WO1999063061A1 - Es cell lines from somatic cells via embryo reconstruction and selective ablation - Google Patents

Abstract

A method of preparing animal stem cells is provided which comprises reconstituting an animal embryo by transferring the nucleus of a donor cell into a suitable recipient cell followed by selective killing of differentiated cells of the reconstituted embryo and culturing the resulting undifferentiated embryonic cells.

Description

ES CELL LINES FROM SOMATIC CELLS VIA EMBRYO RECONSTRUCTION

AND SELECTIVE ABLATION

The present invention relates to a method for generating animal stem cells and cell lines, particularly embryonic stem cells and embryonic germ cells, and to uses of the stem cells produced in medicine.

Stem cells are progenitor cells which have the capacity both to self-renew and to differentiate into mature somatic tissues. Stem cells may be founder cells for embryonic or other cell lineages, for example in haematopoeisis and myogenesis.

Mouse embryonic stem (ES) cells are rapidly dividing cultured cells which retain in culture, the ability to give rise to all embryonic lineages (totipotency). ES cells are isolated from cultured embryos in a process which releases a normally transient cell population from the controlling influence of other lineages within the embryo. Embryos are usually taken at the blastocyst stage immediately following the differentiation of the embryo into trophectoderm and inner cell mass. They can be grown in large numbers in the undifferentiated state and following their reintroduction to an early embryo, will resume their normal programme of differentiation to contribute to all embryonic tissues including the germ cells. ES cells have the innate capacity to proliferate indefinitely without any form of oncogenic transformation. ES cells are therefore widely used as a means to effect genetic changes in the mouse. The ability to apply somatic cell genetics to a cultured cell population allows the use of gene targeting to effect precise gene modifications which can include gene inactivation, subtle sequence changes or the precise placement of new genes into active sites in the genome (Bradley et al Bio /Technology 10 534-539 (1992)). Functionally similar cells to ES cells known as embryonic germ (EG) cells have been generated by the culture of primordial germ cells taken from later stage embryos (Matsui et al Cell 70 841-847 (1992); Labonsky et al Development 120 3197-3204 (1994)).

The standard method of isolating ES lines was devised by Evans et al (Nature 292 154-156 (1981)) and is described in detail by Robertson (Robertson E.J. Embryo- derived stem cells in "Teratocarcinomas and Embryonic stem cells - A Practical Approach" ed. E.J. Robertson, IRL Press Ltd, Oxford (1987)). The method comprises a serial removal of undifferentiated colonies from the influence of other lineages until such time as a stable ES culture is obtained. Fully functional ES lines capable of colonising the germline following their re-introduction to embryos have only been isolated from certain strains of mouse - notably C57BL/6 and strain 129.

Briefly, blastocyst stage embryos are flushed from the uterus and then cultured in medium over a layer of mitotically inactivated fibroblasts, "feeder" cells. Over a few days blastocysts attach and spread onto the feeder layer, exposing cells of the inner cell mass. The inner cell mass of some explants proliferate forming clumps of undifferentiated cells. This portion of each explant is manually isolated, disaggregated and replated onto a new feeder layer. Colonies of cells form, usually composed of several cell types. Any colonies with ES morphology are further isolated, disaggregated and replated. Repeating this through several passages can result in a homogenous population of ES cells.

A vital part of the derivation process is the physical separation of ES cells away from differentiated tissues, e.g. trophectoderm and endoderm. These arise from differentiated cells of the explanted embryo and also from spontaneous differentiation of ES cells. Endoderm is commonly the first product of ES cell differentiation and cells of this type tend to lie closely apposed to undifferentiated ES cells. The proximity of differentiated cells induces further differentiation of ES cells. The presence of feeder cells is believed to be helpful during the first stages of ES cell derivation to reduce spontaneous differentiation of undifferentiated cells. However, the mechanism involved is only partially defined. Established ES cell lines can usually be grown successfully in the absence of a feeder layer (Magin et al. , Nuc. Acids. Res. 14 3795 (1992)), providing that the culture medium is supplemented with the cytokine leukaemia inhibitory factor, LIF (Smith et al , Nature 336 688-689 (1988) and Williams et al. Nature 336684-687 (1988)). However soluble LIF alone is not usually a substitute for feeders during ES cell derivation.

The frequency with which ES lines are derived from embryos varies widely. In skilled hands ES lines can be obtained from 5-10% mouse blastocysts using strain 129Sv (Robertson, E.J., M.H. Kaufman., Bradley, A and M.J. Evans. 1983. "Isolation, Properties and Karyotype Analysis of Pluripotential (EK) Cells from Normal and Parthenogenetic Embryos" in: "Teratocarcinoma Stem Cells", Cold Spring Harbor Conferences on Cell Proliferation 10 647-663. Eds. Martin, G.R., L. Silver and S. Strickland.

There are now several reports of ES-like cells in a number of species (e.g.: Doetschman et al Dev Biol 111 224-227 (1988); Wheeler Reprod. Fertil. Dev. 6 563-568 (1994)) however to date none of these has met the definitive functional test of germline colonisation. Even within the mouse, certain strains are non-permissive for ES isolation by standard techniques, notably strain CBA. The strain barrier to ES isolation in mice can, however, be broken by employing a selective ablation technique which continuously removes differentiating cells from the culture (McWhir et al Nature Genetics 14 223-226 (1996)). This approach suffers, however, from the requirement for genetically modified embryo donors whose embryos express a selectable marker. Recent advances in nuclear transfer technology have allowed the generation of cloned lambs from embryonic cells (Campbell et al Nature 380 64 (1996)) and foetal and adult cells (Wilmut et al Nature 385 810 (1997)) and of cloned calves from foetal cells (Cibelli et al Science 280 1256-1258 (1998)). Cloned lambs have also been generated following nuclear transfer from cells which were genetically modified (Schnieke et al Science 278 2130-2133 (1997)). These advances have been developed to offer an alternative to ES cells which may allow gene targeting strategies to be applied in species other than the mouse. It has also been reported that healthy blastocysts can develop following the transfer of nuclei from sheep, pig, monkey and rat fibroblasts into the bovine oocyte (Dominko et al Theriogenology 385 (1998); Mitalipova et al Theriogenology 389 (1998)), raising the possibility that the bovine or ovine oocytes may act as a universal recipient in nuclear transfer.

It would be highly desirable to extend the use of ES/EG cells to other species with greater commercial utility than mice and to species with greater therapeutic relevance for bio-medical applications. It would also be desirable to develop techniques for the routine isolation of ES/EG lines by selective ablation from any mouse genotype without the requirement for first generating transgenic mouse colonies.

It has now surprisingly been found that nuclear transfer can be employed, not as an alternative to ES or EG cells, but to enhance the opportunities for stem cell isolation, particularly ES/EG isolation, in a broad range of species.

According to a first aspect of the present invention there is provided a method for generating animal stem cells from an animal embryo in culture, the method comprising the steps of reconstituting an animal embryo by transferring the nucleus of a donor cell into a suitable recipient cell followed by selective killing of differentiated cells of the reconstituted embryo.

In the context of the present invention, the animal stem cells may be pluripotent) stem cells, embryonic stem (ES) cells, embryonic germ (EG) cells (primordial germ cell-derived or PGC-derived cells), somatic stem/progenitor cells, haematopoietic stem cells, epidermal stem cells or neuronal stem cells. A totipotent cell can direct the development of a whole animal (when constructing embryos by nuclear transfer from a donor cell into a recipient cell, such as an enucleated oocyte, it is the nucleus of the donor cell which is totipotent). This includes directing the development of extra- embryonic lineages, i.e. the placenta. In this definition, a fertilised zygote and in some species individual blastomeres are also totipotent. In contradistinction, a pluripotent or multipotent cell (i.e. an embryonic stem cell) type has been defined as one which can form all tissues in the conceptus/offspring after injection into the blastocoele cavity. In a preferred embodiment of the present invention, the animal stem cells may be embryonic stem (ES) cells or embryonic germ (EG) cells.

In principle, the invention is applicable to the generation of stem cells from all animals, including mammals, birds, such as domestic fowl, amphibian species and fish species. In practice, however, it will be to mammals, particularly placental mammals, including non-human primates and humans, that the invention is presently envisaged to have an important use in the preparation of stem cells or stem cell lines from which differentiated cells can be produced to treat diseases, particularly diseases characterised by a loss, absence or reduction of normal cell function. The invention is also likely to find application to the generation of stem cells from non-human mammals, e.g. ungulates, particularly economically important ungulates such as cattle, sheep, goats, water buffalo, camels and pigs, both as a means for cloning animals and as a means for generating transgenic or genetically modified animals. It should also be noted that the invention is also likely to be applicable to other economically important animal species such as, for example, horses, llamas or rodents e.g. rats or mice, or rabbits.

It should be noted that the term "transgenic", in relation to animal stem cells, should not be taken to be limited to referring to animal cells containing in their DNA one or more genes from another species, although many transgenic animal stem cells will contain such a gene or genes. Rather, the term refers more broadly to any animal stem cell whose DNA has been the subject of technical intervention by recombinant DNA technology either prior to embryo reconstruction by manipulation of the donor cell or after manipulation by manipulation of a stem cell generated according to a method of the present invention. So, for example, an animal stem cell in whose DNA an endogenous gene has been deleted, duplicated, activated or modified is a transgenic animal stem cell for the purposes of this invention as much as an animal stem cell to whose DNA an exogenous DNA sequence has been added.

In embodiments of the invention in which the animal stem cell is transgenic, the donor nucleus is genetically modified. The donor nucleus may contain one or more transgenes and the genetic modification may take place prior to nuclear transfer and embryo reconstitution. Although micro-injection, analogous to injection into the male or female pronucleus of a zygote, may be used as a method of genetic modification, the invention is not limited to that methodology: mass transformation or transfection techniques can also be used e.g. electroporation, viral transfection or lipofection.

Transgenesis may be achieved, for example, by DNA microinjection or transfection. Microinjection methods involve DNA microinjection into zygotes or early cleavage stage embryos. Methods for transgenic mice are described in detail by Hogan et al , "Manipulating the Mouse Embryo: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1986). DNA microinjection using essentially the same techniques has been successfully applied to other species, including farm animals, albeit with reduced efficiency (reviewed by Wall et al. , Theriogenology 38 337-357 (1992) and Wilmut et al , Reprod. Fert. Suppl. 43 265-275 (1991)). Transgenic animal stem cells may be identified by appropriate DNA analysis of biopsies.

It is not envisaged that such transgene constructs will necessarily be the only transgene constructs integrated into the animal stem cell's DNA, although in many cases they will be the only ones.

Not all nuclear donor cells nor subsequently reconstituted embryos in accordance with this aspect of the invention will be transgenic. An alternative is for an expression construct simply to be introduced into the embryo cell(s) by any suitable means, such as microinjection (see, for example, Burdon et al , Mol. Reprod. and Dev. 33 436-442 (1992)). Although such a construct may not survive for long in the absence of replication sequences (and in the absence of integration) it may persist for long enough for the method of the first aspect of the invention to be practised on the embryo. In any event, the relatively short duration of a non-integrating, non-replicating construct may actually be an advantage, as no particular steps are needed to remove the construct if its continued presence is either not desired, or where its removal is obligatory. The latter case would apply to stem cells containing a toxin gene under the control of a promoter causing specific expression in differentiated cells. Such a construct is normally to be removed before stem cells can participate in embryo development.

A third possibility is that the expression construct may be present in the embryonic cells coupled to sequences that give rise to episomal replication. Episomal vectors of this type have been derived from bovine papilloma virus (Mathias et al. , EMBO J. 2 1487- 1492 (1983)) and adenovirus (Quantin et al , Proc. Nat 'I. Acad. Sci. 89 2581-2584 (1992)). The advantage of using an episomal vector is that most embryos and their cultured derivatives will contain the selective cassette for an extended period after microinjection.

Selection strategies for stem cell, preferably ES cell or EG cell, isolation depend upon expression of stem cell-specific salvage transgenes which preserve the stem cell lineage under generally lethal selection conditions or differentiated cell-specific expression of toxin transgenes. Integration and maintenance of the transgene after ES isolation is probably unimportant and may be undesirable. A short term transfection system such as lipofection (Feigner et al , Nature 337 387-388 (1989)) is therefore suited to this approach in large animals - particularly where transient infection can be effected under near-physiological conditions (Brunette et al, Nucl. Acids Res. 20 1151 (1992)). Lipofection involves the spontaneous association of DNA with a liposome containing a cationic lipid, which then fuses with the cell membrane, leading to internalisation of DNA. Suitable liposome preparations are commercially available.

There are a number of advantages of lipofection as a means of transgenesis in the context of the present invention. First, as a procedure, it takes far less effort and time than microinjection; therefore, large numbers of embryos can be treated. Secondly, lipofection is a relatively benign treatment which can be applied repeatedly without toxicity. Repeated lipofection will therefore help ensure the presence of DNA in a high proportion of explanted embryos. Thirdly, selectable DNA will be present mainly as non-integrated, non-replicating molecules; it should therefore be easy to isolate non- transgenic stem cells once selection is removed.

In the method of the invention described above, a nucleus is transferred from a donor cell to a recipient cell. The use of this method is not restricted to a particular donor cell type. The donor cell may be as described in Wilmut et al Nature 385 810 (1997); Campbell et al Nature 380 64-66 (1996); or Cibelli et al Science 280 1256-1258 (1998). All cells of normal karyotype, including embryonic, foetal and adult somatic cells which can be used successfully in nuclear transfer may in principle be employed in a method according to the present invention. Foetal fibroblasts are a particularly useful class of donor cells. Generally suitable methods of nuclear transfer are described in Campbell et al Theriogenology 43 181 (1995), Collas et al Mol. Reprod. Dev. 38 264-267 (1994), Keefer et al Biol. Reprod. 50 935-939 (1994), Sims et al Proc. Nat 'I. Acad. Sci. USA 90 6143-6147 (1993), WO-A-9426884, WO-A-9424274, WO-A-9807841, WO-A-9003432, US-A-4994384 and US-A-5057420. The invention therefore contemplates the use of an at least partially differentiated cell, including a fully differentiated cell. Donor cells may be, but do not have to be, in culture and may be quiescent. Nuclear donor cells which are quiescent are cells which can be induced to enter quiescence or exist in a quiescent state in vivo. Cultured bovine primary fibroblasts, an embryo-derived ovine cell line (TNT4), an ovine mammary epithelial cell derived cell line (OME) from a 6 year old adult sheep, a fibroblast cell line derived from foetal ovine tissue (BLWF1) and an epithelial-like cell line derived from a 9-day old sheep embryo (SEC1) are described in WO 97/07669. A class of embryo-derived cell lines useful in the invention which includes the TNT4 cell line described in WO 96/07732. An example of a genetically modified donor cell is pOctneol described in McWhir et al (Nature Genetics 14 223-226 (1996)).

Where the donor cells are described as being quiescent, such cells may not be actively proliferating by means of the mitotic cell cycle. The mitotic cell cycle has four distinct phases, Gl, S, G2 and M. The beginning event in the cell cycle, called start, takes place in the Gl phase and has a unique function. The decision or commitment to undergo another cell cycle is made at start. Once a cell has passed through start, it passes through the remainder of the Gl phase, which is the pre-DNA synthesis phase. The second stage, the S phase, is when DNA synthesis takes place. This is followed by the G2 phase, which is the period between DNA synthesis and mitosis. Mitosis itself occurs at the M phase. Quiescent cells (which include cells in which quiescence has been induced as well as those cells which are naturally quiescent, such as certain fully differentiated cells) are generally regarded as not being in any of these four phases of the cycle; they are usually described as being in a GO state, so as to indicate that they would not normally progress through the cycle. The nuclei of quiescent GO cells have a diploid DNA content.

Cultured cells can be induced to enter the quiescent state by various methods including chemical treatments, nutrient deprivation, growth inhibition or manipulation of gene expression. Presently the reduction of serum levels in the culture medium has been used successfully to induce quiescence in both ovine and bovine cell lines. In this situation, the cells exit the growth cycle during the Gl phase and arrest, as explained above, in the so-called GO stage. Such cells can remain in this state for several days (possibly longer depending upon the cell) until re-stimulated when they re-enter the growth cycle. Quiescent cells arrested in the GO state are diploid. The GO state is the point in the cell cycle from which cells are able to differentiate. On quiescence a number of metabolic changes have been reported and these include: monophosphorylated histones, ciliated centrioles, reduction or complete cessation in all protein synthesis, increased proteolysis, decrease in transcription and increased turnover of RNA resulting in a reduction in total cell RNA, disaggregation of polyribosomes, accumulation of inactive 80S ribosomes and chromatin condensation (reviewed Whitfield et al., Control of Animal Cell Proliferation, 1 331-365 (1985)).

Many of these features are those which are required to occur following transfer of a nucleus to an enucleated oocyte. The fact that the GO state is associated with cell differentiation suggests that this may provide a nuclear/chromatin structure which is more amenable to either remodelling and/or reprogramming by the recipient cell cytoplasm. In this way, by virtue of the nuclear donor cells being in the quiescent state, the chromatin of the nuclei of the donors may be modified before embryo reconstitution or reconstruction such that the nuclei are able to direct development. This differs from all previously reported methods of nuclear transfer in that the chromatin of donor cells is modified prior to the use of the cells as nuclear donors. Other nuclear transfer protocols have reported the use of Gl or G0/G1 stage cell nuclei or have stated that the nuclei are from donor cells that are actively dividing. Methods in accordance with the present invention may therefore incorporate the use of any successful nuclear transfer protocol.

The recipient cell to which the nucleus from the donor cell is transferred may be an oocyte or another suitable cell.

Recipient cells at a variety of different stages of development may be used, from oocytes at metaphase I through metaphase II, to zygotes and two-cell embryos. Each has its advantages and disadvantages. The use of fertilized eggs ensures efficient activation whereas parthenogenetic activation is required with oocytes (see below). Another mechanism that may favour the use of cleavage-stage embryos in some species is the extent to which reprogramming of gene expression is required. Transcription is initiated during the second cell cycle in the mouse and no major changes in the nature of the proteins being synthesised are revealed by two-dimensional electrophoresis until the blastocyst stage (Howlett & Bolton J. Embryol. Exp. Morphol. 87 175-206 (1985)). In most cases, though, the recipient cells will be oocytes.

It is preferred that the recipient be enucleate. While it has been generally assumed that enucleation of recipient oocytes in nuclear transfer procedures is essential, there is no published experimental confirmation of this judgement. The original procedure described for ungulates involved splitting the cell into two halves, one of which was likely to be enucleated (Willadsen Nature 320 (6) 63-65 (1986)). This procedure has the disadvantage that the other unknown half will still have the metaphase apparatus and that the reduction in volume of the cytoplasm is believed to accelerate the pattern of differentiation of the new embryo (Eviskov et al, Development 109 322-328 (1990)).

More recently, different procedures have been used in attempts to remove the chromosomes with a minimum of cytoplasm. Aspiration of the first polar body and neighbouring cytoplasm was found to remove the metaphase II apparatus in 67% of sheep oocytes (Smith & Wilmut Biol. Reprod. 40 1027-1035 (1989)). Only with the use of DNA-specific fluorochrome (Hoechst 33342) was a method provided by which enucleation would be guaranteed with the minimum reduction in cytoplasmic volume (Tsunoda eta , J. Reprod. Fertil. 82 173 (1988)). In livestock species, this is probably the method of routine use at present (Prather & First J. Reprod. Fertil. Suppl. 41 125 (1990), Westhusin et al, Biol. Reprod. (Suppl.) 42 176 (1990)).

There have been very few reports of non-invasive approaches to enucleation in mammals, whereas in amphibians, irradiation with ultraviolet light is used as a routine procedure (Gurdon Q. J. Microsc. Soc. 101 299-311 (I960)). There are no detailed reports of the use of this approach in mammals, although during the use of DNA- specific fluorochrome it was noted that exposure of mouse oocytes to ultraviolet light for more than 30 seconds reduced the developmental potential of the cell (Tsunoda et al, J. Reprod. Fertil. 82 173 (1988)).

It is preferred that recipient host cells to which the donor cell nucleus is transferred is an enucleated metaphase II oocyte, an enucleated unactivated oocyte or an enucleated preactivated oocyte. At least where the recipient is an enucleated metaphase II oocyte, activation may take place at the time of transfer. Alternatively, at least where the recipient is an enucleated unactivated metaphase II oocyte, activation may take place subsequently. As described above enucleation may be achieved physically, by actual removal of the nucleus, pro-nuclei or metaphase plate (depending on the recipient cell), or functionally, such as by the application of ultraviolet radiation or another enucleating influence.

Three suitable cytoplast (enucleated oocyte) recipients are:

1. The "MAGIC Recipient" (Metaphase Arrested G1/G0 Accepting Cytoplast) described WO 97/07668.

2. The "GOAT" (G0/G1 Activation and Transfer) - a Mil (metaphase II) oocyte at the time of activation (Campbell et al , Biol. Reprod. 49 933-942 (1993).

3. The "Universal Recipient" (Campbell et al, Biol. Reprod. 649 933-942 (1993), Biol. Reprod. 50 1385-1393 (1994).

All three of these recipients would result in normal ploidy when using donor nuclei in GO in the reconstructed embryo. However, recent reports have suggested that a proportion of the nuclei from quiescent cells are unable to enter the DNA synthetic phase when placed into an S-phase cytoplasm without undergoing disassembly of the nuclear envelope (Leno & Munshi, J. Cell Biol. 127(1) 5-14 (1994)). Therefore, although a proportion of embryos will develop when using the "Universal Recipient" it is postulated that the use of Mil oocytes containing high levels of MPF (M-phase promoting factor or maturation-promoting factor) as cytoplast recipients by either method 1 or 2 will result in a greater frequency of development. Once suitable donor and recipient cells have been identified, it is necessary for the nucleus of the former to be transferred to the latter. Most conveniently, nuclear transfer is effected by fusion.

Three established methods which have been used to induce fusion are:

(1) exposure of cells to fusion-promoting chemicals, such as polyethylene glycol;

(2) the use of inactivated virus, such as Sendai virus; and (3) the use of electrical stimulation.

Exposure of cells to fusion-promoting chemicals such as polyethylene glycol or other gly cols is a routine procedure for the fusion of somatic cells, but it has not been widely used with embryos. As polyethylene glycol is toxic it is necessary to expose the cells for a n inimum period and the need to be able to remove the chemical quickly may necessitate the removal of the zona pellucida (Kanka et al., Mol. Reprod. Dev. 29 110- 116 (1991)). In experiments with mouse embryos, inactivated Sendai virus provides an efficient means for the fusion of cells from cleavage-stage embryos (Graham Wistar Inst. Symp. Monogr. 9 19 (1969)), with the additional experimental advantage that activation is not induced. In ungulates, fusion is commonly achieved by the same electrical stimulation that is used to induce parthogenetic activation (Willadsen Nature 320 (6) 63-65 (1986), Prather et al, Biol. Reprod. 37 859-866 (1987)). In the mouse, activation has also been achieved using a piezo-impact drive unit (Wakayama et al Nature 394 369-374 (1998); Kimura, Y. & Yanigamachi, R. Biol. Reprod. 52 709-720 (1995)). In these species, Sendai virus induces fusion in a proportion of cases, but is not sufficiently reliable for routine application (Willadsen Nature 320 (6) 63-65 (1986)). While cell-cell fusion is a preferred method of effecting nuclear transfer, it is not the only method that can be used. Other suitable techniques include microinjection (Ritchie and Campbell, J. Reproduction and Fertility Abstract Series No. 15, p60).

Before or (preferably) after nuclear transfer (or, in some instances at least, concomitantly with it), it is generally necessary to stimulate the recipient cell into development by parthenogenetic activation, at least if the cell is an oocyte. Recent experiments have shown that the requirements for parthogenetic activation are more complicated than had been imagined. It had been assumed that activation is an all-or- none phenomenon and that the large number of treatments able to induce formation of a pronucleus were all causing "activation". However, exposure of rabbit oocytes to repeated electrical pulses revealed that only selection of an appropriate series of pulses and control of the Ca²⁺ was able to promote development of diploidized oocytes to mid- gestation (Ozil Development 109 117-127 (1990)). During fertilization there are repeated, transient increases in intracellular calcium concentration (Cutbertson & Cobbold Nature 316 541-542 (1985)) and electrical pulses are believed to cause analogous increases in calcium concentration. There is evidence that the pattern of calcium transients varies with species and it can be anticipated that the optimal pattern of electrical pulses will vary in a similar manner. The interval between pulses in the rabbit is approximately 4 minutes (Ozil Development 109 117-127 (1990)), and in the mouse 10 to 20 minutes (Cutbertson & Cobbold Nature 316 541-542 (1985)), while there are preliminary observations in the cow that the interval is approximately 20 to 30 minutes (Robl et al, in Symposium on Cloning Mammals by Nuclear Transplantation (Seidel ed.), Colorado State University, 24-27 (1992)). In most published experiments activation was induced with a single electrical pulse, but new observations suggest that the proportion of reconstituted embryos that develop is increased by exposure to several pulses (Collas & Robl Biol. Reprod. 43 877-884 (1990)). In any individual case, routine adjustments may be made to optimise the number of pulses, the field strength and duration of the pulses and the calcium concentration of the medium.

The reconstituted embryo may be cultured in vitro or in vivo until the next stage of the method which is the derivation of the stem cell line from desired cells of the embryo, e.g. inner cell mass cells. Suitably, the embryo may be cultured to the blastocyst stage, or until the appearance of the primitive streak, i.e. until the end of the period of 14 days beginning with the day on which the nuclear transfer occurred, not counting any time during which the embryo is stored or prior to activation.

The differentiated cells may be derived from the embryo as explanted into the (generally in vitro) culture or may result from the differentiation of ES cells within the embryo. The intention will generally be to selectively kill all differentiated cells. The killing of the differentiated cells may also be termed as being selective ablation.

Explanted embryos will generally contain undifferentiated ES progenitor cells as well as differentiated cells such as trophectoderm and endoderm cells. The explanted embryo may be obtained, as outlined by Robertson (1987) loc. cit. , from an in vitro culture or from an in vivo host by abstracting an embryo from the uterus (for example at the blastocyst stage) and culturing in a suitable medium over a layer of feeder cells, which may be mitotically inactivated fibroblasts, as previously described. The explanted embryo on which the selective killing step of the invention is performed may have gone through one or more passages in which a clump of predominantly undifferentiated cells is isolated, disaggregated and replated onto a new feeder layer. Methods in accordance with the present invention may therefore also include the step of culturing the resulting undifferentiated embryonic cells after the step of selective killing of differentiated cells. In one preferred embodiment of the invention, the differentiated cells are killed by means of a drug selection regime. The regime may be used to kill differentiated cells during early passages of the explanted embryo. Undifferentiated ES cells may express a resistance marker for a toxic drug, so that when the colony is exposed to the drug only the differentiated cells are killed. For this embodiment, the use of a promoter specific or substantially specific for undifferentiated cells is important, and the embryo cells can be made transgenic for, or otherwise be able to express, a construct comprising such a promoter operatively coupled to a DNA sequence encoding the drug resistance marker.

There have been two published reports of genes which are strongly down-regulated on the differentiation of ES cells. These are the transcription factor Oct-3/4 (Okazawa et al , EMBO J. 10 2997-3005 (1991)) and the growth factor FGF-4 (Ma et al Dev. Biol. 154 45-54 (1992)). The examples which appear below use the Oct-3/4 promoter, although the FGF-4 promoter could also be used, as could any other suitable ES- specific promoters.

The transcription factor Oct-3/4 was first identified as present in undifferentiated mouse embryonal carcinoma (EC) cells (closely related to ES cells) but not in their differentiated derivatives (Okamoto et al , Cell 60 461-472 (1990)). Analysis of Oct- 3/4 mRNA expression in mouse showed that it was restricted to undifferentiated ES and EC cells (Ben-Shusan et al , Mol. Cell. Biol. 13 891-901 (1993)), embryo inner cell mass and primordial germ cells (Rosner et al , Nature 345 686-92 (1990)). The Oct- 3/4 promoter is capable of conferring Oct-3/4 specific expression on a heterologous reporter gene in transgenic mouse embryos (Okazawa et al. , EMBO J. 10 2997-3005 (1991)). Several genes are available for conferring drug resistance on non mutant cells, e.g. G418 selection for the neo gene (Colbere-Garapin et al , J. Mol. Biol. 150 1-14 (1981)), hygromycin selection for the hygro gene (Santerre et al Gene 30 147-156 (1984)), histidinol selection for the his gene (Hartman et al , Proc. Nat'l. Acad. Sci. 85 8047-8051 (1988)), methotrexate selection for the dhfr gene (Wigler et al. , Proc. Nat'l. Acad. Sci. USA 77 3567-3570 (1980)), aminopterin/mycophenolic acid selection for the gpt gene (Mulligan et al, Proc. Nat'l. Acad. Sci. USA 78 2072-2076 (1981)), methionine sulphoximine selection for the glutamine synthetase (gs) gene (Hayward et al, Nucl. Acids Res. 14 999-1008 (1986)) and deoxycoformicin selection for the adenosine deaminase (add) gene (Kaufman et al, Proc. Nat'l. Acad. Sci. USA 83 3136-3140 (1986)). The examples below show the use of the neo (aminoglycoside phosphotransferase) gene, although the hygro, his, dhfr, gpt, gs or ada genes could also be used, as could any other suitable drug resistance gene. It should be noted that the use of the term "gene" in this context does not imply that natural genomic DNA has to be used, although that may be preferred. cDNAs may be at least as suitable, as may "iriinigenes'' which contain some, but not all, of the introns which may naturally be present in the gene.

In another important embodiment, a DNA sequence whose expression gives rise to cell death (for example a toxin gene) is selectively expressed in differentiated cells. For this embodiment, the use of a promoter specific or substantially specific for differentiated cells is important, and the embryo cells can be made transgenic for, or otherwise be able to express, a construct comprising such a promoter operatively coupled to the lethal DNA sequence (for example that encoding the toxin).

One suitable promoter is that of the fransforrning growth factor β-2 gene, which is activated on differentiation of ES cells independent of the cell type formed (Mummery et al , Dev. Biol. 137 161-170 (1990)). Genes encoding various toxins may be placed under the control of a promoter active in differentiated cells. For example, the Diphtheria toxin subunit-A gene (Maxwell et al. , Mol. Cell. Biol. 7 1576-1589 (1987)) or the Ricin toxin subunit-A gene (Landel et al, Genes and Dev. 2 1168-1178 (1988)) can be placed under the control of a promoter expressed only in differentiated cells. Again, either the natural gene or a non-natural sequence encoding the toxin may be used.

Embryos which can express either (a) a selectable marker gene under the control of a promoter expressed specifically in undifferentiated ES cells and/or (b) a toxin gene under the control of a promoter expressed specifically in differentiated cells can be generated using standard methods. Such embryos may be transgenic, in that a heterologous DNA construct may be stably integrated in the embryonic genome, or they may simply contain non-integrated expressible DNA. ES cells may then be derived from these transgenic, injected or transfected embryos by culture under selective conditions.

Once a stem cell line has been established the selectable marker or toxin gene construct can, if necessary or desirable, be removed. In the case of non-integrating, non- replicating DNA the removal is, in effect, automatic. For transgenic embryos and embryos in which the expression construct is episomally replicating, more deliberate steps have to be taken. The use of site specific recombination systems to generate precisely defined deletions in cultured mammalian cells has recently been demonstrated. Gu et al. (Cell 73 1155-1164 (1993)) describe how a deletion in the immunoglobulin switch region in mouse ES cells was generated between two copies of the bacteriophage PI loxP site by transient expression of the Cre site-specific recombinase, leaving a single loxP site. Similarly, yeast FLP recombinase has been used to precisely delete a selectable marker defined by recombinase target sites in mouse erythroleukemia cells (Fiering et al , Proc. Nat'l. Acad. Sci. USA 90 8469-8473 (1993)). The Cre lox system is exemplified below, but other site-specific recombinase systems could be used.

A construct used in the Cre lox system will usually have the following three functional elements:

1. The expression cassette;

2. A negative selectable marker (e.g. Herpes simplex virus thymidine kinase (TK) gene) expressed under the control of a ubiquitously expressed promoter (e.g. phosphoglycerate kinase (Soriano et al , Cell 64 693-702 (1991)); and

3. Two copies of the bacteriophage PI site specific recombination site loxV

(Baubonis et al, Nuc. Acids. Res. 21 2025-2029 (1993)) located at either end of the DNA fragment.

This construct can be eliminated from established stem cell lines, for example ES cell or EG cell lines, containing it by means of site specific recombination between the two loxP sites mediated by Cre recombinase protein which can be introduced into the cells by lipofection (Baubonis et al , Nuc. Acids Res. 21 2025-2029 (1993)). Cells which have deleted DNA between the two loxP sites are selected for loss of the TK gene (or other negative selectable marker) by growth in medium containing the appropriate drug (ganciclovir in the case of TK).

Stem cells isolated by the selection procedure can be tested for totipotency by assessing their capacity to form adult tissues, most importantly germ cells. Totipotent stem cells can be used as a means of manipulating an animal's genome either by simple introduction of a transgene or by the more extensive modifications possible using gene targeting technology.

For the production of a genetically modifed cell line, it is necessary that any genetic changes introduced into the animal stem cells are transmitted to the next generation. The preparation of transgenic animal stem cells according to the present invention has several advantages over the use of somatic cells as described in the more traditional nuclear transfer procedures. Firstly, the animal stem cells have the potential to transmit the genetic modification to the next generation by both chimaera formation and nuclear transfer. This is an important advantage as the technique of traditional nuclear transfer is still at an early stage and some animal species may pose particular problems and there is the possibility that results in animals other than sheep and cows may be different. Secondly, somatic cells cannot contribute to the development of a germ line chimaeric animal. Thirdly, somatic cells undergo homologous recombination at a lower rate than ES cells (Arbones et al Nature Genetics 6 90-97 (1994); Thyagaraja et al Nucl. Acids Res. 24 4084-4091 (1996)). Assuming that ES and EG cells have roughly similar targeting frequencies, the animal stem cells of the present invention should target at a higher frequency than somatic cells.

An important application of the invention, however, will be in the generation of human stem lines, preferably ES or EG cell lines, by selective ablation following nuclear transfer of human cells to form animal oocytes. This offers the opportunity to prepare stem cell-derived somatic cells for potential transplantation therapy in a variety of human conditions. The resulting cells would be expected to induce little or no immune response in patients as chromosomally identical lines could be matched to each patient. In many cases, the cells will be differentiated derivatives of the stem cell or stem cell line of the present invention. Zawada et al (Nature Medicine 4 (5) 569-574 (1998)) have shown that transgenic bovine neurons can be generated from bovine embryos reconstituted by nuclear transfer from foetal fibroblasts and used to treat a rat model of Parkinson's disease. They have not, however, sought to engineer the foetal fibroblast to enable subsequent ES isolation.

According to a second aspect of the invention there is therefore provided a stem cell line prepared in accordance with the first aspect of the invention. The cell line may suitably be an embryonic cell line or an embryonic germ cell line. Stem cells and stem cell lines prepared in accordance with a method of the present invention may have widespread uses in medicine in the therapy and/or prophylaxis of diseases. For example, such cells or their differentiated derivatives can be used in the treatment of a diverse range of medical conditions characterised by the native cell population of a patient failing to perform its usual function sufficiently or even at all, e.g. in the treatment of diabetes where a decline or absence in insulin production by the Islets of Langerhans leads to the onset of the disease; or in Parkinson's disease where sufficient dopamine is no longer being released by certain brain cells. Cells prepared in accordance with the present invention may also find application in the treatment of degenerative disorders, e.g. neurodegenerative disorders such as Alzheimer's disease or Motor Neurone Disease. The present invention therefore also includes methods of treatment of diseases in which a viable, therapeutically effective population of stem cells prepared in accordance with the first aspect of the invention or their differentiated derivatives are transplanted into a patient suffering from a disease characterised by a loss of or absence of normal cell function. This aspect of the invention also therefore includes the use of such cells in the transplantation and the use of such cells in the preparation of a therapeutic or prophylactic agent in the treatment of a disease in a patient characterised by a loss of normal cell function. The use of cells in therapy by transplantation is not limited to the preparation of allogeneic cell populations, but also includes the use of autologous cell populations from the patient and xenogeneic cell populations from another animal species. In the situation where a xenogeneic or allogeneic population of cells is used, the cells may also preferably be further transgenically modified by methods known in the art to reduce the risks of immune rejection by the patient.

The stem cell lines and stem cells prepared in accordance with the present invention may also find further application as an alternative source of nuclear donor cells for additional nuclear transfer procedures. These procedures may lead to the production of new embryos which are allowed to develop normally for the production of an animal. Such cells and embryos may also be the subject of further intervention to introduce further transgenes.

It is also contemplated that a new ES cell line of the present invention may act as a source of nuclear donor cells for use in further rounds of nuclear transfer. According to a third aspect of the present invention there is provided a method for preparing an animal, the method comprising:

(a) reconstituting an animal embryo as described above; and

(b) isolating stem cells by selective killing as described above;

(c) reconstituting a further animal embryo from such stem cells as in step

(a) (d) causing an animal to develop to term from the embryo; and

(e) optionally, breeding from the animal so formed.

Steps (a) and (b) have been described in depth above (and by implication also step (c)). The fourth step, step (d) in the method of this aspect of the invention is to cause an animal to develop to term from the embryo. This may be done directly or indirectly. In direct development, the reconstituted embryo from step (a) is simply allowed to develop without further intervention beyond any that may be necessary to allow the development to take place. In indirect development, however, the embryo may be further manipulated before full development takes place. For example, the embryo may be split and the cells clonally expanded, for the purpose of improving yield.

Alternatively or additionally, it may be possible for increased yields of viable embryos to be achieved by means of the present invention by clonal expansion of donors and/or if use is made of the process of serial (nuclear) transfer. A limitation in the presently achieved rate of blastocyst formation may be due to the fact that a majority of the embryos do not "reprogram" (although an acceptable number do). If this is the case, then the rate may be enhanced as follows. Each embryo that does develop itself can be used as a nuclear donor, such as, for example at the morula or 32-64 cell stage; alternatively, inner cell mass cells can be used at the blastocyst stage. Embryos derived from these subsequent transfers could themselves also be used as potential nuclear donors to further increase efficiency. If these embryos do indeed reflect those which have reprogrammed gene expression and those nuclei are in fact reprograrnmed (as seems likely), then each developing embryo may be multiplied in this way by the efficiency of the nuclear transfer process. The degree of enhancement likely to be achieved depends upon the cell type. In sheep, it is readily possible to obtain 55% blastocyst stage embryos by transfer of a single blastomere from a 16 cell embryo to a preactivated "Universal Recipient" oocyte. So it is reasonable to hypothesise that each embryo developed from a single cell could give rise to eight at the 16 cell stage. Although these figures are just a rough guide, it is clear that at later developmental stages the extent of benefit would depend on the efficiency of the process at that stage. In certain instances, where there may be restrictions in the development of a reconstructed embryo to term it may be preferable to generate a chimaeric animal formed from cells derived from a naturally formed embryo and an embryo reconstructed by nuclear transfer. Such a chimaera can be formed by taking a proportion of cells of the natural embryo and a proportion of the cells of the reconstructed embryo at any stage up to the blastocyst stage and foπning a new embryo by aggregation or injection. The proportion of cells may be in the ratio of 50:50 or another suitable ratio to achieve the formation of an embryo which develops to term. The presence of normal cells in these circumstances is thought to assist in rescuing the reconstructed embryo and allowing successful development to term and a live birth.

Aside from the issue of yield-improving expediencies, the reconstituted embryo may be cultured, in vivo or in vitro to the blastocyst stage, or until the appearance of the primitive streak, i.e. until the end of the period of 14 days beginning with the day on which the nuclear transfer occurred, not counting any time during which the embryo is stored or prior to activation.

Experience suggests that embryos derived by nuclear transfer are different from normal embryos and sometimes benefit from or even require culture conditions in vivo other than those in which embryos are usually cultured (at least in vivo). The reason for this is not known. In routine multiplication of bovine embryos, reconstituted embryos (many of them at once) have been cultured in sheep oviducts for 5 to 6 days (as described by Willadsen, In Mammalian Egg Transfer (Adams, E.E., ed.) 185 CRC Press, Boca Raton, Florida (1982)). In the practice of the present invention, though, in order to protect the embryo it should preferably be embedded in a protective medium such as agar before transfer and then dissected from the agar after recovery from the temporary recipient. The function of the protective agar or other medium is twofold: first, it acts as a structural aid for the embryo by holding the zona pellucida together; and secondly it acts as barrier to cells of the recipient animal's immune system. Although this approach increases the proportion of embryos that form blastocysts, there is the disadvantage that a number of embryos may be lost.

If in vitro conditions are used, those routinely employed in the art are quite acceptable.

At the blastocyst stage, the embryo may be screened for suitability for development to term. Typically, this will be done where the embryo is transgenic and screening and selection for stable integrants has been carried out. Screening for non-transgenic genetic markers may also be carried out at this stage. However, because the method of the invention allows for screening of donors at an earlier stage, that will generally be preferred.

After screening, if screening has taken place, the blastocyst embryo is allowed to develop to term. This will generally be in vivo. If development up to blastocyst has taken place in vitro, then transfer into the final recipient animal takes place at this stage. If blastocyst development has taken place in vivo, although in principle the blastocyst can be allowed to develop to term in the pre-blastocyst host, in practice the blastocyst will usually be removed from the (temporary) pre-blastocyst recipient and, after dissection from the protective medium, will be transferred to the (permanent) post- blastocyst recipient.

In optional step (e) of this aspect of the invention, animals may be bred from the animal prepared by the preceding steps. In this way, an animal may be used to establish a herd or flock of animals having the desired genetic characteristic(s). Animals produced by transfer of nuclei from a source of genetically identical cells share the same nucleus, but are not strictly identical as they are derived from different oocytes. The significance of this different origin is not clear, but may affect commercial traits. Recent analyses of the mitochondrial DNA of dairy cattle in the Iowa State University Breeding Herd revealed associated with milk and reproductive performance (Freeman & Beitz, In Symposium on Cloning Mammals by Nuclear Transplantation (Seidel, G. E. Jr., ed.) 17-20, Colorado State University, Colorado (1992)). It remains to be confirmed that similar effects are present throughout the cattle population and to consider whether it is possible or necessary in specific situations to consider the selection of oocytes. In the area of cattle breeding the ability to produce large numbers of embryos from donors of high genetic merit may have considerable potential value in disseminating genetic improvement through the national herd. The scale of application will depend upon the cost of each embryo and the proportion of transferred embryos able to develop to term.

References to transgenic animals according to this aspect of the invention should be construed in conformity with the discussion of the term transgenic as used above in relation to animal stem cells.

By way of illustration and summary, the following scheme sets out a typical process by which transgenic and non-transgenic animals may be prepared. The process can be regarded as involving nine steps:

(1) selection and isolation of suitable donor cells, which may include assessment of karyotype, induction of quiescence (arrest in GO) and/or induction of development;

(2) application of suitable molecular biological techniques for the production of genetically modified cell populations. Such techniques may include gene additions, gene knock-outs, gene knock-ins, and other gene modifications. Optionally, transgenesis, may also be employed by transfection with suitable constructs, with or without selectable markers;

(3) optionally screen and select modified cell populations or clones for the required genotype/phenotype (i.e. stable integrants);

(4) induction of quiescence in modified cell population;

(5) embryo reconstitution by nuclear transfer;

(6) culture, in vivo or in vitro, to blastocyst;

(6a) optionally screen and select for stable integrants - omit if done at (3) - or other desired characteristics;

(7) stem cell isolation under conditions of selective killing of differentiated cells (for example ES or EG cell isolation);

(8) use of cells from stem cells isolated in (8) as fresh nuclear donor cells in further nuclear transfer as in steps (1) to (6), followed by, (9) transfer of blastocyst formed in step (6) if necessary to final recipient.

According to a fourth aspect of the invention, there is provided an animal prepared as described above. The invention is therefore equally applicable in the production of transgenic, as well as non-transgenic animals. Transgenic animals may be produced from genetically altered donor cells.

References to transgenic animals according to this aspect of the invention should be construed in conformity with the discussion of the term transgenic as used above in relation to animal stem cells.

The overall procedure has a number of advantages over conventional procedures for the production of transgenic (i.e. genetically modified) animals which may be summarised as follows: (1) fewer recipients will be required;

(2) multiple syngeneic founders may be generated using clonal donor cells;

(3) subtle genetic alteration by gene targeting is permitted; (4) all animals produced from embryos prepared by the invention should transmit the relevant genetic modification through the germ line as each animal is derived from a single nucleus; in contrast, production of transgenic animals by pronuclear injection or chimerism after inclusion of modified stem cell populations by blastocyst injection, or other procedures, produces a proportion of mosaic animals in which all cells do not contain the modification and the resultant animal may not transmit the modification through the germ line; and (5) cells can be selected for the site of genetic modification (e.g. integration) prior to the generation of the whole animal.

Preferred features of the second and subsequent aspects of the present invention are as for the first aspect mutatis mutandis.

The present invention will now be described by way of example with reference to the following Examples which are provided for the purposes of illustration only and are not to be construed as being limiting.

Example 1: Preparation of Octneol foetal fibroblasts

A line of transgenic mice has been generated previously which carry 1.94kb fragment of the 5' Oct promoter juxtaposed with a fragment encoding neomycin phosphotransferase and designated pOctoneol. Embryos from this line have been shown to be non-permissive for ES isolation by standard techniques and permissive under conditions of selective ablation at a frequency of 10% (McWhir et al Nature Genetics 14 223-226 (1996)). As a consequence, foetal fibroblasts derived from pOctneol mice constitute the ideal nuclear donor as the efficacy of the transgene has already been proven. Hence, inappropriate transgene expression due to site of incorporation effects is not anticipated.

Octneol foetal fibroblasts will be isolated from day 13 foetuses by incubation with trypsin and vigorous pipetting. The resulting cell suspension will be plated onto 10cm plates (1 foetus per plate).

Example 2: Preparation of nuclear donor cells from Octneol foetal fibroblasts and nuclear transfer

Octneol foetal fibroblasts will be prepared as nuclear donor cells at the earliest convenient passage according to the teaching of Campbell et al. (1997. Nature 380,64).

Embryo reconstruction of pOctneol nuclear donor cells with equal numbers of murine and ovine enucleated oocytes will be conducted according to the teachings of Campbell et al., (1997. Nature 380, 64) and of Dominko et al., 1998. Theriogenology 385).

Example 3 : Preparation of ES cell line from reconstructed embryos Reconstructed embryos will be cultured to blastocyst stage and implanted into culture for ES isolation by selective ablation according to the teachings of McWhir et al Nature Genetics 14 223-226 (1996)).

Example 4: Preparation of EG cell line

Reconstructed embryos prepared as described above are transferred to recipient females. Females are sacrificed at stages between 8.5 and 10.5 days of development and the primordial germ cells harvested for explantation into culture and subsequent isolation of EG lines. Culture conditions are described in Labonski et al (1994), with the addition of 50μg/ml G418.

Example 5: Analysis of ES and EG cell lines

ES and EG cell lines isolated as described above are injected into blastocysts as described in Bradley (Bradley, A., Production and analysis of chimaeric mice in "Teratocarcinomas and embryonic stem cells - A practical approach", ed. Robertson, E. J., IRL Press, Oxford (1987)). Injected embryos are transferred to recipient females and the resultant chimaeric pups are test bred or the detection of germline transmission through coat colour markers. Germline pups as indicated by coat colour are tested by Southern analysis for transmission of the pOctneol transgene.

Claims

1. A method for generating animal stem cells from an animal embryo in culture, the method comprising the steps of reconstituting an animal embryo by transferring the nucleus of a donor cell into a suitable recipient cell followed by selective killing of differentiated cells of the reconstituted embryo.

2. A method as claimed in claim 1, in which the animal is a mammalian animal species.

3. A method as claimed in claim 2, in which the mammal is a human.

4. A method as claimed in claim 2, in which the animal is an ungulate species.

5. A method as claimed in claim 4, in which the animal is a cow or bull, pig, goat, sheep, camel or water buffalo.

6. A method as claimed in any one of claims 1 to 5, in which the recipient cell is an oocyte.

7. A method as claimed in any one of claims 1 to 6, in which the oocyte is enucleate.

8. A method as claimed in any one of claims 1 to 7, in which the donor cell is an adult somatic cell.

9. A method as claimed in any one of claims 1 to 7, in which the donor cell is an embryonic somatic cell.

10. A method as claimed in any one of claims 1 to 7, in which the donor cell is a foetal somatic cell.

11. A method as claimed in claim 10, in which the foetal somatic cell is a foetal fibroblast.

12. A method as claimed in any one of claims 1 to 11, in which the differentiated cells are killed by means of a drug selection regime.

13. A method as claimed in claim 12, in which the embryo cells contain a construct comprising a promoter specific or substantially specific for undifferentiated cells operatively coupled to a DNA sequence encoding a drug resistance marker.

14. A method as claimed in claim 12, in which a DNA sequence whose expression gives rise to cell death is selectively expressed in differentiated cells.

15. A method as claimed in claim 14, in which the embryo cells contain a construct comprising a promoter specific or substantially specific for differentiated cells operatively coupled to a DNA sequence whose expression gives rise to cell death.

16. A method as claimed in any one of claims 1 to 15, in which the animal stem cell is an embryonic stem cell.

17. A method as claimed in any one of claims 1 to 15, in which the animal stem cell is an embryonic germ cell.

18. An animal stem cell or cell line prepared by a method as defined in any one of claims 1 to 17, or a differentiated derivative thereof.

19. The use of an animal cell or cell line as defined in claim 18 in medicine.

20. A method of treatment of a patient suffering from a disease characterised by loss or absence of normal cell function, the method comprising the transplantation of a cell as defined in claim 18 into the patient.

21. The use of a cell or a cell line as defined in claim 18 in the preparation of an agent for the treatment of a disease characterised by the loss or absence of normal cell function.