DERIVATION OF PLURIPOTENTIAL EMBRYONIC CELL LINES FROM DOMESTIC ANIMALS

Abstract: The invention relates to pluripotential embryonic stem cells, and such cells derived from porcine and bovine species are exemplified. These cells are useful in the production of transgenic animals. Methods are described for the production of pluripotential ungulate embryonic stem cells, together with details of the morphology enabling recognition of the cells.

Description: DERIVATION OF PLIJRIPOIENT__AL EMBRYONIC CELL LINES FROM DOMESTIC ANIMRLS Mammalian Genetics Traditional genetics depended upon mutations or pre-existing genetic polymorphisms which were discovered in a species. The only experimental approach to widen the scope of genetic variants available for study was irαitagenesis followed by specific screening or fortuitous recovery of relevant alleles. Animal breeding depends upon selection from suitable variation either in or introduced into the stock. The major tool for genetic analysis was breeding segregation studies and direct phenotypic analysis. Notwithstanding the great theoretical and practical interest in mammalian genetics the conventional genetic analysis of experimental mammals, because of their relatively small litter sizes and long life cycles, has been handicapped vis-a-vis other experimental aniiτial systems. Mammalian genetics has, however, benefitted from the study of human genetics where although there is no experimental breeding, detailed observation of a very large population has allowed investigation both of polymorphisms and of very rare mutations, and statistical methods for analysis of data available from human pedigrees of genetic segregation have become highly refined. Added to this, methods for non-meiotic genetic analysis - somatic cell genetics and more recently direct molecular-biological analysis, have been and indeed are being very effectively applied as well as being combined with pedigree analysis so that mammalian genetic maps and knowledge of gene sequence data are advancing rapidly. This is, however, an observational analysis. Our understanding of genetic function and also, in experimental animals, the practical application of our genetic knowledge requires the ability deliberately to modify the genome, preferably in a manner not entirely reliant upon screening the accidents of nature. Thus the concept of a reverse mammalian genetics emerges where the effect of specific genetic modification may be studied in the context of the intact organism. Genes of interest are now not only being identified from the results of the intensifying mammalian genetic analysis but iπportantly also t___rough their molecular biology and by cross hαmology to those of other species. In a great number of cases there are genes which have been identified in mice through their molecular biology, by analogy with those of other species (e.g. human disease syndromes or Drosophila genetics) or from the biochemistry of their protein products but for which there is no lack-of-function allele and thus no rigorous genetic test of function. Neither are these utations of protein structure or of genetic control. These can only be provided by creating such alleles. In the field of practical application to domestic farm animals, potential alteration of normal physiology which may be desirable, deletion or modification of function of controlling genes may be just as inportant as overexpression of others. The technology to undertake such targetted gene deletion or modification has now reached feasibility as exemplified by creation of null alleles at the hy?αxan1_h__ne phosphorbosyl transferase gene - HPRT - locus [1] [2]. The development of methods which will allow targetting and screening for deletion of function or specific modification of any gene whose sequence is known is now well underway and this is clearly a realistic proposal. It will become an available routine technique for mouse cells in the next year or so and with the development of domestic animal embryonic stem cells will be iitimediately applicable to these species and nay well become the transgenic route of choice. Mammalian Transqenesis Transgenic animals possess an alteration in their DNA which has been stably incorporat._d into the genome as a result of intentional experimental intervention. Typically this results from the additional exogenous foreign DNA. or novel DNA constructs. With the advent of specific gene targetting we should not necessarily exclude from the definition of transgenesis specific modification of endogenous gene sequences by direct experimental manipulation. A fully experimental approach to mammalian genetics is very rapidly becoming a reality t_tιrough the use both of conventional zygote injection transgenics and of embryonic stem cells. The latter approach allows extensive in vitro genetic manipulation, selection and screening prior to whole animal reconstruction. Thus both can e_2_perimental molecular genetics and the ability to design genetic changes in animals are available. For practical purposes the mouse has been the species of choice for such studies but it is important to be cable to extend the methods developed in the mouse to larger domestic farm animal species with the intention of their practical application. The new experi_mental mairiτialian genetics allows for testing of genetic modifications in vivo and designing genetic modification of a target species. One of the most important prospects is the construction of e_φeriιnental animal models of disease for pharmaceutical testing and developments. The other is for specific modification of domestic farm animals to create more desirable qualities for food production, disease resistance, and biopharmaceutical protein production. Methods for Transgenesis Although DNA π c_ro-injection is the most oαrπnonly used method of generating transgenic animals, alternatives include embryo infection using re∞irtoinent retroviral vectors innrporating the transgene and also the use of pluripotential embryonic stem (ES) cells. In order to introduce genetic alterations into a π_ammal it is necessary to transform genetically a cell the progeny of which can give rise to all or to the desired part of the intact organism. Zygote micro-injeaction [4] achieves transgenesis by transformation of the embryo's genome at the single cell or very early cleavage stage. As the germ line in mamrials is segregated from scanatic progenitor lineages at the early primitive-streak stage transformation of embryonic cells before this stage for instance by retroviral vector infection of cleavage embryos [5] may provide animals which are transgenic both sαmatically and in the germ line. Genetic transformation of cells after this stage will lead to either germ line or somatic genetic mosaicism. Where stem cells may be isolated from the organism these may be transformed and used to re-colonise their target tissue and the use of such techniques is exeπplified by haematopoetic stem cell manipulation e.g. [7]. This type of approach to somatic transgenesis is likely to be the only ethical route for human gene therapy and could well prove particularly useful for genetic modification of domestic livestock and when the stem cells may be maintained in tissue culture prior to their use to reconstitute their target tissue there is the advantage that selection for the desired transformants may preceed reconstitution. See for example Edwards' use of transient culture of πam ary epithelial stem cells [8], Cells isolated from an embryo before segregation of the germ line care able to provide a genetic vehicle for germ line transgenesis. Whereas embryonic stem cells have been isolated from mice [9] [10] and cells which seem likely to have such properties from hamster, [11] it is by no means apparent that cells of a similar type may be necessarily isolated from other non-rodent embryos. Moreover it is unlikely that the methods as described for mouse and utilised for hamster will be directly applicable to other embryos. Indeed the reported failure (notwithstanding the optimistic title) of some competent researchers in the field [12] to isolate sheep embryonic stem cells by a method based upon that used for mouse embryonic stem cells indicates this. Others have isolated cells but failed to maintain lines or demonstrate their pluripotentiality [15]. Past failures may have been due to the expectation that the cells would be fast-growing and resemble those of the mouse. It was indeed reported that malignant transformation was necessary in order to overcome the inherent quiescence of the embryonic disc [6] . There have also been numerous attempts which have been orally reported at various scientific meetings which have been unsuccessful. When the mouse embryoriic stan cells were first isolated virtually every expected property was predicted and the embryonic stage at which they might be found was clearly identified [13]. None of this background is available for putative ungulate embryonic stem cells. We have discovered that the methods which have been established and described for the isolation of e_τibryonic stem cells from mouse embryos .and successfully applied to hamster embryos are NOT applicable to ungulate embryos as exe plified by bovine and porcine embryos. In particular the most .Important step in embryonic stem cell isolation - identification and isolation of the stan cells from other cell types is quite differently based as is the necessary tissue-culture handling of the cells. Early Maiπnalian Development: Theoretical Consideration for the Isolation of Stem Cells from Ungulates In mammals, the pattern of embryonic development from fertilisation to implantation is broadly similar between species: fertilisation of the oocyte occurs in the oviduct, and the zygote is transported to the uterus whilst undergoing a series of mitotic divisions. At each division cell size decreases, and so the volume of the aribryo remains constant. A blastocyst is formed at a certain stage, when a cavity appears within the embryo. At this time the cells have differentiated into two types, the trophαblast and the inner cell mass, destined to became the fetal portion of the placenta and the fetus, respectively. However, by the time of iπplantation great differences are observed between species. In particular in the mouse, iπplantation into the uterine epithelium is an invasive and rapid process (14) . In ungulates, in particular in cattle, sheep and pigs, implantation occurs only after a considerable delay during which the trophectoderm proliferates rapidly and the inner cell mass forms a quiescent embryonic disc. In these cases implantation involves loose association between the fetal cells and the maternal cotyledons (14) . Thus early development at the tiine when stem cells may be isolated is very significantly different in many species including ungulates from the mouse. Procedures for the isolation of murine embryonic stem cell lines care now well established. Success in the isolation of embryonic stem cells fran the mouse depended on the recognition of the need for careful timing, so that ICM cells are committed to the ICM lineage and yet are ree from the influence of differentiated derivatives [9] . Although, by extrapolation, it can be argued that stem cells may be isolated similarly from the ungulates and other species, we anticipated that problems would arise in that exactly analogous stages do not exist in the embryos of mice and ungulates owing to difference in their development. We in erred that a different strategy will be required for the ungulates as, for example, the rate of development is much slower and the early embryonic ectoderm is present in a discoid arrangement and not as a solid mass as in the 5 day mouse embryo. These considerations led us to predict that the embryonic cells of the pre-implanting embryo, owing to the obligatory period of metabolic quiescence of the embryonic disc, would not be culturable with facility in vitro; and that stem cells, if isolated, would not necessarily resemble mouse €_mbryonic steam cells in morphology or growth characteristics. In these situations recognition of the stem cell types would be difficult. Nevertheless, we are able to define those conditions which are sufficient for the isolation of stem cells from ungulates, and for preventing cell differentiation sufficiently for cell lines to be established. It is likely that the cell type required and the means of its isolation may prove more general than that of embryonic steam cell isolation in the mouse as many other mammalian embryos - for example primate embryos - develop through an embryonic disc more similar to the structure found in ungulates than the epiblast and egg cylinder seen in the mouse and some other rodents. Method of Isolation and Culture of Embryonic Stem Cells from Ungulate Embryos 1. Tissue Culture Ifedium Dulbecco's modified EMEM culture medium supplimented with 5 to 10 percent of both foetal and new born calf serum, non-essential amino acids to the Eagles formula and 0.1 Millimolar 2-mercaptoethar_ol is used. Particular attention to selection of the sera is essential and unlike most sera used for mouse embryonic stem cell derivation, it was found that heat-inactivation of the serum at 56 degrees centigrade for 30 minutes was necessary. 2. Explantation of the Embryo Both species of embryo behave differently in culture from the mouse. In the case of the bovine embryos the procedure is as follows: Either fresh 6-day embryos flushed from in vivo or preferably embryos which have been grown in vitro from in vitro fertilised in vitro matured oocytes (supplied by Animal Biotechnology Cambridge Ltd. ) at 6-7 days of development - unhatched, fully expanded blastocysts - are grown from 1 or two extra days in tissue culture medium. They either hatch spontaneously or are freed from their zona pellucida mechanically and allowed to explant upon the bottom of a petri dish containing an STO fibroblast feeder layer as previously described [9]. Unlike the case with a mouse embryo the inner-cell-mass derived cells do not form a central egg cylinder. Their derivatives which may be isolated as the precursors of the embryonic stem cells are found on the periphery of the explant. These are isolated by careful trypsinisation using trypsin/EGTA/polyvinyl alcohol (0.25%:0.1ιrM:10 ug/ml respectively) and replated as below. For porcine embryos the procedure is: Embryos from the stage of hatching (6.5 days) to trophoblast expansion ( 11 days) are either explanted intact in which case most of the trophoblast layer dies, or preferably dissected to isolate the embryonic disc before this is explanted onto a fibroblast feeder layer typically inactivated STO fibroblasts. Primary outgrowths may be recognised to be the precursors of the embryonic stem cells and these are disaggregated and passaged. These primary stem cell outgrowths are different from the established cell lines and appeaar as more translucent and flatter tightly-packed epithelial colonies. 3. Feeder Cells and Growth Factors It is sufficient to use STO fibroblasts as feeder cells for either species. It is not necessary to use exogenous growth factors or conditioned media. 4. Recognition and Isolation of the Stem Cells Unlike those of the mouse and hamster, the ungulate embryonic stan cells do not form multilayered colonies but grow in distinctive flat polarised epithelial colonies which eventually spread to form monolayers. The cells are larger than those of the mouse, have large clear nuclei, several prominent nucleoli and relatively little cytoplasm. The cell size is found to vary from isolation to isolation and with growth conditions but the general morphology and appearance is distinctive. Other non-epithelial cell types may be observed and isolated but these do not have the diffeare tiative properties described. 5. Maintenance of Cells in Culture Cells are passaged 1 in 4-5 by trypsinisation onto fresh feeder cells at 3-4 day intervals or just before they attain confluency. Failure to passage prior to confluency results in the onset of spontaneous differentiation which if allowed to continue leads to loss of the cell line. Undifferentiated colonies may be able to be recovered after partial differentiation of the culture. The cells will grow without feeders but their ability to form e bryoid bodies becomes ccπpramised. 6. Verification of the Stan Cells These cells differentiate readily in culture either spontaneously or in response to morphogeans such as dimethyl sulphoxide or retinoic acid. The main differentiated derivatives are fibroblast, nerve endoderm and muscle which are representative of the three germ layers and verifies the pluripotentiality of the cells. Iforeover these cells will form tumours when transplanted beneath the kidney capsule of an irradiated nude mouse, these tumours being teratocarcinαmas showing a variety of differentiated cell types (fig. 7a and 7b). On aggregation in vitro anbryoid bodies are formed. These are distinctly different from those of the mouse by virtue of the fact that they show a polarisation akin to that seen in those formed by human teratocarcino a cells. Explanation of these anbryoid bodies into a tissue culture dish results in rapid and extensive differentiation. All these observations deamonstrate that the cultures described are indeed embryonic stem cells. The present invention can provide stem cells, and a general method as exemplified above for isolation of embryonic stem cells from all embryos in which development is via an embryonic disc in particular ungulate embryos (such as porcine embryos and bovine embryos) . The invention can provide for the derivation of such cells from embryos carrying a particular genetic background or specific utations. For example derivation of such cells from high-pedigree agricultural stock. It can also provide a method for preparation and use of such cells for differentiation and developmental studies in vitro, together with a method of use of such cells as a source of any other differentiated cell in vitro or in vivo. Furthearmore, the invention can provide for the use of such cells to repopulate an embryo of the same species thus giving rise to a chimaeric animal, particularly a chimaearic animal in which same or all of the germ cells are derived frcm the tissue-culture cells; for example a chimaeric animal in which sane or all of the germ cells are derived from the tissue-culture cells where the embryonic stan cells have been genetically modified or selected for genetic modification in culture. Stan cells according to the invention can be cultured either transiently or maintained as a cell line to provide nuclei for nuclear transfer into enucleated oocytes or other embryonic cells, e.g. -using cells with specific genetic properties either by virtue of their provenance from specific embryos or otherwise by specific genetic modification. The invention can allow development of embryos from cells which have received a necleus from an embryonic stem cell cultured in vitro. It can allow the use of stem cells genetically transformed in such a way as to introduce a novel protein production in a specific part (e.g. the mammary gland, the liver) of a subsequently derived dimaeric animal or the offspring of such a chimaeric animal, or a subsequently derived nuclear-cloned animal or the offspring of such an animal to provide nuclei for neclear transfer into enucleated oocytes or other embryonic cells. Steam cells of the inveantion may be used in techniques of genetic transformation and may be used in the creation of embryos to produce a genetically transfoπred living animal by embryo transfer. The scope of this invention eaxteands to cover not only the steam cells per se, but to embryos and genetically transformed animals derived therefrom. The invention also covers essentially non-biological methods for production of stem cells, embryos and animals. According to one aspect of the invention there is provided a bovine stem cell line, and a method for its production. This aspect of the inveantion also provides a cell culture system comprising bovine stem cells. Bovine stem cells are conveniently produced by growing bovine blastocysts (fertilisead in vivo or in vitro) in suitable tissue culture growth medium. One preferred medium consists of a mixture of 75 parts of Dulbecco's Modified Eagles Medium (EMEM) to 25 parts of Ham's F12M Medium, supplemented with non essential amino acids (Eagle's) (about 1% by volume), beta mercapto ethanol 10 _4 M, 10% new born calf serum and 10% foetal calf serum, both sera having beaen beat inactivatead by treatment at 56°C for 30 minutes before use. After hatching the blastocysts are desirably treated to cause disaggregation. This .is preferably effected by treating the blastocysts about 1 day after hatching by soaking for about 10 minutes in trypsin (0.25% Difco trypsin 1 in 250) supplemeanted with 10 ~4 molar EGTA (ethylene glycol tetracetic acid) and lOug/ml polyvinyl alcohol, followed by physical treatment to cause disaggregation, e.g. by sucking and blowing through a small pipette. Hence in a preferred aspect the present invention provides a method of obtaining bovine stem cells, involving the steps of growing bovine blastocysts in tissue culture growth meadium, and soaking the blastocysts about 1 day after hatching for about 10 minutes in trypsin suppleamented with EGIA and polyvinyl alcohol to cause disaggregation of the blastocysts. After disaggreagation, the disrupted blastocyst cells may be replated and grown in a furthear supply of the growth meadium together with inactivated STO cells, resulting in the growing Lip of colonies of cells of at leaast 2 different types. After a suitable incubation line colonies of steam cells can be selectead by morphology, as described below. The cells are grown up in the growth medium plus inactivated STO cells, with the cells passaged about once a week. After the 4th or 5th passage then cells can be subjeacted to further treatment of different types. For example, the cells can be frozen if required for storage. Alternatively the fresh cells can be introduced to a host blastocyst e.g. using conventional ir crαmanipulation techniques. Typically between 1 and 15 cells are introducead to a host blastocyst. The blastocyst can then be introduced to the uterus of a pseudopregnant foster mother where it may develop into a chimaeric animal. Prior to introduction to a host blastocyst, the cells can if desiread by manipulated in culture by known teachniques, e.g. by DNA transformation, targetted mutation by homologous recombination, or infection with retroviral vectors, so enabling modification of the resulting animals and enabling production of an animal having desiread characteristics. In a further aspect the present invention provides a method of obtaining bovine stem cells, comprising growing bovine blastocysts in tissue culture growth meadium; causing disaggregation of the blastocysts after hatching; growing the disaggregated cells in tissue culture growth medium; selecting stem cell colonies by morphological characteristics; and growing the selected stem cells in tissue culture growth medium. The invention also provides a blastocyst to which has beaen introduced one or more bovine stem cells of the inveantion, and the chimaeric progeny of such a blastocyst. The invention will be further describead, by way of illustration, in the following example and with reference to the accompanying figures. Figure 1 is a photograph of colonies of stem cells growing in cell culture; and Figure 2 is a photograph of a monolayer of bovine stan cells. Example Bovine embryos are treated by the following procedure: The embryos may be fertilisead in vivo, in which case they are obtained by flushing from a cow by known techniques at 5-6 days embryonic development. However, it is preferred to use embryos fertilisead in vitro and the following work was carriead out using fresh expanded bovine blastocysts derived by in vitro fertilisation and obtainead frαn Animal Biotechnology Cambridge limited, Cambridge, England. Similar material may also be obtained from other commercial sources. The blastocysts may also be obtained in frozean form, but it is preferred to use fresh blastocysts. The blastocysts are grown on a culture dish, in tissue culture growth meadium on a feaeadear layear of n tαmycin inactivatead STO cells. The medium used consists of a mixture of 75 parts of Dulbecco's Modified Eagles medium (DMEM) to 25 parts of Ham's F12M Medium, supplemented with non essential amino acis (Eagle's) (about 1% by volume), beta mercapto ethanol to 10 molar, 50 units/ml penicillin G, 10% new born calf serum and 10% foetal calf searum, both sera having beaen heat inactivated at 56°C for 30 minutes before use. The blastocysts are incubated at 37°C in a carbon dioxide gas humidified incubator in an atmosphere of about 5% carbon dioxide in air. The incubating blostocysts are periodically examined using a microscope, say twice daily, until hatching is observead. The timing of this varies with different embryos, but will typically be after about 2 days. The hatched blastocysts are then treated in one of two alternative ways. 1) In a first approach, about 1 day after hatching the blastocysts are treated by being soaked for about 10 minutes in trypsin (0.25% Difco trypsin 1 in 250) supplemented with 10 -4 molar EGIA (ethylene glycol tetracetic acid) and 10 ug/ml polyvinyl alcohol. This mixture acts to disaggregate the cells of the blastocysts while maintaining cell viability. Using a small pipette, with a tip about 50 to 100 microns in diameter the cells are then physically disaggregated by sucking and blowing, causing the cells to fall into clusters, breaking down the blastocyst structure. The disrupted blastocyst is immeadiately subjectead to further treatment, describead below. 2) In an alternative approach the hatched blastocysts are left in the tissue culture meadium and allowed to develop further. After about 3-4 days development it is observed that the inner cell mass of the blastocyst is found in clusters of roundead cells located around the edge of the explant. At this stage the trypsin mixture used in 1) .above is added to the culture dish and left for about 10 minutes. It is found that the cell clusters at the edge of the explant loosen more easily than others, and these cells are picked off using a small pipette with a tip about 20 to 30 microns in diameter, and transferred for further treatment. After treatment by method 1) or 2) the disrupted blastocyst cells are replated into a tissue culture dish surface treated with a gelatin solution. A further supply of the growth meadium described above is added, together with about 10 ~ ° inactivated STO cells, and the dish placed in an incubator at 37°C. The cells are periodically examinead by microscope, e.g. daily. Colonies of cells of at least 2 different types are observed growing up. After a suitable incubation time colonies believed to be stem cells are selectead by morphology: steam cells have the follcwing features:- a) They are round cells, tightly packed with large nuclei in relation to cytoplasm, and fairly prαmineant nucleoli. b) They grow in tightly adherent colonies. As the colonies get larger the cells tend to flatten out in the centre of the colony, with the colony having an outer rim of cells of the form described in a). c) On trypsinisation of such a colony using the trypsin mixture describead in 1) above it may be seen that the outer, less flattened cells of a larger colony or all the cells of a smaller colony without central flattening are radily disaggregated into small spherical cells which have a bright phase contrast appearance, and if αbservead after a short time of incubation at 37°C show lobular pseudopodia. Such colonies are illustrated in Figure 1. Suitable colonies, believed to be of stem cells, are. selected and transferred to another dish, to which is added a further supply of the growth meadium together with inactivatead STO cells. The dish is placed is an incubator at 37°C to allow colonies of the cells to grow up. The cells are passaged about once a week, i.e. subjected to the trypsin treatment as described in 1) above and replated onto a new dish. This is to stop differentiation. If the cells care to be stored they care forzen after the 4th or 5th passage. Otherwise, cells at this stage can be introduced to another, host, bovine blastocyst: typically between 1 and 15 cells are introduced to a blastocyst using conventional micromanipulation techniques. The blastocyst can then be introduced to the uterus of the pseudopregnant foster mother in known manner, or maintainead in an artificial environment, in the uterus develop into a chimaeric animal containing DNA from both the host blastocyst and the introduced cells. Chimaerism can be detectead in known ways, e.g. by use of genetic markers, or possibly simply by visual inspection. Prior to introduction to a host blastocyst, the cells can be manipulated in culture by known techniques, e.g. by DNA transformation or infection with retroviral vectors, so enabling modification of the resulting animals and enabling production of an animal having desired characteristics. According to another aspect of the inveantion there is provided a porcine stem cell line, and a method for its production. This aspect of the inveantion also provides a cell culture system comprising porcine stem cells. This aspect of the inveantion will now be further described, by way of illustration in the following example and with reference to the accompanying figures in which are: Fig. 3. Appearance of primary colony resulting from attachment of innear cell mass from 8 d blastocyst. The flattened, translucent colonies are arrowed: Morphologies of colonies resulting from disaggregated primary outgrowths of inner cell masses. Fig. 4a; colony of cells producing large, trophoblast-like cells, which care visible at the perimeter. This culture was derived from a 7 d blastocyst. Fig. 4b; and 4c; colonies of stem-like cells, which ∑are epithelial, adherent and have large nuclei and prominent nucleoli. The colony shown in Fig. 4b derived from a 7 d blastocyst, and that in 4c f αn an 8 d blasocyst. Fig. 5. Established porcine cell line showing morphological differentiation. Fig. 5a. Nest of un ifferentiated cells. Fig. 5b. Conflueant monolayear of cells. Fig. 5c. Confluesnt monolayer showing morphological differentiation into neuron-like cells. Fig. 6a. Aggregates formed by porcine cell line following culture for 7 d on a non-adhesive substratum. Fig. 6b to 6d. Outgrowths of cells from aggregates which were permittead to reattach to a substratum. Several differentiated cell types are visible; 6b epithelial, 6c muscle and fibroblastic and 6d nerve-like. Hatched blastocysts were recovered by retrograde uterine flushing from Large British White gilts at 7-9 days post oestrus. The animals were naturally mated and not superovulated. Either intact blastocysts or the inner cell masses manually dissectead from than were explanted onto STO fibroblast πύt_otically-__ractivated feeder cells in a manner similar to that described by Evans & Kaufman (1981) [9] for mouse blastocysts. The medium used was Dulbecco's odifiead Eagle's medium supplemented with 10% new born calf serum and 5% to 10% foetal calf serum, and 0.1 milliirolar 2-mercaptoethanol, and neither conditioned meadium nor exogenous growth factors were added. In order to stimulate organised differentiation, cells were disaggregated by trypsinisation and then seaeaded onto culture dishes which had been coated with a layer of 0.5% agarose as describead by Magrane (1982) [3] for stimulation of formations of anbryoid bodies by human teratocarcinαma cell cultures. To enhance cell differentiation both mercaptoethanol and foetal calf serum were omitted from the medium. Establishment of Cultures We have by these methods of cultures been able routinely to establish cultures from explanted porcine embryos. Although the success rate is variable it is often high with as many as 6 successful cultures being derived from 8 explanted blastocysts in one experiment. When blastocysts or embryonic discs are brought into culture, they attach within one day. The primary outgrowths consist of colonies of large flat, highly-translucent epithelial cells (Figure 3). These are clearly of very different appearance and culture morphology to murine EK cells. Portions of trophectoderm dissectead rom blastocysts between the ages of 7-10 days and cultured in the same way were unable to form colonies or outgrowths. The primary outgrowths were dissaggregated 7-14 days after explantation and passagead to fresh feeder layers. Pregressively growing colonies were formed which grew as a monolayer with very distinct colony boundaries. The cells are epithelioid with large clear nuclei containing 2-4 prominent nucleoli, and relatively sparse cytoplasm. Some differences in the appearance of these cells have been noticed between different isolates which is principally related to cell size. Figure 4 shows small colonies of large, undifferentiated cells which continuously produce cells with morphological characteristics of trophoblast giant cells. Such cultures have beaen maintained for 4 months in continuous culture. Cultures of this type have only beaean observed to arise from 7 day anbryos. Figure 4b shows a cell type which is more stable and able to grow in larger colonies. These cells are the most common isolate. Several cell lines of the type shown in figure 4b have been derived frcan both 7 and 9 day embryos. One cell line has been maintainead in continuous culture for more than one year with passaging 1 in 4 every 5-7 days without change of cell phenotype. It therefore appears to be immortal, diffeareantiation of these cells occurs spontaneously when the cells are pear itted to reaach high density (figure 5) . Overtly differentiated cells fail to reattach on passage leaading to regeneration of undifferentiated cultures. Figure 4c shows a colony of smaller cells which are a more rarely isolated form. All of these cell types grow more slowly than and differ in appearance to mouse embryonic steam cells. They show spontaneous differentiation in culture mainly into trophoblast-like cells or endoderm-like cells. Other differentiated (mainly fibroblastoid) cell types are also formed. Differentiation into Embryoid Bodies As a test of differentiation potential (Martin & Evans 1975) [16] cells from the cell line which had been maintained for 12 months were induced to form aggreagates by seeding onto a non-adhesive substratum. After several days an outer smooth layer of cuboidal epithelial cells appeared at one end of the aggregates and at the other pole there were more loosely attached cells of a more ragged appearance. Exteansive differentiation occurred when the anbryoid bodies were permitted to attach to the substratum, by replating onto tissue-culture dishes (Fig. 6b). Cells migrated and multiplied to form dense cultures, with several types visible, including epithelium, endoderm, muscle and neural cells. These differentiated cells are representative derivatives of all three embryonic germ layers, and suggest that the stem-cell-like culture represents a primary ectodermal lineaage of the pre-sαmite embryo. Discussion The cell lines isolated have an appearance considerably different although slightly reminiscent of marine embryonic steam cells. In appearance and form of growth they are more similar to some cell lines derived rom human testicular teratocarcinαmas. The form of development of their aggreagates when maintainead in suspension is very similar to that of a human teratocarcinαma cell line Hutt KEB (as describead in Magrane 1982) [3]. We tentatively conclude that these differentiating structures care indeaed homologous to the murine anbryoid bodies and this conclusion is strengthened by the observations resported here of a more extensive in vitro differentiation following their re-explanation onto a tissued-culture surface. One of us (MJE) has previously speculated that the assymetric form of the Hutt KEB erribryoid bodies reflects the developneant of the human embiryo via an eambryonic disc in contrast to the mouse egg cylinder. It is interesting to note here that in another speacies where early development is via an embryonic disc with a clearly epithelioid eambryonic epiblast, the isolated cells grow more as a monolayer than in the piling colonies typical of mouse EC and EK cells and differentiation of their eambryoid bodies is clearly assymetric. This distinctly different behaviour from that of mouse EK cells may be a general feature of those non-rodeant embryos where embryonic develop-tteant is via an embryonic disc. We conclude that pluripotent embryonic lineages may be derived from the pig and can be maintainead in culture. These are very different both in appearance, growth characteristics and beabaviour to those previously described in the mouse. The clear similarity between porcine cell lines and those seen from bovine embryos strongly supports the suggestion that this type of embryonic cell lineaage is the form of cell line which will be obtained from marπmalian species in genearal developing via an embryonic disc, examples of such species being e.g. ungulates. We are curreantly evaluating the potential of our embryonic cells, to determine (a) their relationship to normal eambryonic cells, and to determine their distinctive features compared to the murine EK lineaages, and (b) whether there exist restricted potency stem cell populations which may be transitory in nature, and (c) the origin and nature of those cells which are capable of expressing pluripotency under certain conditions. It is notable that the rate of proliferation of undifferentiated, stem-like cells in eaxplants of porcine embryos is slower compared with those derived from murine embryos. This may reflect another important difference in pre-implantational development in these species, that is the period of quiescence of the inner cell mass in ungulates up to the time of gastrulation. As these new cell lines may be considered homologous to murine EK cells they have the potential as a vector for genetic manipulation by their incorporation into a normal fertile pig via embryo chimaerism leading to their contribution to the germ cell line. References [1] M R Kuehn, A Bradley, E J Robertson and M J Evans. A potential animal model for Iasc±i-Nyhan syndrome through introduction of HPRT mutations into mice. NATURE 32£ 295 (1987) ' [2] M L Hooper, K Hardy, A Handyside, S Huntear and M Monk. HPRT-deficient (Lesc -Nyhan) mouse embryos derived from gemline colonisation by cultured cells. NATURE 326292 (1987) [3] Magrane G.G. (1982) A comparative study of Human and Mouse Teratocarcinαmas. PhD Thesis University of London. [4] Palmiter R D & Brinstear R L Germline transformation of mice. ANN.REV. GENETICS 20465 (1986) [5] Van der Putten H., Botteari F.M., Miller A.D., Rosenfield M.D., Fan H., Evans R.M. & Verma I.M. Efficient insertion of genes into the mouse germ line via retroviral vectors. Proc Natl Acad Sci 826148-6152 (1985) [6] McWhir J. Ph.D. thesis "Embryonic cell lines in farm animals". University of Calgary (1989) [7] Williams D.A. , Lemishka I.R. , Nathan D.G. & Mulligan R.C. Introduction of new genetic material into pluripotential haematopoetic stem cells of the mouse. NATURE θ 476-480 (1984) [8] Edwards P.A.W., Ward J.L. & Bradbury J.M. Alteration of morphogenesis by the v-myc oncogene in transplants of mammary gland. Oncogene 2 407-412 (1988) [9] Evans M J & Kaufman M H Establishment in culture of pluripotential cells from mouse embryos. NATURE 292 154-156 (1981) [10] Martin G.R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditionead by teratocarcinoma steam cells. Proc Natl Acad Sci 787634-7638 (1981) [11] Doetschman T., Williams P. & Maeda N. Establishment of hamster blastocyst-derived eambryonic ste (ES) cells. Dev. Biol 127 224-227 (1988) [12] Handyside A., Hooper M.L., Kaufman M.H. & Wilmut I. Towards the isolation of stem-cell lines from the sheep. Roux archive of developmeantal biology 196 185-190 (1987) [13] Evans M. Origin of mouse embryonal carcinoma cells and the possibility of their direct isolation into tissue culture. J. Reprod. Fert. 62 625-631 (1981) [14] Amoroso E.C. Placeantation. In "Marshall's Physiology of Reproduction", 3rd edn., Vol. 2. pp 127-311. Ed. A.S. Parkes, Longmans, Green & Co., London. [15] Pieadrahita J.A. , Anderson G.B., Martin G.R., Bon Durant R.H. Pashen R.L. Theriogenology 29286 (1988). [16] Martin G.R. & Evans M.J. (1975) Differentiation of clonal lines of teratocarcinαma cells: formation of embryoid bodies in vitro. Proc. Natl. Acad. Sci. USA 72, 1441-1445.

Claims: Claims 1. Pluripotential anbryonic stan cells isolated from in vitro treatment of non-human and non-rodent blastocysts. 2. Stan cells according to claim 1 from ungulate species. 3. Stem cells according to claim 1 from bovine speacies. 4. Steam cells according to claim 1 from porcine species. 5. A blastocyst to which has been __nt_roducead one or more stem cells according to cany of claims 1 to 4. 6. An embryonic cell to which has been introduced by nuclear transfer a nucleus of a stem cell according to any of claims 1 to 4. 7. A chimeric animal which is the progeny of a blastocyst according to claim 5 or an eambryonic cell according to claim 6. 8. A method of obtaining pluripotential embryonic stem cells according to any of claims 1 to 4, comprising growing blastocysts in tissue culture growth medium which includes both new born calf searum and foetal calf serum (both sera having beaen heat inactivated before use); causing disaggreagation of the blastocysts either after spontaneous hatching or after mechanical removal of the zona pellucida; growing the disaggregated cells in tissue culture growth medium; selecting stem cell colonies by morphological characteristics; and growing the selectead stem cells in tissue culture growth meadium; wherein the morphologically selected cells grow in distinctive flat polarised epithelial colonies which teand to spread to form monolayers and the cells are round, have relatively large clear nuclei, have prominent nucleoli and relatively little cytoplasm. 9. A method according to claim 8, further including passaging the selectead stem cells by trypsinisation onto fresh tissue culture growth medium at intervals to prevent differentiation of the cells and to maintain a cell line in culture. 10. A method which comprises introducing into a blastocyst one or more stem cells made according to the method of claim 8. 11. A method which comprises introducing by nuclear transfer into an embryonic cell a nucleus of a stem cell made according to the method of claim 8. 12. A method which comprises introduction to the uterus of a pseudo-pregnant foster mother animal, a viable embryo obtained using a technique involving any of the methods of claim 8 to 11, so as to produce progeny in the form of a chimeric animal.

Inventors: EVANS MARTIN JOHN, MOOR ROBERT MICHAEL, NOTARANNI ELENA, EVANS, MARTIN, JOHN, MOOR, ROBERT, MICHAEL, NOTARANNI, ELENA
Applicants: ANIMAL BIOTECH CAMBRIDGE LTD, INST ANIMAL PHYSIOLOGY GENET
Application year: 1989
Main classification: C12N5
IPC codes: A01K67, C12N5