WO1995016770A1 - Cellules souches embryonnaires derivees de cellules du stade preblastocyste d'ongule et leur utilisation pour produire des ongules chimeres et transgeniques clones - Google Patents

Cellules souches embryonnaires derivees de cellules du stade preblastocyste d'ongule et leur utilisation pour produire des ongules chimeres et transgeniques clones Download PDF

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WO1995016770A1
WO1995016770A1 PCT/US1994/014587 US9414587W WO9516770A1 WO 1995016770 A1 WO1995016770 A1 WO 1995016770A1 US 9414587 W US9414587 W US 9414587W WO 9516770 A1 WO9516770 A1 WO 9516770A1
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cells
cell
embryo
embryonic stem
preblastocyst
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PCT/US1994/014587
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Nick Strelchenko
Steven L. Stice
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Abs Global, Inc.
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Priority to AU14392/95A priority Critical patent/AU1439295A/en
Publication of WO1995016770A1 publication Critical patent/WO1995016770A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
    • C12N15/877—Techniques for producing new mammalian cloned embryos
    • C12N15/8771—Bovine embryos
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/235—Leukemia inhibitory factor [LIF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"

Definitions

  • the present invention is generally directed to embryonic stem cell cultures derived from ungulate preblastocyst stage ungulate embryos, preferably bovine preblastocyst stage embryos, methods for producing said embryonic stem cell cultures, methods of using the embryonic stem cells contained in said cultures for nuclear transplantation of recipient oocytes, methods of using the embryonic stem cells contained in said cultures to produce transgenic embryonic stem cells, methods of using said transgenic embryonic stem cells for nuclear transplantation in recipient oocytes for the production of transgenic embryos, methods of using the resultant transgenic embryos to produce transgenic ungulate animals, and transgenic embryonic stem cell cultures and transgenic ungulate animals produced using transgenic preblastocyst derived embryonic stem cell lines.
  • Embryo multiplication by nuclear transfer involves the transplantation of living nuclei from embryonic cells, or the whole embryonic cells themselves, into recipient cells, typically unfertilized eggs, followed by the fusion of the donor and recipient. Such transfers are typically made in order to increase the number of genetically identical embryos which can be obtained from elite genetic stock.
  • the earliest research relating to nuclear transfer of nuclei of embryonic cells into unfertilized eggs was performed in amphibians. Specifically, embryonic frog blastomere cells were separated and the nuclear material was reintroduced into frogs' eggs which had been enucleated. See "Transplantation of Living Nuclei from Blastula Cells into Enucleated Frog Egg", Briggs, R.
  • cleavage stage which simply means that the embryo comprises at least two cells
  • recipient oocytes which have been enucleated
  • recipient oocytes which have been enucleated
  • blastomeres isolated from pregastrulation embryos comprise the source of such donor nuclei.
  • other known sources of donor nuclei include embryonic stem cells.
  • oocytes are recovered from the ovaries or reproductive tracts of cows. These oocytes are then selected for a particular stage of development, and are enucleated by physical aspiration through a transfer pipette, resulting in an enucleated oocyte which still retains its external membranes and which functions as a recipient donor nucleus or as a donor embryonic cell.
  • a donor embryo at the proper stage of development typically at the cleavage or morula stage, is manipulated so that one or more cells or blastomeres are removed from the embryo.
  • the donor cell which, of course, contains its nucleus, is then inserted into the perivitelline space of a recipient oocyte.
  • An electronic pulse is than applied in order to fuse the membranes of the donor cell and the recipient oocyte, thus creating an activated, fused single cell embryo.
  • the resultant single cell nuclear transfer embryo is then cultured in vitro, or in the oviduct of a mammal, until it reaches a stage of development wherein it may successfully be implanted into a recipient cow.
  • This methodology results in a significant number of fused embryos which are viable, and which when transplanted surgically or non- surgically into the uteri of cows result in viable pregnancies and the production of genetically identical calves.
  • totipotent cells are cells which have not as yet significantly differentiated and thus may be used as donor nuclei or nucleated donor cells in nuclear transplantation.
  • ICM inner cell mass
  • blastocyst stage bovine embryos embryo of at least about 64 cells
  • totipotent embryonic cell lines which, under proper growth conditions, remain both totipotent and undifferentiated until these cell lines are induced to differentiate.
  • stem cell lines have been isolated from mice, and have putatively been obtained from bovine, ovine, hamster, mink and porcine blastocyst ICM cells. (See, e.g., Doetschman et al., "Establishment of hamster blastocyst-derived embryonic stem cells", Devel. Biol.
  • embryonic stem cells for the production of cloned animals which are genetically identical. This, of course, is of great importance, especially in the agricultural industry for the production of identical animals having specific desirable traits, e.g., enhanced milk production or increased size.
  • embryonic stem cell cultures are also important application of embryonic stem cell cultures. Such transgenic cells lines may be used for .in vitro research, or may be used as nuclear or cell donors for nuclear transfer to obtain transgenic embryos which, in turn, may be used for implantation into maternal recipients to produce transgenic animals.
  • Transgenic animals have tremendous research and commercial potential. For example, animals may be produced which contain disease causing genes and used as animal models for drug research. Also, transgenic animals may be produced which contain genes providing for desirable traits, e.g., growth hormone genes.
  • the production of transgenic animals, and in particular transgenic ungulates has been reported in the literature. For example, Simons et al., Bio/Technology. 6, 179-183, 1988 reports the production of transgenic sheep by a microinjection technique. Also, Rexroad et al. , Mol. Reprod. Dev.. 1, 164-169, 1989 reports the integration of growth- regulating genes in sheep by microinjection of pronuclei.
  • Theriogenology. 33(4), 819-828, 1990 report the microinjection of very early stage embryos (one and two cell containing bovine ova) to create transgenic cattle. Further, Wilmut et al., Theriogenology. 33(1), 113-123, 1990 report gene transfer in order to modify milk production by microinjection of bovine pronuclei with foreign DNA. The reference also discusses retroviral and stem cell transfer to blastocysts, and the use of sperm containing foreign DNA to fertilize eggs.
  • a significantly improved method to produce transgenic nonhuman animals entails microinjection of embryonic cells with desired genetic material, e.g., a homologous or heterologous DNA, and then use of the injected cells as nuclear donors for enucleated eggs.
  • desired genetic material e.g., a homologous or heterologous DNA
  • the injected cell and egg are fused, cultured to a requisite stage of development, and then implanted in a maternal animal for the production of genetically transformed animals.
  • This method has been reported to provide for much greater efficiencies than pronuclear injection. Hill et al., Theriogenolog . 37, 222, 1992, and Bondioli et al., "Transgenic Animals", First, N. and Haseltine F. (eds.), Butterworth- Heinemann, Stoncham, MA., pp. 265-273, 1991.
  • It is another object of the invention to provide a method for producing in vitro cultures containing totipotent ungulate embryonic stem cells comprising; (i) removing the zona pellucida from a preblastocyst stage ungulate embryo; (ii) disaggregating the cells of said preblastocyst stage embryo; and (iii) culturing the resultant disaggregated blastomeres in a cell culture medium under culturing conditions which prevent differentiation until embryonic stem cell colonies are obtained.
  • -li ⁇ lt is another object of the invention to provide a method for the continuous production of in vitro cultures containing totipotent ungulate embryonic stem cells comprising; (i) removing the zona pellucida from a preblastocyst stage ungulate embryo; (ii) disaggregating the cells of said preblastocyst stage embryo; (iii) culturing the resultant disaggregated blastomeres in a cell culture medium under culturing conditions which prevent differentiation until stem cell colonies arise; and (iv) passaging the individual embryonic stem cells onto a new culture medium which prevents differentiation and provides for the growth of said embryonic stem cells and the production of embryonic stem cell colonies.
  • It is another object of the invention to provide a novel method for nuclear transplantation in ungulates comprising introducing into a recipient ungulate oocyte a totipotent embryonic stem cell derived from an embryo cell of an ungulate preblastocyst stage embryo. It is a more specific object of the invention to provide a novel method for nuclear transplantation in bovines comprising introducing into a recipient bovine oocyte a totipotent embryonic stem cell derived from a embryo cell of a bovine preblastocyst stage embryo.
  • transgenic bovine embryonic stem cells comprising transfecting totipotent embryonic stem cells derived from a bovine preblastocyst-stage embryo cell with a desired polynucleotide.
  • It is another object of the invention to provide a novel method for producing transgenic ungulate animals comprising: (i) introducing into a recipient ungulate oocyte a totipotent transgenic embryonic stem cell derived from an embryo cell of an ungulate preblastocyst-stage embryo; (ii) fusing the membranes of the donor cell and the recipient oocyte; (iii) culturing the resultant fused embryo either jLn vitro or in vivo until it develops to such extent that it can be implanted into a maternal recipient ungulate; and, (iv) implanting the cultured embryo into a recipient ungulate.
  • It is a more specific object of the invention to provide a novel method for producing transgenic bovines comprising: (i) introducing into a recipient bovine oocyte a totipotent transgenic bovine embryonic stem cell derived from a preblastocyst stage bovine embryo; (ii) fusing the membranes of the donor cell and the recipient oocyte; (iii) culturing the resultant bovine embryo either in in vitro or in vivo until it develops to such extent that it may be implanted into a recipient bovine maternal recipient; and (iv) implanting the transgenic bovine embryo into a maternal bovine recipient.
  • It is another object of the invention to provide for the production of an ungulate chimeric embryo comprising introducing one or more embryonic stem cells derived from an ungulate preblastocyst stage embryo cell, which embryonic stem cells may or may not comprise a heterologous DNA, into an early stage embryo, so as to produce a chimeric embryo which expresses the genotype of the embryonic stem cells in some or all of its cells.
  • Cleavage embryo refers to an embryo having at least two cells which may be disaggregated into individual cells.
  • Preblastocyst stage embryo will refer to any ungulate embryo having less than or equal to about 64 cells, which has not as yet differentiated, and does not contain an inner cell mass and trophectoderm.
  • Blastocyst stage embryo refer to an embryo having at least about 64 cells which comprises a discernable inner cell mass and trophectoderm.
  • Embryonic stem cells refers to cells derived from preblastocyst stage embryos which lack the zona pellucida and which when cultured under appropriate conditions do not differentiate until they are induced to differentiate.
  • Trophoblast cells refers to specialized cells in the embryo which have flattened out and surround the blastocoel. These cells ultimately become the placental membranes.
  • Blastocoel refer to a fluid filled cavity formed during the development of the trophoblast cells which varies in size during embryo development.
  • Inner Cell Mass refers to the cells which are compact and form the inner cell mass of the blastocyst which ultimately become the fetus.
  • Vitelline Membrane refers to the membrane which surrounds the cytoplasm of the individual cells.
  • Perivitelline Space is the space located between the vitelline membrane and the zona pellucida.
  • Zona pellucida refer to the protective covering of the embryo which provides for the aggregation of early embryonic cells.
  • Zygote refers to the one cell embryo which is surrounded by a smooth vitelline membrane.
  • Late Morula refers to the embryo stage having approximately 32 cells. The cells are adhered together to provide a compact cell mass. Late Morula refers to the embryo, at about 64 cells into embryo development. The cells at that stage have adhered into a tightly compacted mass, having a distinguishable scalloped edge that appears on the outside of the cell mass and exhibits a dappled cytoplasmic appearance.
  • Feeder Cells refers to any cells which prevent the differentiation of blastomeres and enable the formation of stem cells colonies.
  • these cells will comprise fibroblast cells.
  • Blastomeres refers to cells obtained from preblastocyst embryos which are cultured for the production of embryonic stem cell colonies.
  • Figure 1 illustrates eight- to sixteen-cell-stage blastomeres (cells) which have just been placed underneath a mouse fibroblast feeder layer.
  • Figure 2 illustrates eight-to sixteen-cell-stage blastomeres (cells) 24 hours after they had been placed underneath a mouse fibroblast feeder layer.
  • the Figure also illustrates the much larger cytoplasmic volume in comparison with the nuclear volume of the blastomeres.
  • Figure 3 illustrates an initial ES-cell colony derived from a preblastocyst-stage embryo and demonstrates that the cells of the colony are considerably smaller in size than the blastomeres shown in Figures 1 and 2.
  • Figure 4 illustrates a blastomere that degenerated and was unsuitable for producing an ES cell line.
  • Figure 5 illustrates endoderm-like cells which are generated from blastomeres and undesirable for ES- cell-line production.
  • Figure 6 illustrates embryonic stem cells which were produced from preblastocyst-stage embryos.
  • the Figure demonstrates that the ES cells grow as a monolayer on top of the feeder layer and are epithelial-like in appearance.
  • the Figure demonstrates further that, unlike in blastomeres, most of ES cell volume is nuclear instead of cytoplasmic and there are nucleoli in ES cells.
  • the present invention is directed to a culture of ungulate embryonic stem (ES) cells derived from preblastocyst stage embryo cells.
  • ES embryonic stem
  • these cells will be obtained from bovine preblastocyst stage embryos.
  • suitable ungulates include, e.g., cows, sheep, deer, pigs, horses, goats, antelope, etc.
  • the cells which are to be used as progenitors of the subject ES cells lines will comprise cells derived from an ungulate embryo, e.g., a bovine embryo, that has not as yet reached the blastocyst developmental stage. At this stage, all of the embryo cells are totipotent. Therefore, the invention embraces embryos up to about the early to late morula stage which immediately precedes the blastocyst stage. Ungulate embryos as a group differentiate at a similar rate. Generally, the blastocyst stage of development occurs at about 64 cells into embryo development. The late morula stage, which is the stage which precedes the blastocyst stage and comprises about 64 cells. The subject invention accordingly embraces ES cells derived from any preblastocyst embryo of less than or equal to about 64 cells.
  • an ungulate embryo e.g., a bovine embryo
  • all of the embryo cells are totipotent. Therefore, the invention embraces embryos up to about the early to late morula stage which immediately precede
  • a blastocyst stage embryo has undergone significant differentiation.
  • it comprises an inner cell mass (ICM) of differentiated trophectoderm cells surrounding a liquid-filled blastocyst.
  • the trophectoderm cells have differentiated to such extent that they are not totipotent. Consequently, such cells are not a suitable source of ES cells.
  • the ICM cells (which eventually give rise to the fetus) are totipotent and may be used to produce ES cells.
  • blastocyst stage embryos are disadvantageous sources of ES cells since they comprise both differentiated and non-differentiated (totipotent) cells which necessitates the separation of these cells (specifically separation of ICM from the trophectoderm) and the dissociation of the ICM prior to culturing which steps have proven to be relatively difficult.
  • preblastocyst stage embryos only comprise totipotent cells; therefore, there is no need to separate totipotent from differentiated cells.
  • the present invention provides an improvement over previous methods for generating ES cell lines since there is no risk that the ES cultures will be contaminated by differentiated cells from the trophectoderm, which cells may not be used to produce ES cells.
  • Preblastocyst cells e.g., bovine preblastocyst cells are not only functionally distinct from blastocyst cells (in that they are all totipotent) ; they are morphologically distinct as well.
  • preblastocyst embryo cells are easier to dissociate than blastocyst (ICM) cells.
  • ICM blastocyst
  • Cells that have the above-noted characteristics may be isolated from any ungulate preblastocyst stage embryo, including, but not limited to, naturally occurring in vivo fertilized cultured embryos, embryos derived from an earlier nuclear transfer procedure, or transgenic embryos derived from an earlier transfection procedure.
  • bovine embryos The developmental rate of bovine embryos, has been well characterized, particularly with respect to size, shape, and generalized appearance. See, for example, Dorn, C.G. and D.C. raemer, Bovine Embryo Grading. 1982, Texas, A. & M. University, College of Veterinary Medicine, Department of Physiology and Pharmacology, College Station, Texas.
  • ES efficiency which is defined as the number of ES cell lines established relative to the number of embryos used to produce ES cell lines, appears to vary depending upon the number of cells contained in the preblastocyst embryo used as a source of blastomeres.
  • embryos having 8-16 cells provided for greater ES efficiencies than did preblastocyst embryos having 17-64 cells. Sixteen cell stage embryos appear to provide for the best ES efficiencies.
  • the present inventors also attempted to produce ES cell lines from preblastocyst cleavage stage embryos having only four cells, but have been unsuccessful to date. It is theorized that the development of 4-cell cleavage embryos may still be under the control of maternal message. By contrast, it is theorized that 8-cell cleavage embryos, especially late 8-cell cleavage embryos, have developed to a sufficient extent that they develop into ES cell lines autonomously, i.e., in the absence of maternal message. Consequently, it may be possible to obtain ES cells from 4-cell cleavage embryos (and even 2-cell or 1-cell embryos) if the culture medium is supplemented with appropriate maternal factors, e.g.
  • the subject medium contains glucose which increases ES cell efficiency but may inhibit 4-cell embryos from developing to such extent that ES cells may be obtained.
  • the preblastocyst stage embryo cells used as a progenitor for ES cells will comprise zygotes which have undergone from about 3 to 6 cell divisions. This corresponds to embryos whose cell number ranges from about 8 to 64 cells. More preferably, preblastocyst embryos which have undergone about 3 to 5 cell divisions will be utilized, and most preferably zygotes which have undergone 4 cell divisions and comprise about 16 cells will be utilized.
  • ES cell line from a preblastocyst stage embryo
  • the zona pellucida will be removed by treating the preblastocyst stage embryo with pronase at a concentration of about 1 mg/ml for a time ranging from about 1 to 30 minutes. After the zona pellucida is removed, it is preferable, but not essential to the invention, that the embryo then be placed on a culture medium which produces for reduction of the compact structure of the embryo. This facilitates the later disaggregation of the embryo cells.
  • any culture medium which provides for the maintained viability of the embryo and which facilitates reduction of compaction may be used.
  • this culture medium will be MEM- ⁇ . containing cytochalasin B at 7.5 mg/ml. Ca ++ or Mg ++ free medium may also reduce compaction of the embryo.
  • pretreatments are not essential since the embryo may simply be mechanically disaggregated without previous culturing using well known micromanipulation techniques.
  • Disaggregation of the preblastocyst stage embryo cells will preferably be effected mechanically by repeatedly passing the embryo through a fine bore pipette.
  • This technique which results in the eventual production of blastomeres, is described in Bondioli et al., "Production of Identical Bovine Offspring by Nucleus Transfer", Theriogenology 33(1), 165-174, 1990.
  • treatment of the embryo with a proteolytic enzyme such as trypsin may enhance disaggregation.
  • proteolytic enzymes may adversely affect the properties of the resultant blastomeres.
  • the cells After disaggregation is completed (which gives rise to individual cells referred to as blastomeres) , the cells will then be placed in a culture medium which prevents the differentiation of the blastomeres and gives rise to ES cell lines and ES cell colonies.
  • the blastomeres will be co-cultured with a feeder cell layer.
  • This feeder cell layer will comprise any cell layer which provides for the growth of blastomeres and the production of ES cell lines and colonies, but which prevents the differentiation of the cultured blastomeres until later induction. Suitable feeder cell layers include, e.g., fibroblasts (either primary fibroblasts or fibroblast cell lines) and buffalo rat liver cells.
  • the feeder cell layer will comprise fibroblasts.
  • the type of fibroblasts used does not appear to be essential to the invention.
  • any species fibroblast may be used, these cells may be immortal or normal, and primary cultures or cell lines may be used.
  • STO cells cells of an immortal embryonic murine fibroblast cell line which is derived from SIM embryonic fibroblasts and is 6-thioguanine and ouabain resistant
  • primary mouse fibroblast feeder cell layers primary mouse fibroblast feeder cell layers
  • bovine fibroblast cells have all been proved to be suitable for generating ES cell lines and colonies.
  • a feeder cell layer e.g., a fibroblast feeder layer
  • LIF leukemia inhibitor factor
  • feeder layers which likewise produce differentiating inhibiting factors.
  • the selection of other suitable feeder cells may be effected by one having ordinary skill in the art.
  • Cell layers which provide for the production of ES cell lines and ES colonies may be identified by routine screening, using the methodology described herein, to select for other cell layers which facilitate ES cell line deve1opment.
  • the blastomeres may be cultured in a cell culture medium which contains factors which inhibit differentiation and which enables the production of ES cell lines and ES cell colonies.
  • the blastomeres may be cultured in a LIF containing culture medium, or any other factor which prevents the differentiation of blastomeres.
  • the individual blastomeres will be placed in contact with the feeder layer, most preferably fibroblasts.
  • Such feeder cell layers may be produced according to well known methods.
  • mouse fibroblast feeder layers may be prepared as follows. Mouse fetuses are obtained at 10-20 days of gestation. The head, liver, heart, and alimentary tract are then removed and the remaining tissue washed in phosphate buffered saline and incubated at 37°C in a solution of 0.05% trypsin - 0.02% EDTA. The mouse cells are then placed in tissue culture flasks containing a culture medium that provides for the support of the feeder layer and the blastomeres.
  • a suitable medium comprises a modified Eagle's Medium containing non-essential amino acids (alanine, asparagine, aspartic acid, glutamic acid, glycine, proline and serine) , ribonucleoside and 2'- deoxyribonucleosides (hereinafter, MEM-c supplemented with 100 IU/ml penicillin, 50 /xg/ml streptomycin, 10% fetal calf serum (FCS) and 0.1 mM ⁇ -mercaptoethanol.
  • MEM-c modified Eagle's Medium containing non-essential amino acids (alanine, asparagine, aspartic acid, glutamic acid, glycine, proline and serine)
  • MEM-c ribonucleoside and 2'- deoxyribonucleosides
  • FCS fetal calf serum
  • the invention is not limited to the use of this specific culture medium.
  • one or more of these moieties may be non-essential to the growth of the blastomeres and generation of ES cells.
  • the amount of FCS may be reduced to about 5% without detrimental effects to growth of ES cells.
  • ⁇ -mercaptoethanol may be essential to the culture medium.
  • the cells are preferably treated with mitomycin C, e.g., at a concentration of about 10 mg/ml for about three hours.
  • Mitomycin C treatment inhibits DNA synthesis, and therefore inhibits cell division of the fibroblasts, yet still provides for the cell layer to support the growth of co-cultured blastomeres.
  • a suitable feeder cell layer e.g., a mouse fibroblast feeder cell layer, or a cell culture medium which provides for the prevention of blastomere differentiation and the formation of ES cell colonies
  • the blastomeres will be cultured for a sufficient time to provide for the formation of embryonic stem cell colonies.
  • this will be effected by placing the preblastocyst derived blastomeres in contact with a fibroblast feeder layer.
  • a fibroblast feeder layer As discussed supra. it has been found that by providing significant cell-to-cell contact between the blastomeres and feeder layer, it is easier to generate ES cell lines and to prevent the differentiation of the blastomeres. This is theorized to be attributable to membrane associated differentiating inhibiting factors produced by fibroblasts which apparently prevent the differentiation of blastomeres as they develop into ES cell lines. Interestingly, blastomeres do not appear to go through an ICM stage as they multiply into ES cells. This may be another result of the cell-to-cell contact. In the absence of such contact, the preblastocyst derived blastomeres differentiate into trophoblast vesicles.
  • blastomeres be placed underneath the feeder layer.
  • feeder cell layer e.g., fibroblasts
  • the blastomeres be placed underneath the feeder layer.
  • the culture medium will be replaced as necessary, typically every two to three days. After the blastomeres have been cultured for a requisite period of time, generally on the order of seven to ten days after initiation of culturing, the cells will then be passaged. However, this will vary dependent upon the particular feeder cell layer, the orientation of cells on the cell layer, the stage of the preblastocyst blastomeres, the composition of the culture medium, among other factors.
  • the determination as to when passage should be effected will be determined on the basis of when the cells start to differentiate and exhibit an embryoid- like appearance.
  • the cells will then be passaged to another feeder cell layer or a culture medium which prevents differentiation and provides for the growth of ES cells.
  • passage will be effected without chemicals or proteases such as trypsin which may be traumatic to the ES cells.
  • trypsin may denature ES protein and cell receptors.
  • Mechanical means are the preferred means for effecting passage. For instance, a fine glass needle may be used to cut an ES cell colony from the feeder layer into smaller cell clusters. These clusters may be further broken down by repeated pipetting. Because of the apparently non-degradative nature of this method, the cells may be passaged at higher dilutions such as
  • the subject ES cells may be passaged indefinitely using the described methodology, thus providing for an essentially unlimited number of ES cells, which, at this stage, may be considered to be a embryonic stem cell line.
  • the present inventors currently have an actively growing ES cell line produced according to the present invention which has been passaged for about 13 months.
  • the preblastocyst cells used to produce the subject ES cell lines are functionally and morphologically distinct from blastocyst cells.
  • the ES cell lines produced using preblastocyst progenitors are morphologically different from blastocyst-derived ES cells.
  • preblastocyst derived ES cells are larger, and typically contain more cytoplasm and more lipid vesicles than blastocyst derived ES cells.
  • the doubling time of the ES cells of the present invention ranges from about 24 to 28 hours.
  • the ungulate ES cells produced according to the invention may be used as donor cells in nuclear transfer protocol such as described in United States Patents 4,994,384 and 5,057,420 to Prather et al. and Massey respectively, or may be used in any other known method of nuclear transfer.
  • an oocyte typically at metaphase II, is enucleated.
  • a membrane-bound nucleus typically in the form of a donor cell, is then inserted into the enucleated oocyte.
  • the two cells are then fused, e.g. by electrofusion, PEG fusion, or viral-induced fusion to provide a single-cell nuclear transfer embryo.
  • the nucleus transfer embryo is then cultured in vitro or in vivo to the morula or blastocyst stage according to known methods, at which time it will be implanted into a maternal recipient where it will give rise to a full term fetus.
  • the ES cells of the invention will preferably be disaggregated to provide single cells. This may be effected by chemical or mechanical means, however, mechanical means is again preferred.
  • a pronase treatment at about 0.1 to 5.0 mg/ml may also be effected to facilitate breaking down the outer layers of the ES cells.
  • the individual cells may be incorporated directly as donors into recipient oocytes which have previously been enucleated.
  • the production of donor ES cells and suitable recipient enucleated oocytes may be synchronized before fusion, according to known methods.
  • the donor and recipient will be fused according to known methods such as are identified supra.
  • the resultant fused nuclear transfer embryos will then be cultured according to known methods to such stage that these embryos may be successfully implanted into maternal recipients, typically to the morula or blastocyst stage.
  • Suitable methods and culture medium for culturing embryos in vitro and in vivo are known to the art, and may be utilized to culture the subject ungulate embryos preferably bovine embryos.
  • a preferred post-fusion culture medium is exemplified in United States Patent No. 5,096,822 by Rosenkratz, Jr. et al.
  • a preferred post-fusion culture method will comprise culturing the nucleus transfer embryos for about three to four days in CR1 medium supplemented with amino acids (CRlaa) and 3 mg/ml bovine serum albumin (BSA) followed by culturing in CRlaa plus 10% fetal calf serum for about three to four days.
  • CRlaa medium, variations thereof, and use of these medium to culture post- fusion embryos is disclosed in U.S. patent No. 5,096,822. However, this medium is only exemplary, many other media and protocols for culturing post- fusion embryos are known and should be suitable for use in the present invention.
  • the embryos which develop to the blastocyst or morula stage will be transferred to recipient maternal ungulates, e.g., bovines which will give rise to identical ungulate fetuses.
  • recipient maternal ungulates e.g., bovines which will give rise to identical ungulate fetuses.
  • in vivo culture may be effected short term in a sheep oviduct prior to implantation as an alternative in vitro culturing.
  • the subject ES stem cells will also provide materials which may be used for the production of transgenic or genetically altered ES cells, which in turn may be used to produce transgenic or genetically altered ungulates, e.g., bovines.
  • Methods for introduction of polynucleotides, i.e., desired DNA and/or RNA's into cells in culture are well known in the art. Such methods include, e.g., electroporation, retroviral vector infection, particle acceleration, transfection, and microinjection.
  • the cells which contain the desired polynucleotide, e.g., a desired gene, which may be homologous or heterologous to the host cell will be selected according to known methods.
  • the cells may be co-transfected with a marker gene which provides for selection.
  • a culture of transgenic ES cells is obtained which contains a desired polynucleotide integrated in its genome, the individual cells may be used as nuclear transfer donors.
  • the transgenic ES cell will facilitate the production of many identified animals having an identical genetic modification, e.g., a desired gene integration. These animals will provide a means of in vivo study of the effects of the particular gene.
  • animals may be obtained which express a disease causing gene and may be used to assay potential drug therapies for treatment of the particular disease condition.
  • the transfection of ES cells in vitro with a desired polynucleotide will permit most of the genetic modification steps to be performed in vitro. This avoids the need to perform repeated breeding to introduce a desired gene into a particular genetic background. Also, one is able to characterize the donor cell genome in vitro (e.g., by use of suitable probes) that is impossible when the donor is a primary embryo cell. Consequently, this procedure may be accomplished faster than previous breeding methods. Also since one has a high expectation that the offspring will express the desired gene, the need to screen animals is substantially reduced. Therefore, the invention should significantly reduce the cost of animal breeding, and the production of transgenic animals, particularly among larger domesticated animals, which have relatively prolonged gestation periods and which bear only one or a few offspring per pregnancy.
  • preblastocyst embryos may themselves be used as a source of blastomeres and the production of ES cells. Also, these genetically altered ES cells may be subjected to further genetic modifications.
  • preblastocyst- derived ES cells are in the production of chimeric animals.
  • an ES cell derived from preblastocyst stage embryo cells may be genetically altered, as desired, before it is used to repopulate an early stage embryo. After culture in vitro and/or in vivo, such repopulated embryos will give rise at some frequency to animals which express in some or all of their cells, the genetic material of the inserted genetically altered ES cells used for repopulation.
  • Preblastocyst stage bovine embryos were obtained from in vivo fertilizations and in vitro fertilizations. These embryos ranged in size from the 8-cell stage to the 64-cell stage, and were selected such that they were between 2 and 5 days from time of fertilization. These embryos were then treated with 1 mg/ml of pronase to remove the zona pellucida. Some of these embryos were then placed in phosphate buffered saline (PBS) medium containing 7.5 ⁇ g/ml of cytochalasin B to reduce the compactness of the embryo structure and therefore facilitate the disaggregation of cells. However, some embryos were disaggregated without a cytochalasin B pretreatment.
  • PBS phosphate buffered saline
  • mice fibroblast feeder cell layer prepared according to the method described infra.
  • the feeder cell layer was prepared from mouse fetuses at ten to twenty days of gestation.
  • the head, liver, heart and alimentary tract were removed from the fetuses and the remaining tissue was washed and incubated at 37°C in 0.05% trypsin-0.02% EDTA.
  • Loose cells were then cultured in tissue culture flasks containing MEM- ⁇ supplemented with penicillin, streptomycin, 10% fetal calf serum and 0.1 mM ⁇ mercaptoethanol.
  • the feeder cell cultures were established over a two- to three-week culture period at 37°C, 5% C0 2 and at 100% humidity. These cells were then treated with mitomycin C at 10 ⁇ g/ml prior to their usage as feeder cells.
  • the mitomycin C-pretreated fibroblast layer was then used as a feeder cell layer for the blastomeres.
  • the individual blastomeres were placed on top of the feeder cell layer.
  • ES cell lines were more readily established, and differentiation was better inhibited, when the blastomeres were placed beneath the feeder layer.
  • this enhanced cell-to-cell contact provides for the blastomeres to be more in contact with membrane associated differentiating inhibiting factors such as LIF.
  • Cells placed on top of the feeder layer had lesser cell-to-cell contact between the blastomeres and feeder cells.
  • the blastomeres occasionally differentiated into trophoblast vesicles.
  • the MEM- ⁇ plus 10% FCS growth medium was replaced. After the cells had been cultured for a total of about seven to ten days, embryonic stem cell monolayers were obtained. Also, around this time into the culturing, the blastomeres (which now had become ES cell lines) start to differentiate and exhibit an embryoid-like appearance. Accordingly, the cells are passaged at this time onto new feeder layers. This was effected mechanically using a fine glass needle to cut the ES cell monolayer into smaller cell clusters. These cell clusters were then repeatedly pipetted at a 1:100 dilution onto fresh fibroblast feeder layers.
  • This method has resulted in the generation of numerous ES cell lines from preblastocyst-derived embryos from the 8- to 64-cell stage, and has provided for both male and female ES cell lines.
  • Table 1 The ES cell data obtained by the above-described method is presented in Table 1 below. This Table also contains data relating to the use of the subject ES cells as nuclear transfer donors. This use is discussed in Example 2 below.
  • ES cell lines obtained according to the present invention have also been used as nuclear donors in the nuclear transfer protocols taught by
  • NT embryos that had developed to the blastocyst stage were transferred to recipient females and pregnancies were established.
  • NT embryos fused embryos
  • 11/483 developed to the blastocyst stage and 2/11 of the implanted blastocysts resulted in pregnancy. This translates roughly into a 2% blastocyst success rate and a 20% pregnancy success rate.
  • Figures 1-6 of this application illustrate 8- to 16-cell stage blastomeres which have been placed underneath a mouse fibroblast feeder layer.
  • Figure 2 in particular illustrates the large differential between nuclear and cytoplasmic volume of the blastomeres.
  • Figure 3 illustrates an initial ES cell colony derived from a preblastocyst-stage embryo. The illustration demonstrates that the ES cells of the colony are much smaller than the blastomeres illustrated in Figures 1 and 2.
  • Figure 4 illustrates a degenerated blastomere which did not result in an ES cell line.
  • Figure 5 illustrates endoderm-like cells generated from blastomeres. These cells are undesirable for ES cell line production.
  • Figure 6 illustrates ES cells produced from preblastocyst-stage embryos and demonstrates that the ES cells grow as a monolayer which is epithelial in appearance. Also, unlike in blastomeres, the majority of the cell volume of the
  • ES cells is nuclear rather than cytoplasmic and the
  • ES cells comprise nucleoli. It is believed that these figures provide further evidence as to the advantages obtained by the present invention.

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Abstract

Lignées de cellules souches embryonnaires totipotentes d'ongulés, par exemple d'origine bovine, dérivées de cellules précurseurs du stade préblastocyste. Ces lignées cellulaires sont utilisées pour cloner des ongulés, pour produire des lignées cellulaires transgéniques, et pour produire des ongulés transgéniques et chimères.
PCT/US1994/014587 1993-12-17 1994-12-16 Cellules souches embryonnaires derivees de cellules du stade preblastocyste d'ongule et leur utilisation pour produire des ongules chimeres et transgeniques clones WO1995016770A1 (fr)

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WO1996007732A1 (fr) * 1994-09-05 1996-03-14 Roslin Institute (Edinburgh) Cellules totipotentes pour un transfert nucleaire
WO1997047734A1 (fr) * 1996-06-14 1997-12-18 The Regents Of The University Of California Differenciation et culture in vitro de cellules souches multipotentes de primates et usages therapeutiques
WO1998030683A2 (fr) * 1997-01-10 1998-07-16 University Of Massachusetts, A Public Institution Of Higher Education Of The Commonwealth Of Massachusetts Transfert nucleaire au moyen de cellules donneuses foetales et adultes differentiees
WO1998039416A1 (fr) * 1997-03-06 1998-09-11 Infigen, Inc. Methode de clonage d'animaux
EP0930009A1 (fr) * 1995-08-31 1999-07-21 Roslin Institute (Edinburgh) Population de cellules quiescentes pour transfert nucléaire
US5942435A (en) * 1993-05-14 1999-08-24 The Board Of Trustees Of The University Of Illinois Transgenic swine compositions and methods
WO1999046982A1 (fr) * 1998-03-16 1999-09-23 Relag Pty Ltd Transfert de noyau de cellules porcines
US6235969B1 (en) * 1997-01-10 2001-05-22 University Of Massachusetts Cloning pigs using donor nuclei from non-quiescent differentiated cells
US6331659B1 (en) 1998-01-21 2001-12-18 University Of Hawaii Cumulus cells as nuclear donors
US6395958B1 (en) 1997-03-06 2002-05-28 Infigen, Inc. Method of producing a polypeptide in an ungulate
US6753457B2 (en) 1993-02-03 2004-06-22 Tranxenogen Nuclear reprogramming using cytoplasmic extract
US6906238B2 (en) 2000-03-15 2005-06-14 University Of Georgia Research Foundation, Inc. Effective nuclear reprogramming in mammals using CDK2 inhibitors
WO2006036164A1 (fr) * 2004-09-27 2006-04-06 Reproductive Genetics Institute Cellules souches embryonnaires derivees de morula
EP2479256A1 (fr) * 2004-11-04 2012-07-25 Advanced Cell Technology, Inc. Dérivation de cellules de souche embryonnaire
TWI392736B (zh) * 2006-06-23 2013-04-11 Academia Sinica 由單一分裂球取得多能幹細胞之方法
US8742200B2 (en) 2004-11-04 2014-06-03 Advanced Cell Technology, Inc. Derivation of embryonic stem cells and embryo-derived cells
US8796021B2 (en) 2007-02-23 2014-08-05 Advanced Cell Technology, Inc. Blastomere culture to produce mammalian embryonic stem cells
US9617515B2 (en) 2006-02-27 2017-04-11 Moraga Biotechnology Corporation Non-embryonic totipotent blastomere-like stem cells and methods therefor
WO2019140260A1 (fr) * 2018-01-12 2019-07-18 The Regents Of The University Of California Dérivation efficace de cellules souches embryonnaires pluripotentes stables de bovins

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Cited By (33)

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Publication number Priority date Publication date Assignee Title
US6753457B2 (en) 1993-02-03 2004-06-22 Tranxenogen Nuclear reprogramming using cytoplasmic extract
US5942435A (en) * 1993-05-14 1999-08-24 The Board Of Trustees Of The University Of Illinois Transgenic swine compositions and methods
US7071373B1 (en) 1993-05-14 2006-07-04 The Board Of Trustees Of The University Of Illinois Transgenic ungulate compositions and methods
WO1996007732A1 (fr) * 1994-09-05 1996-03-14 Roslin Institute (Edinburgh) Cellules totipotentes pour un transfert nucleaire
EP0930009A1 (fr) * 1995-08-31 1999-07-21 Roslin Institute (Edinburgh) Population de cellules quiescentes pour transfert nucléaire
WO1997047734A1 (fr) * 1996-06-14 1997-12-18 The Regents Of The University Of California Differenciation et culture in vitro de cellules souches multipotentes de primates et usages therapeutiques
US6235969B1 (en) * 1997-01-10 2001-05-22 University Of Massachusetts Cloning pigs using donor nuclei from non-quiescent differentiated cells
US5945577A (en) * 1997-01-10 1999-08-31 University Of Massachusetts As Represented By Its Amherst Campus Cloning using donor nuclei from proliferating somatic cells
EP1808484A1 (fr) * 1997-01-10 2007-07-18 University of Massachusetts, a Public Institution of Higher Education of The Commonwealth of Massachusetts, Transfert nucléaire avec cellules donatrices adultes et fýtales différenciées
US6235970B1 (en) * 1997-01-10 2001-05-22 University Of Massachusetts, Amherst Campus CICM cells and non-human mammalian embryos prepared by nuclear transfer of a proliferating differentiated cell or its nucleus
CN1248288B (zh) * 1997-01-10 2011-11-09 马萨诸塞大学，马萨诸塞州高等教育公立研究所 用分化胎儿和成年供体细胞进行的核转移
WO1998030683A2 (fr) * 1997-01-10 1998-07-16 University Of Massachusetts, A Public Institution Of Higher Education Of The Commonwealth Of Massachusetts Transfert nucleaire au moyen de cellules donneuses foetales et adultes differentiees
WO1998030683A3 (fr) * 1997-01-10 1998-09-17 Univ Massachusetts Transfert nucleaire au moyen de cellules donneuses foetales et adultes differentiees
US6603059B1 (en) 1997-03-06 2003-08-05 Infigen, Inc. Method of cloning animals
US6395958B1 (en) 1997-03-06 2002-05-28 Infigen, Inc. Method of producing a polypeptide in an ungulate
WO1998039416A1 (fr) * 1997-03-06 1998-09-11 Infigen, Inc. Methode de clonage d'animaux
US6331659B1 (en) 1998-01-21 2001-12-18 University Of Hawaii Cumulus cells as nuclear donors
WO1999046982A1 (fr) * 1998-03-16 1999-09-23 Relag Pty Ltd Transfert de noyau de cellules porcines
US6906238B2 (en) 2000-03-15 2005-06-14 University Of Georgia Research Foundation, Inc. Effective nuclear reprogramming in mammals using CDK2 inhibitors
US7547818B2 (en) 2000-03-15 2009-06-16 The University Of Georgia Research Foundation, Inc. Metaphase donor cells for effective nuclear reprogramming in mammals
WO2006036164A1 (fr) * 2004-09-27 2006-04-06 Reproductive Genetics Institute Cellules souches embryonnaires derivees de morula
EP2479256A1 (fr) * 2004-11-04 2012-07-25 Advanced Cell Technology, Inc. Dérivation de cellules de souche embryonnaire
US8642328B2 (en) 2004-11-04 2014-02-04 Advanced Cell Technology, Inc. Derivation of embryonic stem cells
US8742200B2 (en) 2004-11-04 2014-06-03 Advanced Cell Technology, Inc. Derivation of embryonic stem cells and embryo-derived cells
EP2960328A1 (fr) * 2004-11-04 2015-12-30 Ocata Therapeutics, Inc. Dérivation de cellules souches embryonnaires
US9550974B2 (en) 2004-11-04 2017-01-24 Astellas Institute For Regenerative Medicine Derivation of embryonic stem cells
US9617512B2 (en) 2004-11-04 2017-04-11 Astellas Institute For Regenerative Medicine Derivation of embryonic stem cells and embryo-derived cells
US10072243B2 (en) 2004-11-04 2018-09-11 Astellas Institute For Regenerative Medicine Derivation of embryonic stem cells and embryo-derived cells
US9617515B2 (en) 2006-02-27 2017-04-11 Moraga Biotechnology Corporation Non-embryonic totipotent blastomere-like stem cells and methods therefor
TWI392736B (zh) * 2006-06-23 2013-04-11 Academia Sinica 由單一分裂球取得多能幹細胞之方法
US8796021B2 (en) 2007-02-23 2014-08-05 Advanced Cell Technology, Inc. Blastomere culture to produce mammalian embryonic stem cells
US10584313B2 (en) 2007-02-23 2020-03-10 Astellas Institute For Regenerative Medicine Method of producing a differentiated mammalian cell comprising culturing a single mammalian blastomere
WO2019140260A1 (fr) * 2018-01-12 2019-07-18 The Regents Of The University Of California Dérivation efficace de cellules souches embryonnaires pluripotentes stables de bovins

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