
differentiate into germ cells. These pluripotential cells are referred to as
embryonic germ cells ( EG cells). Each of these types of pluripotential cell
has
the same developmental potential with respect to differentiation into
alternate
cell types.
It is important to note that an intact embryo cannot be produced from a single
pluripotential cell ( eg either an ES or EG cell). Therefore a pluripotential
cell
has an increased commitment to terminal differentiation when compared to a
totipotent cell.
For the sake of clarity where the term pluripotential cell is used it will
refer
equally to ES and/or EG cells.
The establishment of in vitro cultures of ES/EG cells has proven to be
problematic. It has only recently be shown that in vitro cultures of ES/EG
cells
derived from non-murine species can be established ( please see US 5 453 357
and US 5 690 926). Typically the ES/EG cultures have well defined
characteristics. These include, but are not limited to;
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i) maintenance in culture for at least 20 passages when maintained on
fibroblast feeder layers;
ii) produce clusters of cells in culture referred to as embryoid bodies;
iii) ability to differentiate into multiple cell types in monolayer culture;
iv) can form embryo chimeras when mixed with an embryo host;
v) express ES/EG cell specific markers.
Until very recently, in vitro culture of human ES/EG cells was not possible.
The first indication that conditions may be determined which could allow the
establishment of human ES/EG cells in culture is described in WO 96/22362.
The application describes cell lines and growth conditions which allow the
continuous proliferation of primate ES cells which exhibit a range of
characteristics or markers which are associated with stem cells having
pluripotent characteristics.
For example, and not by way of limitation, the expression of specific cell
surface markers SSEA-3 (+), SSEA-4 (+), TRA-1-60 (+), TRA-1-81 (+)
Shevinsky et al 1982; Kannagi et al 1983; Andrews et al 1984a) and alkaline
phosphatase (+). In addition the established primate cell lines disclosed in
WO 96/22362 have stable karyotypes and continue to proliferate in an
undifferentiated state in continuous culture. The primate ES cell lines also
retain the ability, throughout their continuous culture, to form tissues
derived
from all three embryonic germ layers (endoderm, mesoderm and ectoderm).
More recently Thomson et al 1998 have published conditions in which human
ES cells can be established in culture. The above characteristics shown by
primate ES cells are also shown by the human ES cell lines. In addition the
human cell lines show high levels of telomerase activity, a characteristic of
cells which show the ability to divide continuously in culture.
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The establishment of human EG cell cultures is disclosed in WO 98/43679.
This application describes the isolation of EG cells from the gonadal or
genital
ridges of human embryos. EG cells described in WO 98/43679 exhibit features
in common with primate and human ES cells, (eg expression of cell surface
markers, continuous proliferation in culture in an undifferentiated state,
normal karyotype and the ability to differentiated into selected tissues under
defined conditions).
It is evident that the use of in vitro cultures of pluripotential stem cells,
especially human cells, has important ramifications for both basic research
(eg as a model fcr studying gene expression and/or tissue differentiation) and
in transplantation and/or replacement therapies for tissues which have been
damaged either through injury or disease. The establishment of in vitro
cultures of human ES and EG cells is a major step toward realising the full
potential of this technology; because of their pluripotent nature ES and EG
cells may be capable of differentiating under controlled conditions into a
variety of cell types and/or tissues and organs that could have a wide variety
of
applications. For example, and not by way of limitation, replacement of
damaged and/or diseased coronary and/or major arteries; replacement of
damaged and/or diseased organs ( eg as a result of kidney disease, (eg
cirrohosis), diabetes, various autoimmune diseases); replacement of damaged
neurones ( eg Alzhiemers disease, Parkinsons disease, spinal injuries) or
cancer. It will also be apparent to one skilled in the art that diseases such
as
AIDS may benefit from from tissues derived from ES or EG cells. The
depletion of T-cells through virus induced cell death is the major
contributory
factor to the immuno-compromised state of AIDS suffers.
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However, there are practical and ethical difficulties associated with the use
of
material derived from human embryos. Morever, such allogeneic material, if
transplanted into another human, may illicit a severe immune reaction in the
host and be thus destroyed.
5
It has been known for many years that amphibian somatic cell nuclei retain
their ability to give rise to entire organisms when they are transplanted into
egg cells which have had their nucleus removed or inactivated (Gurdon 1974).
Thus determination of the pluripotent of these cells must be controlled by the
egg cytoplasm which was able to in effect reprogramme the somatic cell
nucleus into a totipotent state.
Mammalian somatic cell nuclei have also been shown to retain this placicity
and can be reprogrammed when transferred to enucleated oocytes, (Campbell
et al 1996; Wakayama et al 1998)
Moreover nucleated mouse ES cells have been shown to be able to
reprogramme somatic cell nuclei, although in this case, a heterokaryon was
produced containing the cytoplasm and nuclei from both types of cells so it is
difficult to determine the actual mechanism of action of the reprogramming
state.
In all these examples, althought the material produced is genetically
identical
to the somatic cell donor, these somatic cells were reprogrammed by cellular
elements are derived from either ooctyes or ES cells and again, in human this
poses practical and ethical concerns.
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Methods that promote the fusion of cells are well known in the art (Kennett et
~al 1979). However, although it is relatively easy to fuse cells to form
hybrid
cells, nuclear fusion results in a cell containing two sets of chromosomes.
This
has enabled scientist to study the dominant expression of cell markers
characteristic of each cell type and indeed enabled some to study mitotic
chromosome stability in cross species hybrids. It is also well known in the
art
that cell hybrids may be formed by fusing the cytoplasm of a cell ( in which
the nucleus has been removed) with a selected intact cell to form a so called
cybrid ( Ege et al 1973; Veomett et al 1974; Wright and Hayflick et al 1975)
This has enabled investigation into nucleo-cytoplasmic interactions and, in
particular, the influence of cytoplasmic determinants on nuclear gene
expression.
It has been known for several years that selected chemical treatments of cells
in culture can result in cells extruding nuclei resulting in the formation of
separate nuclear and cytoplasmic parts termed karyoplasts and cytoplasts,
respectfully. These sub-cellular components have been used in fusion
experiments. For example, and not by way of limitation, as mentioned, it is
possible to produce a cytoplast from one cell and fuse the cytoplast to a
selected cell to form a cytoplasmic hybrid or cybrid. In addition it is also
possible to fuse the karyoplast or cell with a selected cell to form a nuclear
hybrid. The nuclei fuse after nuclear membrane breakdown during mitosis and
reconstitute after cytokinesis to form a polyploid or anueploid nucleus. The
afore described techniques are well known in the art and will not be detailed
extensively at this stage.
We have prepared cytoplasts, or parts thereof, derived from ES/EG cells and
fused said cytoplasts with selected somatic cells to form cybrids. The aim of
this approach is to re-programme the differentiated somatic cell nucleus
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through contact with factors located in the ES/EG cytoplasm, so that the
cybrid de-differentiates and so takes on the characteristic features of a
pluripotential cell. This then provides the basis for the establishment of
pluripotential cell lines which, upon exposure to various differentiation
factors, can lead to the production of selected differentiated tissue for use,
inter alia, transplantation therapy. The pluripotential cells so formed retain
the
nucleus of the somatic cell and at least part of the cytoplasm of the ES/EG
cell ( the mitochondrial genome would be retained and replicated by the
cybrid). Ideally, the somatic nucleus is derived from a patient requiring
transplant tissue so that the tissue produced by the aforementioned method is
immunologically compatible with the patient requiring the transplant. The use
of ES/EG cells directly in the production of tissue means the tissue is not
entirely immunologically "silent" due to the presence of a complete set of
male or female chromosomes from one of the parents of the embryo formed
for the purpose of providing the ES/EG cells.
It is therefore an object of the invention to provide a pluripotential cell
and
corresponding cell line.
It is a further object of the invention to provide a differentiated tissue for
use
in transplantation therapy.
According to a first aspect of the invention there is provided a cell
comprising
at least part of the cytoplasm derived from at least one embryonal stem cell
or
embryonal germ cell combined with the at least the nucleus of at least one
somatic cell.
In a preferred embodiment of the invention said cell, ideally a cybrid, is
characterised by the possession of at least one pluripotential characteristic.
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We believe that the acquisition of this pluripotential characteristic is as a
result
of the re-programming of said somatic nucleus.
It will be apparent to those skilled in the art that the cell of the invention
may
be derived, most preferably, by the creation of a cybrid; but an alternative
option involves the fusion of a somatic cell with an ES/EG cell. Clearly this
latter option is not preferred because subsequent mitosis will result in a
hybrid
having an abnormal karyotype.
Ideally said pluripotential characteristic includes the ability to
differentiate
into at least one selected tissue type, preferably upon exposure to at least
one
differentiation factor.
Alternatively, or additionally, said pluripotential characteristic includes
the
ability of said cell to proliferate in culture in an undifferentiated state.
In yet a further preferred embodiment of the invention said cell has the
capacity to proliferate in continous culture in an undifferentiated state for
at
least 6 months and ideally 12 months.
Alternatively or additionally, said pluripotential characteristic includes the
expression of at least one selected marker of pluripotential cells.
It is well known in the art that pluripotential cells express a number of
genes
not typically expressed by differentiated cells. These are valuable tools to
monitor whether the ESBG cytoplasm has re-programmed a somatic cell
nucleus. One such example is Oct4.
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In a preferred embodiment of the invention said selected marker is expression
of the Oct4 gene.
In yet still a further preferred embodiment of the invention said selected
marker is a cell surface marker. Preferably said cell surface marker is
selected
from the group including : SSEA-1 (-);and/or SSEA-3 (+); and/or SSEA-4 (+);
and/or TRA-1-60 (+); and/or TRA-1-81 (+); and/or alkaline phosphatase (+).
Alternatively or additionally said pluripotential characteristic includes the
presence of telomerase activity in said pluripotential cell. Ideally said
telomerase activity is correlated with extension of telomeres.
For the sake of clarity, telomerase enzymes add, de novo, repetitive DNA
sequences to the ends of chromosomes. These ends are referred to as
telomeres. For example the telomeres of human chromosomes contain the
sequence '5 TTAGGG 3' repeated approximately 1000 times at their ends. In
young, dividing cells the telomeres are relatively long. In aging, or non-
dividing cells, the telomeres become shortened and there is a strong
correlation between telomere shortening and capacity to proliferate. Methods
to increase the length of telomeres to increase proliferative capacity are
known
in the art and are described in W09513383.
Alternatively or additionally said pluripotential characteristic includes the
presence of a chromosomal methylation pattern characteristic of pluripotential
cells.
It is well known in the art that the genome of eukaryotic organisms is
variably
methylated through the addition of methyl ( -CH3) groups attached to cytosine
residues in DNA to form 5'methylcytosine ( 5'-mC). Methylation is correlated
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with the control of gene expression. Typically genes that are hypomethylated
tend to be highly expressed. Hypermethylation is correlated with reduced gene
expression. It will be apparent to one skilled in the art that pluripotential
cells
will have a typical methylation pattern. This pattern may be analysed at a
5 genomic level or at the level of a specific gene. Methods to analyse the
extent
of methylation are well known in the art and include, by example and not by
way of limitation, restriction enzyme digestion of DNA with methylation
sensitive restriction endonucleases followed by Southern blotting and probing
with suitable gene probes ( Umezawa et al 1997).
Alternatively or additionally said pluripotential characteristic includes the
ability to induce tumours when introduced into an animal, ideally a rodent
experimental model. More ideally still said animal is immunosupressed
According to a second aspect of the invention there is provided a cell-line
comprising cells according to the invention. Ideally, said cell- line are of
human origin.
According to a third aspect of the invention there is provided a method for
preparing a cytoplast, or part thereof, for use in the production of the cell
or
cell line of the invention comprising;
i) providing at least one ES/EG cell;
ii) separating at least part of the cytoplasm from the nucleus of said ESlEG
cell;
iii) isolating said cytoplasmic part; and, optionally
iv) storing said isolated cytoplasmic part prior to use.
In a preferred method of the invention said cytoplasmic part is a cytoplast.
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It will be apparent to one skilled in the art that said cytoplast may be
provided
either as an aliquot isolated from at least one ES/EG cell ( eg an aliquot
extracted from an intact ES/EG cell via micromanipluation techniques) or
alternatively and preferably, said cytoplasmic part may be provided as an
isolated cytoplast.
In a preferred method of the invention said cytoplast is separated from said
nucleus by exposure to a pharmacologically effective amount of a
cytochalasin. Ideally, cytochalasin B.
It is well known in the art that cytochalasin B is an example of a chemical
effective at separating the nucleus of a cell from the cytoplasm to form a
karyoplast and cytoplast respectively, (Methods in Enzymology Vol 151,
p221-2371987).
According to a fourth aspect of the invention there is provided a method for
preparing a cell or cell line in accordance with the invention comprising;
i) combining at least one ES/EG cell with at least one somatic cell;
ii) removing from said combined cell, the ES/EG cell nucleus;
iii) culturing said cell under conditions conducive to proliferation and
expansion of said cell; and, optionally
iv) storing said cell culture under suitable storage conditions.
It will be apparent to one skilled in the art that methods of
micromanipulation
exist that facilitate the removal of nuclei from selected cells. It will be
apparent that this method of the invention advantageously provides that ;
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i) the factors produced by the ES/EG cell are continually produced
thereby maintaining a steady-state level of factors necessary to
reprogramme the somatic cell nucleus; and
ii) the ES/EG cell nucleus is removed from the combined cell prior to
mitosis ensuring nuclear fusion does not occur.
It will be apparent to those skilled in the art that the nature of the somatic
cell
selected is not critical to the operation of the invention although the cell-
type
will be selected so as to optimise or maximise success in terms of production
of a cell or cell-line of the invention.
According to a fifth aspect of the invention there is provided a method for
preparing a cell or cell line in accordance with the invention comprising;
i) providing at least part of the cytoplasm of an ES/EG cell;
ii) combining said cytoplasmic part with at least one somatic cell;
iii) growing said combined cell in culture; and, optionally
iv) storing said combined cell under suitable storage conditions.
In a preferred method of the invention said cytoplasmic part is provided as a
cytoplast.
In yet a further preferred method of the invention said cytoplast is combined
with said somatic cell via cytoplast/somatic cell fusion.
In the above described methods the ES/EG cell and somatic cell are, ideally of
human origin.
According to a sixth aspect of the invention there is provided a cell culture
comprising at least one cell according to the invention.
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According to a seventh aspect of the invention there is provided a method for
inducing differentiation of at least one cell of the invention comprising:
i) providing a cell according to the invention;
ii) culturing said cell under conditions conducive to the differentiation of
said cell into at least one tissue; and,optionally
iii) storing of said differentiated tissue prior to use under suitable storage
conditions.
Ideally said culture conditions are selected from so as to provide a tissue
type,
by example and not by way of limitation, that is neuronal, muscle (eg smooth,
striated, cardiac), bone, cartilage, liver, kidney, respiratory epithelium,
haematopoietic cells, spleen, skin, stomach, intestine.
According to a eighth aspect of the invention there is provided at least one
tissue type or organ comprising at least one cell according to the invention.
It will be apparent to one skilled in the art that differentiated tissue
according
to the invention may have extensive application with respect to
transplantation
therapy. For example, and not by way of limitation, replacement of damaged
and/or diseased coronary and/or major arteries; replacement of damaged
and/or diseased organs ( eg as a result of kidney disease (cirrohosis),
diabetes,
various autoimmune diseases); replacement of damaged neurones ( eg
Alzhiemers disease, Parkinsons disease, spinal injuries), or cancer. It will
also
be apparent to one skilled in the art that diseases such as AIDS may benefit
from from tissues derived from the cells of the invention. The depletion of T-
cells through virus induced cell death is the major contributory factor to the
immuno-compromised state of AIDS suffers. The provision of a non-
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exhaustive supply of T-cells derived from a non-infected somatic cell from the
_patient has obvious benefits. Moreover, tissue rejection due to a host cell
immune responses are likely to be negligible since the tissue is derived from
the host into which the tissue is to be transplanted.
According to a nineth aspect of the invention there is provided a therapeutic
composition comprising at least one cell of the invention including a suitable
excipient, diluant or carrier.
In a preferred embodiment of the invention said therapeutic composition is
provided for use in tissue transplantation.
According to a tenth aspect of the invention there is provided a method to
treat
conditions or diseases requiring transplantation of tissue comprising;
i) providing at least one tissue type or organ according to the invention;
ii) surgically introducing said tissue or organ into a patient to be treated;
iii) treating said patient under conditions which are conducive to the
acceptance of said transplanted tissue by said patient.
According to an eleventh aspect of the invention there is provided a kit
comprising; at least one cell according to the invention; instructions with
respect to the maintenance of said cell in culture; and, optionally, factors
required to induce differentiation of said cell to at least one desired
tissue, type
or organ.
Embodiments of the invention will now be described, by example only and
with reference to the following materials and methods and Figure.
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Figure 1 shows PCR amplification of Oct4 mRNA from a human EC x
somatic cell ( thymocyte) heterokaryon.
Materials and Methods
This experiment exploits human tetratocarcinoma (EC) cells fused to mouse
thymocytes. We reasoned that. EC cells have many of the properties of ES/EG
cells and are therefore a useful tool to analyse re-programming of somatic
cell
nuclei.
Preparation of Mouse Thymocytes
The thymocytes were obtained by mincing a thymus removed from a 4-6 week
old male mouse (Swiss strain) and suspending the released cells in 10 ml
medium (DMEM) with 10% foetal calf serum (FCS). After standing for 2-3
minutes to allow large fragments of thymus to settle, the supernatant was
removed and centrifuged at 1500 rpm for 5 min to pellet the suspended
thymocytes. The thymocytes were resuspended in fresh medium without FCS,
and pelletted again by centrifugation; this was repeated a second time after
which the cells were resuspended in fresh serum free medium and counted.
Human EC cells were obtained by trypsinisation of confluent cultures as
previously described (Andrews et al., 1980; 1982). After washing two times in
serum free DMEM, and counting, the human EC cells were mixed with the
mouse thymocytes in a ratio of 1 EC cell to 10 thymocytes. The mixed cells
were pelletted by centrifugation at 1500 rpm for 5 min.
Heterokaryon Fusion of Human EC cells and Mouse Thymocytes & Extraction
of RNA
The cells were fused using polyethylene glycol (PEG) (Kennett, 1979). The
pellet (in Experiment l, 2 x 106 EC cells and 2 x 107 thymocytes; in
Experiment 2, 3 x 106 EC cells and 3 x 107 thymocytes) was resuspended in
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200 ~.l 50% (w/v) PEG 1500 in 75 mM HEPES, pH8.0 (Boehringer
Mannheim) and incubated at 37° C for 1.5 min. Serum free medium,
pre-
warmed to 37° C, was then added gradually over 5 min. The cells were
then
pelletted by centrifugation at 1500 rpm for 5 min. and resuspended in 5 ml
DMEM with 20% foetal calf serum. These cell were then plated into a T25
flask and placed in a humidified incubator (10% COZ in air) at 37°C for
2
days.
After 2 days, the non-attached cells were aspirated. The remaining attached
cells were harvested by trypsinisation, and washed two times in DEPC-treated
PBS to remove the serum. The pellet was then resuspended into Tri reagent (1
ml) to isolate RNA (Sigma-Aldrich Chemical Co., as described in Sigma
Technical Bulletin MB-205). The isolated RNA was quantified by optical
density measurements and the absence of contaminating DNA was determined
by PCR using ~i-actin and HPRT primers in separate samples (Wakeman et al.,
1998). If free of DNA, the RNA was then used for RT.PCR analysis of Oct4
expression-
PCR Amplification of Oct4 from Human EC x Mouse Thymocyte Heterokaryon
In one experiment (2102Ep with thymocytes), a control was prepared,
consisting of cells treated as for fusion except that the incubation with PEG
was omitted - thus it was anticipated that no 2102Ep x thymocyte
heterokaryons would be formed. In another experiment RNA was isolated
from thymocytes alone and also from a mouse EC line (PCC4 azal, clone 3),
to provide further negative and positive controls for mouse Oct4 expression.
cDNA was then produced from the samples using reverse transcriptase (RT)
(Wakeman et al., 1998). PCR was then performed using oligonucleotide
primers specific for human and mouse Oct 4, a marker of pluripotent cells
under the standard PCR conditions described in Wakeman et al. (1998) with
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an annealing temperature of 61°C. These products were then subjected to
electrophoresis and separated DNA fragments detected by ethidium bromide
staining (Figure 7). Molecular size of the amplified fragments was determined
by using a lkb DNA step ladder.
PCR Primers for human and mouse Oct 4
Species AnnealinSequence Bp GenBank Accession
g Temp No. and primer
C location
61.4 573 X52437
onward 5'-cgaccatctgccgctttgag-3' 120-139
Reverse 3'-ccccctgtcccccattccta-5' 534-S15
60.4 41 211899
S
onward 5'-gtccgcccgcatacgagttc-3' 361-3 80
Reverse 3'-aggggccgcagcttacacat-3' 937-918
These primers were designed using the PrimerSelect module of the Lasergene
suite of
programs (DNAStar Inc., USA). The mouse primers would not be expected to
amplify
human Oct4.
Enucleation of cells to yield 'cytoplasts' and 'karyoplasts' or 'mini-cells'.
One of the techniques that is employed in our method for producing Re-
programmed Embryonic Stem cells (ItPES cells) is the use of cytochalasin B
to generate enucleated ES/EG cells (ES/EG cytoplasts) as the cytoplasm
donor, and 'karyoplasts' (also called 'mini-cells') from the differentiated or
committed cells as the nucleus donor. Cytochalasin B is well-known to induce
cells to extrude their nuclei (Carter, 1967) and has been employed by
numerous authors to induce enucleation of a wide range of cells of a variety
of
species including both mouse and human cells (Poste 1972; Prescott et al
1972; Goldman et al 1973; Wright and Hayflick 1973; Ege and Ringertz
1974a; Wigler and Weinstein 1975). Such enucleation results in a cell lacking
a nucleus, but is otherwise intact and viable for a number of days (Goldman et
al 1973); these enucleated cells have been called anucleate cells (Poste 1972)
or cytoplasts (Veomett et al 1974). The nucleus that is extruded from the cell
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retains a thin rim of cytoplasm and is surrounded by a plasma membrane;
_these structures have been called 'karyoplasts' (Veomett et al 1974) or 'mini-
cells' (Ege and Ringertz 1975). Enucleation of cells to yield both cytoplasts
and karyoplasts may be achieved by well-established techniques in which cells
growing attached to a plastic disc are inverted over a solution of
cytochalasin
B in a centrifuge tube and centrifuged; the cytoplasts remain attached to the
plastic disc, while the karyoplasts are pelleted at the bottom of the
centrifuge
tube (Prescott et al 1972). Alternatively, cells in suspension may be
centrifuged through a density gradient, typically composed of Ficoll,
containing cytochalasin B (Wigler and Weinstein 1975). In this case,
cytoplasts and karyoplasts are formed and may be recovered from different
parts of the gradient after centrifugation.
Methods for combining (fusing) the cytoplasm of one cell with the nucleus
of another.
The methods for creating hybrid cells by fusing two or more cells of different
origins together are very well established and widely known. For a review of
the commonly used methods based upon Sendai virus induced cell fusion, or
cell fusion induced by polyethylene glycol (PEG), see Kennett (1979).
Briefly, mixtures of cells that it is desired to fuse are incubated with a
fusogenic agent, such as Sendai virus or PEG, often with centrifugation or
agitation to encourage clumping and close apposition of the cell membranes;
variables such as time, temperature, cell concentration and fusogenic agent
concentration are optimised for each cell combination. These techniques have
also been shown to allow fusion of cytoplasts, prepared by cytochalasin B
induced enucleation, with whole cells or karyoplasts, also derived by
cytochalasin B induced enucleation (Poste and Reeve 1971; Ege and Ringertz
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1975; Ege et al 1973, 1974; Veomett et al 1974; Wright and Hayflick 1975;
Shay 1977)).
Another technique that is now well established and widely used for inducing
cell fusion, 'electrofusion', involves passing short electric pulses through
mixtures of cells (Neil and Zimmermann 1993).
Production of RPES cells
The production of RPES cells requires several steps:
1. the selection of appropriate differentiated cells (the Nucleus
Donor) and, if necessary, the isolation of their nuclei,
2. the selection of ES/EG cells (the Cytoplasm Donor),
3. the fusion of the differentiated cell nuclei with the ES/EG cells,
and
4. the removal of the ES/EG cell nucleus, either before or after
fusion.
The production technique may, in some cases, be optimised by pre-treatment
of the differentiated cells, or contemporaneous treatment of the
differentiated
cell/ ES/EG cell fused products, with various agents such as, but not limited
to, inhibitors of DNA methylation, to enhance the ability of the
differentiated
cell nucleus to be re-programmed. After the production of the RPES cells
additional methods are required to propagate the cells, to characterise their
properties and to induce them to differentiate into required somatic cell
types.
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Differentiated cells to be used as Nuclear Donors
A large range of somatic cells derived from any tissue or organ of an adult
mammal or human, or from embryos or foetuses, or from extra-embryonic
tissues such as the trophoblast or yolk sac may be used as a source of nuclei
5 for reprogramming. Particular somatic cell types include but are not limited
to
thymocytes, peripheral blood lymphocytes, epidermal cells such as from the
bucal cavity, cumulus cells, or other stem cells isolated from biopsies of
various tissues, such as the bone marrow, the nervous system and the gut. The
technique may also be applied to various established cell lines, such as those
10 derived from various tumours including, for example, but not limited to
lymphoblastoid cell lines. The selected somatic cells used for the re-
programming procedure may be used directly upon isolation or they may be
cultured for a short time before further manipulation. In some instances such
somatic cells may be combined entirely with ES/EG cells as described below,
15 or nuclei or karyoplasts may first be isolated from them, for example using
agents such as cytochalasin B, as discussed above, or by other methods. For
example, nuclei may also be isolated using established micromanipulation
procedures, or other established cell fractionation procedures.
20 Fusion of parental differentiated cells and parental ES/EG cells to yield
RPES cells:
Several methods may be used to combine the cytoplasm of an ES/EG cell and
the nucleus of a differentiated cell to yield an RPES containing the nuclear
genome of the differentiated cell but not the ES/EG cell.
A. Cells may be fused by use of chemical agents such as polyethylene
glycol (PEG) or viruses such as Sendai virus, or by passing an electric
current through a mixture of cells. As discussed above, these methods
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21
are well known and may be readily applied. These methods may be
used to fuse:
1. a differentiated cell with an ES/EG cell, or
2. a karyoplast from a differentiated cell with an ES/EG cell, or
3. a differentiated cell with one or more cytoplasts isolated from
ES/EG cells, or
4. a karyoplast from a differentiated cell with one or more
cytoplasts isolated from ES/EG cells.
In cases ( I ) and (2), the result will initially be a heterokaryon
containing two nuclei, one from each parental cell. If this heterokaryon
were allowed to divide the result would be a hybrid cell containing a
single nucleus with a complete or partial genome from each parental
cell. However, in our method of producing RPES cells, the ES/EG
nucleus is removed prior to cell division of the hybrid cell, so that the
derivative dividing cell population retains only the genome of the
parental differentiated cell.
In cases (3) and (4) the ES/EG nucleus is removed from the ES/EG cell
before fusion, for example by enucleation with cytochalasin B as
discussed above, so that the resulting product contains only the
differentiated cell nucleus and cytoplasm from the ES/EG cell parent.
In any of these cases, the resulting RPES cells that continue to
proliferate retain only the nuclear genome of the differentiated parental
cell, which is now reprogrammed to express a new pattern of gene
activity.
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In cases ( 1 ) and (2) the ES/EG cell nucleus is removed from the
heterokaryon in one of several ways that include, but are not limited to,
partial enucleation using drugs such as cytochalasin B, applied in the
same manner as described above for enucleating ES/EG cells and
generating cytoplasts for fusion. In the present case in which
enucleation is carried out after fusion, some heterokaryons lose both
nuclei, in which case they do not proliferate, some heterokaryons lose
the differentiated cell nucleus, in which case they retain the parental
ES/EG nucleus and continue proliferating, some heterokaryons lose the
ES/EG cell nucleus, in which case they continue proliferating as RPES
cells, and some heterokaryons retain both nuclei and eventually
continue proliferating as hybrid cells. Several methods are used to
select the RPES cells and to eliminate any of the cells retaining an
ES/EG cell genome or to eliminate any cells retaining a somatic
nucleus that has failed to undergo re-programming. In one method, the
proliferating cells are cloned by established techniques (e.g. by picking
single cells with a micropipette - see Andrews et al 1982, 1984b), and
individual clones are screened using genetic markers for those that
retain an ES/EG genome. The latter cells are discarded, whereas those
that retain only a differentiated cell genome but not an ES/EG cell
derived genome, and express an RPES phenotype, are retained.
Standard DNA genotyping techniques using well established DNA
fingerprinting technology (Jeffreys et al 1985, 1988; Yan et al 1996)
may be used to identify whether the nuclear genome of any
proliferating cells is derived from either the ES/EG cell or
differentiated cell parent, or both.
In another method, before use as a fusion partner, the ES/EG cell parent
is genetically marked by insertion of a gene that will allow selection
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23
against any cell carrying that gene; for example, the ES/EG cell can be
stably transfected with a vector encoding the Herpes Simplex Virus-1
Tk gene (HSV 1-Tk), such that any cells carrying that gene can be killed
by culture in the presence of a number of drugs including acyclovir (9-
[(2-hydroxyethoxy)methyl]guanine) or FIAU (1-(2-deoxy-2-fluoro-~3-
D-arabinofuranosyl)-5-iodouracil) (Borrelli et al 1988; Hasty et al
1991), or gancyclovir (Rubinstein et al 1993; McCarrick and Andrews
1992). In this method, following partial enucleation, the remaining
heterokaryons are cultured in medium containing this drug, and only
those that have lost the ES/EG cell nucleus survive. Other selectable
genetic systems can also be similarly used. Persisting parental
differentiated cells that have not been reprogrammed are removed by
cloning the surviving cells, or by selecting RPES cells by virtue of their
expression of specific surface antigen markers that include, but are not
limited to, SSEA3, SSEA4, TRA-1-60 or TRA-1-81, as discussed
above as characteristic markers of ES/EG cells. For the latter approach,
fluorescence activated cell sorting (FACS), a widely used method for
separating subsets of cells can be used (e.g. Andrews et al 1982, 1987;
Ackerman et al 1994; Williams et al 1988).
In another method, the ES/EG cell parent is incubated prior to fusion,
with a drug that irreversibly inactivates its nucleus and prevents its
replication, for example, topoisomerase inhibitors such as etoposide
(Downes et al 1991; Fulka and Moor 1993). The resulting
heterokaryon naturally eliminates this treated nucleus prior to cell
division, so that the resulting dividing cell population only contains the
genome derived from the parental differentiated cell. This approach
may also be combined with the preceding 'partial enucleation of
heterokaryons' approach to ensure complete loss of the ES/EG genome.
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In another method, after cell fusion to produce a heterokaryon, the
ES/EG cell nucleus is removed by micro-manipulation.
B. Rather than chemical; viral or electrically induced fusion, the
nucleus of the differentiated cell is combined with an ES/EG cell parent
by micro-manipulation. In this method, the nucleus of the
differentiated cell is withdrawn using a micropipette inserted through
the cell membrane. It is then injected either into an inoculated ES/EG
cell, or into an intact ES/EG. In the later case the ES/EG cell nucleus is
then removed by a similar technique, or by one of the techniques
described above, before nuclear fusion and cell division occurs.
Growth and selection of RPES cells
Following fusion to combine a differentiated cell and an ES/EG cells, with
prior or subsequent removal of the ES/EG cell nucleus, it is necessary to
provide appropriate conditions for the re-programming of the differentiated
cell nucleus and for the subsequent proliferation of the resulting RPES cells.
Several methods are used to enhance the efficiency of reprogramming:
1. prior to fusion the differentiated cell and ES/EG cell are
synchronised with respect to position in the cell cycle, by use of
reversible inhibitors that arrest the cell cycle at specific stages (e.g.
nocodazole), or by the use of conditions such as low serum to arrest
cells in Gl, or by selection of cells at specific stages of the cell
cycle by using vital DNA stains and flow microfluorimetry
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(Fluorescence Activated Cell Sorting) (Ashihara and Baserga 1979;
Andrews et al 1987; Crissman 1995; Stein et al 1995).
2. the differentiated cell or the immediate fusion product is cultured in
the presence of drugs that inhibit methylation or promote
5 demethylation (e.g. 5-azacytidine) (e.g. Taylor and Jones 1979;
Jones 1985; Keshet et al 1986), or alter the structure of chromatin,
for example butyrate, spermine, trichostatin A or trapoxin which
inhibit deacetylation and promote acetylation of histones, which
plays a role in X chromosome inactivation, gene imprinting and
10 regulation of gene expression (Caldarera et al 1975; McKnight et al
1980; Stein et al 1997; Hu et al 1998; Wolffe and Pruss 1996;).
3. the period of time between production of heterokaryons and the
removal of the ES/EG cell nucleus is made as long as possible
without permitting nuclear fusion. This period can be elongated by
15 culturing the heterokaryons under conditions that reversibly inhibit
progress through the cell cycle (e.g. thymidine block - Stein et al
1995), or by altering growth conditions, such as serum starvation or
lowered temperature, that retard cell division but permit
reprogramming to proceed.
20 4. any, or all combinations of these methods.
In all these experiments the cells are cultured in standard cell culture media
that include but are not restricted to Dulbecco's modified Eagle's Medium
(DME, high glucose formulation) or Ham's F 12, supplemented in some cases
25 with foetal bovine serum or with other additives (e.g. see Andrews et al
1980,
1982, 1984, 1994). Subsequent to fusion and re-programming, the growth of
the resulting cells may be optimised culture on feeder layers of cells that
include, but are not restricted to, irradiated or mitomycin C treated STO
cells,
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or embryonic fibroblasts of various species, including humans (see Robertson
1987a; Thomson et al 1998). The cells may be cultured in the presence of
various growth factors or other tissue culture additives, that include but are
not
restricted to LIF, FGF, SCF
Differentiation of the RPES cells
In the best cases, the RPES cells acquire pluripotent properties that closely
resemble those of embryonic stem cells, so that the RPES cells are able to
differentiate and to initiate differentiation pathways that result in the
formation
of any cell type that may be found in the adult, embryo or in extra-embryonic
tissues, given appropriate conditions. The maintenance of an ES/EG cell state
can be monitored by assay of various markers that include the cell surface
antigens SSEA3, SSEA4, TRA-1-60, TRA-1-81, by their expression of
alkaline phosphatase and by expression of Oct3/4, as discussed above. The
RPES cells typically retain their stem cell phenotype when cultured on
appropriate feeder cells. However, they can initiate differentiation under a
variety of circumstances.
Thus removal from feeder cells, or culture in suspension, followed by
replating in the absence of feeder cells in appropriate tissue culture flasks
results in differentiation of stem cells into a variety of cell types that
include
neurons, muscle of various sorts and haematopoietic cells (see descriptions in
Robertson 1987a). Differentiation of pluripotent stem cells may also be
initiated by altered conditions affecting cell density and aggregation (e.g.
seeding at low cell densities or trypsinisation) or by forcing growth
suspensions by exposure to various agents that include but are not restricted
to
retinoic acid, and other retinoids, hexamethylene bisacetamide, and the bone
morphogenetic proteins (see Robertson 1987a; Andrews 1984; Andrews et al
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1982, 1990, 1994, 1996; Thomson et al 1998). The type of cells that arise
depend upon the nature of the inducing agent, and the culture conditions
including the presence or absence of specific growth factors or other
molecules.
Discussion
Although pluripotent stem cell lines have been derived from early embryos
(Robertson, 1987b; Thomson et al 1995, 1998), primordial germ cells (Matsui
et al 1992; Shamblott et al 1998) and from germ cell tumours (reviewed,
Andrews, 1998) of various species, including the laboratory mouse, rhesus
monkeys and humans, and nuclei from differentiated somatic adult cells have
been re-programmed to yield embryonic stem cells by transplantation to
enucleated oocytes (Campbell et al 1996; Wakayama et al 1998), there are no
reports that pluripotent stem cells, resembling embryonic stem cells with the
capacity to differentiate into a variety of functional somatic cell types, can
be
produced by the re-programming of differentiated or committed embryonic or
adult somatic cells, or extra-embryonic cells, without the use of oocytes.
We now describe methods by which ES/EG cells can be used to re-program
various somatic, differentiated cells, or other embryonic or extra-embryonic
cell types, to a state from which they can then be induced to differentiate
into
one or more functional differentiated cell types that are distinct from the
parental cells. In the best cases, but not necessarily in all cases, the re-
programmed cells produced by this technique, called 'Re-programmed
Embryonic Stem Cells' (RPES cells), resemble embryonic stem cells derived
directly from early embryos, and can be induced to differentiate into a broad
range of functional, differentiated cell types that include, but are not
limited
to, neurons, muscle (including skeletal and cardiac muscle) and
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haematopoietic cells. These RPES cells are diploid with a normal karyotype,
and isogenic with the differentiated parental cells from which they are
derived.
They may be used to generate differentiated cells for transplantation and use
in
cell and tissue replacement therapies.
In some cases, only partial reprogramming occurs with, for example, the
activation of several genes that are not active in the parental differentiated
nuclear donor cell. Such cells are also of use in a variety of these same
circumstances.
An example of such a gene is Oct4. Oct4 has previously been reported to be
characteristically expressed by undifferentiated EC and ES cells (Brehm et
al.,
1998). Therefore, to test the ability of human EC cell cytoplasm to reprogram
somatic cells, isolated mouse thymocytes were fused with human EC cells,
(2102Ep, clone 4D3 (Andrews et al., 1982) or TERA1 (Fogh and Trempe,
1975; Andrews et al., 1980)), to produce heterokaryons which were tested
after 2 days for activation of Oct4 expression from the thymocyte genome.
Evidence for such activation would indicate, not only that human EC cells are
capable of re-programming a somatic cell nucleus to an ES/EC cell like state,
but also that the regulatory factors involved are capable of working between
different mammalian species. Thus if human EC cells can reprogram a mouse
somatic cell, we would anticipate not only that they would be able to
reprogram a human somatic cell, but also that mouse EC cells would be able
to reprogram human somatic cells as well. Similarly, given the resemblance
of EC and ES cells, it would be expected that ES cells could reprogram
somatic cells in the same way as EC cells.
In Experiment l, as anticipated, an amplified band (573 bp), corresponding to
human Oct4 expression was detected similarly in RNA preparations from the
2102Ep x thymocyte fusion in the presence of PEG, and in the mock fusion in
the absence of PEG, consistent with its expression by 2102Ep human EC cells.
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However, a band corresponding to mouse Oct4 (415 bp) was only detected in
the RNA preparation from the 2102Ep x thymocyte fusion in the presence of
PEG, when heterokaryons were expected to be present. The corresponding
absence of mouse Oct4 from the mock fusion indicates both the absence of
Oct4 expression from mouse thymocytes in this experiment, and the
requirement for formation of heterokaryons for its activation from the
thymocyte genome by the 2102Ep cytoplasm. No products were seen in the
'water' control, indicating absence of contamination.
In a second experiment, in which 2102Ep and TERM human EC cells were
fused with mouse thymocytes in the presence of PEG, mouse Oct4 was only
detected in the 2102Ep fusion, again confirming the ability of 2102Ep cells to
reprogram mouse thymocytes with activation of Oct4 expression, but
suggesting in this experiment that TERA 1 cytoplasm did not achieve
reprogramming. In both cases, human Oct4 was detected as expected,
consistent with its expression by 2102Ep or TERA1 human EC cells.
In further controls, no mouse Oct4 expression was detected in RNA prepared
from isolated mouse thymocytes not used for fusion. However, a similar sized
PCR band to that detected in the 2102Ep x thymocyte fusion samples,
corresponding to mouse Oct4, was detected in mouse PCC4 EC cells as
expected.
In our method, RPES cells are created by combining the nucleus from a
differentiated or committed cell (the Nuclear donor), whether from adults or
from embryos, with the cytoplasm from an ES/EG cell (the Cytoplasm donor),
from which the nucleus is removed. Several methods can be used to combine
the nucleus from the differentiated cell and the cytoplasm from the ES/EG
cell; in some methods the ES/EG cell nucleus is removed prior to
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combination of the cytoplasm with the donated nucleus, and in other methods
the ES/EG cell nucleus is removed after combination. If ES/EG cells and
differentiated cells from the same species are used, then the resulting RPES
cells retain cytoplasmic genetic determinants (e.g. the mitochondrial genome)
5 and a nuclear genome from the same species. By contrast, embryonic stem-
like cells produced by transplantation of somatic cells into enucleated
oocytes
of other species will continue to harbour mitochondria of that other species.
Especially for the production of human RPES cells and their differentiated
derivatives for transplantation into a human host, the maintenance of a human
10 nuclear and human cytoplasmic genome could be a distinct advantage.
The method that we describe incorporates the techniques for maintaining and
propagating the RPES cells produced, and the techniques for inducing them to
differentiate into a range of differentiated, functional cell types.
20
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References
Andrews P.W. and Goodfellow P.N. (1980) Antigen expression by somatic
cell hybrids of a murine embryonal carcinoma cell with thymocytes and L
cells. SOOOOomat. Cell Genet. 6: 271-284.
Andrews P.W., Bronson D.L., Benham F., Strickland S. and Knowles B.B.
(1980) A comparative study of eight cell lines derived from human testicular
teratocarcinoma. Int. J. Cancer 26: 269-280.
Andrews P.W., Goodfellow P.N., Shevinsky L., Bronson D. L. and Knowles
B.B. (1982) Cell surface antigens of a clonal human embryonal carcinoma cell
line: Morphological and antigenic differentiation in culture. Int. J. Cancer
29:
523-531.
Andrews P.W. (1982) Human embryonal carcinoma cells in culture do not
synthesize fibronectin until they differentiate. Int. J. Cancer 30: 567-571.
Andrews P.W., Damjanov L, Simon D., Banting G., Carlin C., Dracopoli N.C.
and Fogh J. ( 1984b) Pluripotent embryonal carcinoma clones derived from
the human teratocarcinoma cell line Tera-2: Differentiation in vivo and in
vitro. Lab.Invest.50: 147-162.
Andrews P.W., Meyer L.J., Bednarz K.L. and Harris H. (1984c) Two
monoclonal antibodies recognizing determinants on human embryonal
carcinoma cells react specifically with the liver isozyme of human alkaline
phosphatase. Hybridoma 3: 33-39.
Andrews P.W. (1984) Retinoic acid induces neuronal differentiation of a
cloned human embryonal carcinoma cell line in vitro. Dev. Biol. 103: 285-
293.
CA 02371900 2001-08-17
WO 00/49137 PCT/GB00/00576
32
Andrews P.W., Banting G.S., Damjanov I., Arnaud D. and Avner P. (1984a)
_Three monoclonal antibodies defining distinct differentiation antigens
associated with different high molecular weight polypeptides on the surface of
human embryonal carcinoma cells. Hybridoma 3: 347-361.
Andrews P.W., Gonczol E., Plotkin S.A., Dignazio M. and Oosterhuis J.W.
(1986) Differentiation of TERA-2 human embryonal carcinoma cells into
neurons and HCMV permissive cells: Induction by agents other than retinoic
acid. Differentiation 31: 119-126.
Andrews, P.W., Oosterhuis J.W. and Damjanov I. (1987) Cell lines from
human germ cell lines. In: Teratocarcinomas and embryonic stem cells: A
practical approach (E.J. Robertson, ed.). IRL Press, Oxford, pp 207-248.
Andrews P.A., Nudelman E., Hakomori S. -i. and Fenderson B.A. (1990).
Different patterns of glycolipid antigens are expressed following
differentiation of TERA-2 human embryonal carcinoma cells induced by
retinoic acid, hexamtehylene bisacetamide (HMBA) or bromodeoxyuridine
(BUdR). Differentiation 43, 13 I -13 8.
Andrews P.W., Damjanov L, Berends J., Kumpf S., Zappavingna V. Mavilio
F. and Sampath K. (1994). Inhibition of proliferation and inductionof
differentiation of pluripotent human embryonal carcinoma cells by osteogenic
protein-1 (or bone morphogenetic protein-7). Laboratory Investigation,7l,
243-251.
Ashihara, J., and Basergu, R. (1979). Cell Synchronisation Methods.
Enzymology, 58, 248-262.
Bernstine, E.F., Hooper, M.L., Grandchamp, S., Ephrussi, B. (1973). Alkaline
phosphatase activity in mouse teratoma. Proc Natl Acad Sci USA 70, 3899
3902.
CA 02371900 2001-08-17
WO 00/49137 PCT/GB00/00576
33
Brehm, A., Ovitt, C.E., Scholer, H.R. (1978). Oct 4: more than just a
_.POUerful marker of the mammalian germline? Acta Path. Mirobiol, Immunol,
Scand. 106, 114-126.
Bronson, D.L., Andrews, P.W., Solter, D., Cervenka, J., Lange, P.H., and
Fraley, E.E.(1980). A cell line derived from a metastasis of a human
testicular
germ-cell tumour. Cancer Res. 40, 2500-2506.
Bronson, D.L., Ritzi, D.M., Fraley, E.E., and Dalton, A.J. (1978).
Morphologic evidence for retrovirus production by epithelial calls derived
from a human testicular tumour metastasis. J.Natl. Cancer Inst. 60, 1305-1308.
Bronson, D.L., Andrews, P.W., Vessella, R.L., and Fraley, E.E. (1983). In
vitro differentiation of human embryonal carcinoma cells. In
"Teratocarcinoma Stem Cells" (L.M. Silver, G.R. Martin and S. Strickland,
eds.), Cold Spring Harbor Conferences on Cell Proliferation, Vol. 10, pp. 597-
605. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Bronson, D.L., Saxinger, W.C., Ritzi, D.M., Fraley, E.E. (1984). Production of
virions with retrovirus morphology by human embryonal carcinoma cells in
vitro. J. Gen. Virol 65, 1043-1051.
Campbell, K.H.S., McWhir, J., Ritchje, W.A., Wilmut, I. (1996). Sheep
cloned by nuclear transfer from a cultured cell line. Nature, 380, 64-66.
Carter, S.B. (1967). Effects of cytoccholasins on mammalian cells. Nature,
213, 261-264.
CA 02371900 2001-08-17
WO 00/49137 PCT/GB00/00576
34
Downes, C.S., Mullinger, A.M., Johnson, R.T. (1991). Inhibitions of DNA
_topoisomerase II prevent sistem chromatid separation in mammalian cells but
do not prevent exit from mitosis. Proc. Nat. Acad. Sci (USA). 88, 8895-8899.
Ege, T., and Ringertz, N.R., ( 1974). Preparation of microcells by enucleation
of micronucleate cells. Exp. Cells Res. 87, 378-382.
Ege, T., Krondahl, U., Ringertz, N.R. (1974). Introduction of nuclei and
micronuclei into cells and enucleated cytoplasms by Sendai virus induced
fusion. Ex.p Cell Res. 88, 428-432.
Ege, T., and Ringertz, N.R. (1975). Viability of cells reconstituted by virus-
induced fusion of mini cells with enucleate cells. Expl. Cell Res., 94, 469-
473.
Ege, T., Zeuthen, J., Ringertz, N.R. (1973). Cell fusion with enucleated
cytoplasms. Nobel, 23, 189-194.
Engstrom, W., Tally, M., Granerus, M., Hedley, E.P., and Schofield, P.
( 1991 ). Growth factors and the control of human teratoma cell proliferation.
Recent Res. Cancer Res. 123, 145-153.
Fenderson, B.A., Andrews, P.W., Nudelman, E., Clausen, H., and Hakomori,
S.-L, (1987). Glycolipid core structure switching from clobo- to facto- and
ganglio-series during retinoic acid-induced differentiation of TERA-2 derived
human embryonal carcinoma cells. Dev. Biol. 122, 21-34
Fogh, J., and Trempe, G. (1975). New human tumor cell lines. In "Human
Tumor Cells in vitro" (J.Fogh, ed.), pp. 115-159. Plenum Press, New York.
CA 02371900 2001-08-17
WO 00/49137 PCT/GB00/00576
Fulka, J., Moor, R.M. (1993). Non invasive chemical enucleation of mouse
oocytes. Mol. Reprod. Devel., 34, 427-430.
Goldman, R.D., Plllack, R., Hopkins, N.H. (1973). Presrvation of normal
5 behaviour by enucleated cells in culture. Proc. Nat. Acid. Sci. (USA). 70,
750-
754.
Gonczol, E., Andrews, P.W., and Plotkin, S.A. (1984). Cytomegalovirus
replicates in differentiated but not undifferentiated human embryonal
10 carcinoma cells. Science 224, 159-161.
Gurdon J. The control of gene expression in animal development (Oxford
University Press 1974).
15 Hogan, B., Fellous, M., Avner, P., and Jacob, F. (1977). Isolation of a
human
teratoma cell line which expresses F9 antigen. Nature (London) 270, 515-518.
Jakob, H., Boon, T., Galliard, J., Nicolas, J.F., Jacob, F. (1973).
Teratocarinome de la souris: isolement, culture et properietes de cellules a
20 potentialities multiples. Ann Microbiol (Paris) 124b 269-282.
Jeffreys, A.J., Wilson, V., Thein, S.L. (1985). Individual specific
fingerprints
of human DNA. Nature, 316, ~76-79
25 Jeffreys, A.J., Wilson, V., Newmann, R., Keyte, J. (1988). Amplification of
human minisatellites by the polymerise chain reaction - towards DNA
fingerprinting of single cells.
CA 02371900 2001-08-17
WO 00/49137 PCT/GB00/00576
36
Kennett, R.H. (1979). Cell Fusion in: Cell Culture, Methods in Enzymology.
_ (eds. Jakoby, W.B., and Pastan, LH.) Academic Press San Diego, 58, 345-359.
Matsui, Y., Szebo, K., and Hogan, B.L.M. ( 1992). Derivation of pluripotential
embryonic stem cells for murine primoridal germ cells in culture. Cell, 70,
841-847.
Mavilio F., Simeone A., Boncinelli E. and Andrews P.W. (1988) Activation
of four homeobox gene clusters in human embryonal carcinoma cells induced
to differentiate by retinoic acid. Differentiation, 37,73-79.
Miller., W.H., and Dmitrovsky, E. (1991). Growth factors in human germ cell
cancer. Recent Res.Cancer Res. 123, 183-189.
Neil, G.A., and Simmermann, U. (1993). Electrofusion, Methods in
Enzymology, 220, 174-196.
Poste, G., and Reeve, P. (1971). Formation of hybrid cells and heterokaryous
by fusion of enucleated and nucleated cells. Nature New Biology, 229, 122-
125.
Poste, G. ( 1972). Enucleation of mammallian cells by cytochalasin B. Exp.
Cell Res. 73, 273-286.
Prescott, D.M., Myerson, D., Wallace, J. ( 1972). Enucleation of mammalian
cells with cytochalasin B. exp. Cell Res. 71, 480-485.
Robertson, E.J. (Editor) (1987). Teratocarcinomas and embryonic stem cells:
A practical approach. IRL Press, Oxford.
CA 02371900 2001-08-17
WO 00/49137 PCT/GB00/00576
37
Robertson, E.J., (1987b). Embryo-derived stem cell lines in (Robertson, E.J.,
Editor) Teratorcarcinomas and embryonic stem cells: A practical approach.
IRL Press, Oxford, pp 71-112.
Rosfjord, E., and Rizzino A. (1994). The octaner motif present in the Rex-1
promoter binds Oct-1 and Oct-3 expressed by EC cells and ES cells. Biochem.
Byophys. Res. Counc., 203, 1795-1803.
Shamblott, M.J., Axelman, J., Wang, S., Bugg, E.M., Littlefield, J.W.,
Donovan, P.J., Blumenthal, P.D., Huggins, G.R., Gearhart, J.D. ( 1998).
Derivation of pluripotent stem cells from cultured human primordial germ
cells. Proc. Nat. Acad. Sci. USA, 95, 13726-13731.
Shay, J.W. (1997). Selection of recontributed cells from karyplasts fused to
chloramphenical resistant cytoplasts. Proc. Nat. Acad. Sci. USA. 74, 2461-
2464.
Shevinsky, L.H., Knowles, B.B., Damjanov, I., Solter, D. (1982). Monclonal
antibody to murine embryo defines a stage specific embryonic antigen
expression on mouse embryos and human teratocarcinoma cells. Cell 30, 697-
705.
Simeone, A., Acampora, D., Arcioni, L., Andrews, P.W., Boncinelli, E., and
Mavilio, F. (1990). Sequential activation of human HOX2 homeobox genes by
retinoic acid in human embryonal carcinoma cells. Nature (London) 346, 763-
766.
CA 02371900 2001-08-17
WO 00/49137 PCT/GB00/00576
38
Softer, D., and Knowles, B.B. (1978). Monoclonal antibody defining a stage
specifice mouse embryonic antigen (SSEA-1). Proc Natl Acad Sci (USA). 75,
5565-5569.
Teshima, S., Shimosato, Y., Hirohashi, S., Tome, Y., Hayashi, I., Kanazawa,
H., and Kakizoe, T., ( 1988). Four new human germ cell tumor cell lines. Lab.
Invest. 59, 328-336.
Thomson, J.A., Itskovitz-Eldor, J., Shapiral, S.S., Waknitz, K.A., Swiergiel,
J.J., Marshall, V.S., Jones, J.M. (1998). Embryonic stem cell lines derived
from human blastocysts. Science, 282, 1145-1147.
Thompson, S., Stern, P.L., Webb, M., Walsh, F.S., Engstrom, W., Evans, E.P.,
Shi, W.K., Hopkins, B., and Graham, F.F. (1984). Cloned human teratoma
cells differentiate into neuron-like cells and other cell types in retinoic
acid. J.
Cell Sci. 72, 37-64.
Veomett, G., Prescott, D.M., Shay, J., Porter, K.R. (1974). Reconstruction of
mammalian cells from nuclear and cytoplasmic components separated by
treatment with cytocholasin B. Proc Nat Acad Sci, 71, 1999-2002.
Vogelzang, N., Andrews, P.W., and Bronson, D. (1983). An extragonadal
human embryonal carcinoma cell line 1618K. Proc. Am. Assoc. Cancer Res.
24, 3.
Von Keitz, A.T., Riedmiller, H., Neuman, K., Grutschart, W., Fonatsch, C.
(1994). Establishment and characterisation of a seminoma cell line (S2).
Invest. Urol (Berlin). 5, 28-31.
CA 02371900 2001-08-17
WO 00/49137 PCT/GB00/00576
39
Wakayama, T., Perry, A.C.F., Succoth, M., Johnson, K.R., Yanagimachi, R.
X1998). Full term development of mice from enucleated oocytes injected with
cumulus cell nuclei. Nature, 394, 369-374.
Wakeman, J.A., Heath, P.R., Pearson, R.C.A., Andrews, P.W. (1997) MAL
mRNA is induced during the differentiation of human embryonal carcinoma
cells into neurons, and is also localised within specific regions of the human
brain. Differentiation 62:97-105.
Wakeman, J.A., Walsh, J., Andrews, P.W., (1998). Human Wnt-13 is
developmentally regulated during the differentiation of NTERA-2 pluripotent
human embryonal carcinoma cells. Oncogene 17:179-186
Wang, N., Trend, B., Bronson, D.L., and Fraley, E.E. (1980). Nonrandom
abnormalities in chromosome 1 in human testicular cancers. Cancer Res. 40,
796-802.
Wang, N., Perkins, K.L., Bronson, D.L., and Fraley, E.E. (1981). Cytogenetic
evidence for pre-meiotic transformation of human testicular cancers. Cancer
Res. 41, 2135-2140.
Wenk, J., Andrews, P.W., Casper, J., Hata, J-L, Pera, M.F., von Keitz, A.,
Damjanov, L, Fenderson, B.A. 1994. Glycolipids of germ cell tumours:
extended globo-series glycolipids are a hallmark of human embryonal
carcinoma cells. Int. J. Cancer., 58, 108-115.
Wigler, M.H., and Weinstein, LB. (1975). A preparative method for obtaining
enucleated mammalian cells. Biochem Biophys Res Comm. 63, 669-674.
CA 02371900 2001-08-17
WO 00/49137 PCT/GB00/00576
Williams B.P., Daniels G.L., Pym B., Sheer D., Povey S., Okubo Y., Andrews
P.W. and Goodfellow P.N. (1988) Biochemical and genetic analysis of the
OKa blood group antigen. Immunogenetics, 27, 322-329.
5 Wright, W.E., Hayflick, L. (1973). Enucleation of cultured human cells.
Proc.
Soc. Exp. Biol. and Med. 144, 587-592.
Wright, W.E., and Hayflick L. (1975). Use of biochemical lesions for
selection of human cells with hybrid cytoplasms. Proc. Nat. Acad. Sci (USA).
10 72, 1812-1816.
Yan, R., Ottenbreit, K., Hukku, B., Mally, M., Chou, S.R., Kaplan, J. (1996).
DNA fingerprinting of human cell lines using PCR amplification of fragment
length polymorphisms. In Vitro Celular and Developmental Biology, 32, 656
15 662.
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