Classifications

• • 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
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2217/00—Genetically modified animals
  • A01K2217/07—Animals genetically altered by homologous recombination
  • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
• • 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
  • C12N2510/00—Genetically modified cells
• • 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
  • C12N2517/00—Cells related to new breeds of animals
  • C12N2517/04—Cells produced using nuclear transfer

Definitions

• the present invention generally relates to the production of embryonic or stem-like cells by transplantation of cell nuclei derived from animal or human cells into enucleated animal oocytes of a species different from the donor nuclei.
• the present invention more specifically relates to the production of primate or human embryonic or stem- like cells by transplantation of the nucleus of a primate or human cell into an enucleated animal oocyte, e.g., a primate or ungulate oocyte and in a preferred embodiment a bovine enucleated oocyte.
• the present invention further relates to the use of the resultant embryonic or stem-like cells, preferably primate or human embryonic or stem-like cells for therapy, for diagnostic applications, for the production of differentiated cells which may also be used for therapy or diagnosis, and for the production of transgenic embryonic or transgenic differentiated cells, cell lines, tissues and organs.
• the embryonic or stem-like cells obtained according to the present invention may themselves be used as nuclear donors in nuclear transplantation or nuclear transfer methods for the production of chimeras or clons, preferably transgenic cloned or chimeric animals.
• ES cells can be passaged in an undifferentiated state, provided that a feeder layer of fibroblast cells (Evans et al., Id.) or a differentiation inhibiting source (Smith et al., Dev. Biol., 121 :1-9 (1987)) is present.
• ES cells have been previously reported to possess numerous applications. For example, it has been reported that ES cells can be used as an in vitro model for differentiation, especially for the study of genes which are involved in the regulation of early development.
• ES cells can give rise to germline chimeras when introduced into preimplantation mouse embryos, thus demon- strating their pluripotency (Bradley et al, Nature, 309:255-256 (1984)). In view of their ability to transfer their genome to the next generation, ES cells have potential utility for germline manipulation of livestock animals by using ES cells with or without a desired genetic modification. Moreover, in the case of livestock animals, e.g., ungulates, nuclei from like preimplantation livestock embryos support the development of enucleated oocytes to term (Smith et al., Biol. Reprod., 40: 1027-1035 (1989); and Keefer et al, Biol. Reprod., 50:935-939 (1994)).
• ES cells from livestock animals are highly desirable because they may provide a potential source of totipotent donor nuclei, genetically manipulated or otherwise, for nuclear transfer procedures.
• mice embryonic fibroblast feeder layers without the use of conditioned medium. These cells reportedly differentiate into several different cell types during culture (Gerfen et al., Id.). Further, Saito et al, Roux's Arch. Dev. Biol., 201 : 134-141 (1992) report bovine embryonic stem cell-like cell lines cultured which survived passages for three, but were lost after the fourth passage. Still further, Handyside et al., Roux's Arch. Dev. Biol., 196:185-190 (1987) disclose culturing of immunosurgi- cally isolated inner cell masses of sheep embryos under conditions which allow for the isolation of mouse ES cell lines derived from mouse ICMs.
• Van Stekelenburg et al reported, however, that their cell lines resembled epithelial cells more than pluripotent ICM cells. (Id.) Still further, Smith et al., WO 94/24274, published October 27, 1994, Evans et al, WO 90/03432, published April 5, 1990, and Wheeler et al, WO 94/26889, published November 24, 1994, report the isolation, selection and propagation of animal stem cells which purportedly may be used to obtain transgenic animals. Also, Evans et al., WO 90/03432, published on April 5, 1990, reported the derivation of purportedly pluripotent embryonic stem cells derived from porcine and bovine species which assertedly are useful for the production of transgenic animals.
• primate embryonic stem cells that are (i) capable of proliferation in an in vitro culture for over one year; (ii) maintain a karyotype in which all chromosomes characteristic of the primate species are present and not noticeably altered through prolonged culture; (iii) maintains the potential to differentiate into derivatives of endoderm, mesoderm and ectoderm tissues throughout culture; and (iv) will not differentiate when cultured on a fibroblast feeder layer.
• These cells were reportedly negative for the SSEA- 1 marker, positive for the SEA-3 marker, positive for the SSEA-4 marker, express alkaline phosphatase activity, are pluripotent, and have karyotypes which include the presence of all the chromosomes characteristic of the primate species and in which none of the chromosomes are altered. Further, these cells are respectfully positive for the TRA-1-60, and TRA-1-81 markers.
• the cells purportedly differentiate into endoderm, mesoderm and ectoderm cells when injected into a SCID mouse. Also, purported embryonic stem cell lines derived from human or primate blastocytes are discussed in Thomson et al., Science 282: 1145-1147 and Proc. Natl. Acad.
• a different approach to treating Parkinson's which promises to have broad applicability to treatment of many brain diseases and central nervous system injury, involves transplantation of cells or tissues from fetal or neonatal animals into the adult brain. Fetal neurons from a variety of brain regions can be incorporated into the adult brain. Such grafts have been shown to alleviate experimentally induced behavioral deficits, includ- ing complex cognitive functions, in laboratory animals. Initial test results from human clinical trials have also been promising. However, supplies of human fetal cells or tissue obtained from miscarriages is very limited. Moreover, obtaining cells or tissues from aborted fetuses is highly controversial. There is currently no available procedure for producing "fetal-like" cells from the patient.
• allograft tissue is not readily available and both allo- graft and xenograft tissue are subject to graft rejection.
• many cells or tissues wherein such modification would be desirable do not divide well in culture and most types of genetic transformation require rapidly dividing cells.
• OBJECTS OF THE INVENTION It is an obj ect of the invention to provide novel and improved methods for producing embryonic or stem-like cells.
• It is another object of the invention to provide a novel method for producing lineage-defective non-human primate or human embryonic or stem- like cells which involves transplantation of the nucleus of a non-human primate or human cell, e.g., a human adult cell into an enucleated non-human primate or human oocyte, wherein such cell has been genetically engineered to be incapable of differentiation into a specific cell lineage or has been modified such that the cells are "mortal", and thereby do not give rise to a viable offspring, e.g., by engineering expression of anti-sense or ribozyme telomerase gene.
• a DNA construct that provides for the expression of genes that inhibit apoptosis, e.g., Bcl-2 or Bcl-2 family members and/or by the expression of antisense ribozymes specific to genes that induce apoptosis during early embryonic development.
• a detectable marker e.g., one that encodes a visualizable (e.g., fluorescent tag) marker protein.
• Such therapies include by way of example treatment of diseases and injuries including Parkinson's, Huntington's, Alzheimer's, ALS, spinal cord injuries, multiple sclerosis, muscular dystrophy, diabetes, liver diseases, heart disease, cartilage replacement, burns, vascular diseases, urinary tract diseases, as well as for the treatment of immune defects, bone marrow trans- plantation, cancer, among other diseases.
• transgenic or genetically engineered embryonic or stem-like cells produced according to the invention for gene therapy, in particular for the treatment and/or prevention of the diseases and injuries identified, supra. It is another object of the invention to use the embryonic or stem-like cells produced according to the invention or transgenic or genetically engineered embryonic or stem-like cells produced according to the invention as nuclear donors for nuclear transplantation. It is still another object of the invention to use genetically engineered ES cells produced according to the invention for the production of transgenic animals, e.g., non-human primates, rodents, ungulates, etc.
• FIGURES are photographs of a nuclear transfer (NT) unit produced by transfer of an adult human cell into an enucleated bovine oocyte.
• Figures 2 to 5 are photographs of embryonic stem-like cells derived from a NT unit such as is depicted in Figure 1.
• the present invention provides a novel method for producing embryonic or stem-like cells, and more specifically non-human primate or human embryonic or stem-like cells by nuclear transfer or nuclear transplantation.
• nuclear transfer or nuclear transplantation or NT are used interchangeably .
• the isolation of actual embryonic or stem-like cells by nuclear transfer or nuclear transplantation has never been reported. Rather, previous reported isolation of ES-like cells has been from fertilized embryos.
• successful nuclear transfer involving cells or DNA of genetically dissimilar species, or more specifically adult cells or DNA of one species (e.g., human) and oocytes of another non-related species has never been reported.
• human embryonic or stem-like cells and cell colonies may be obtained by transplantation of the nucleus of a human cell, e.g., an adult differentiated human cell, into an enucleated animal oocyte, which is used to produce nuclear transfer (NT) units, the cells of which upon culturing give rise to human embryonic or stem-like cells and cell colonies.
• a human cell e.g., an adult differentiated human cell
• NT nuclear transfer
• NT units used to produce ES-like cells will be cultured to a size of at least 2 to 400 cells, preferably 4 to 128 cells, and most preferably to a size of at least about 50 cells.
• embryonic or stem- like cells refer to cells produced according to the present invention.
• the present application refers to such cells as stem-like cells rather than stem cells because of the manner in which they are typically produced, i.e., by cross-species nuclear transfer. While these cells are expected to possess similar differentiation capacity as normal stem cells they may possess some insignificant differences because of the manner they are produced. For example, these stem-like cells may possess the mitochondria of the oocytes used for nuclear transfer, and thus not behave identically to conventional embryonic stem cells.
• the present discovery was made based on the observation that nuclear transplantation of the nucleus of an adult human cell, specifically a human epithelial cell obtained from the oral cavity of a human donor, when transferred into an enucleated bovine oocyte, resulted in the formation of nuclear transfer units, the cells of which upon culturing gave rise to human stem-like or embry- onic cells and human embryonic or stem-like cell colonies.
• This result has recently been reproduced by transplantation of keratinocytes from an adult human into an enucleated bovine oocyte with the successful production of a blastocyst and ES cell line.
• bovine oocytes and human oocytes and likely mammals in general must undergo maturation processes during embryonic development which are sufficiently similar or conserved so as to permit the bovine oocyte to function as an effective substitute or surrogate for a human oocyte.
• oocytes in general comprise factors, likely proteinaceous or nucleic acid in nature, that induce embryonic development under appropriate conditions, and these functions that are the same or very similar in different species. These factors may comprise material RNAs and/or telomerase.
• human cell nuclei can be effectively transplanted into bovine oocytes, it is reasonable to expect that human cells may be transplanted into oocytes of other non-related species, e.g., other ungulates as well as other animals.
• other ungulate oocytes should be suitable, e.g. pigs, sheep, horses, goats, etc.
• oocytes from other sources should be suitable, e.g. oocytes derived from other primates, amphibians, rodents, rabbits, guinea pigs, etc.
• the present invention involves the transplantation of an animal or human cell nucleus or animal or human cell into the enucleated oocyte of an animal species different from the donor nuclei, by injection or fusion, to produce an NT unit containing cells which may be used to obtain embryonic or stem-like cells and/or cell cultures.
• the invention may involve the transplantation of an ungulate cell nucleus or ungulate cell into an enucleated oocyte of another species, e.g., another ungulate or non- ungulate, by injection or fusion, which cells and/or nuclei are combined to produce NT units and which are cultured under conditions suitable to obtain multicellular NT units, preferably comprising at least about 2 to 400 cells, more preferably 4 to 128 cells, and most preferably at least about 50 cells.
• the cells of such NT units may be used to produce embryonic or stem-like cells or cell colonies upon culturing.
• the preferred embodiment of the invention comprises the production of non-human primate or human embryonic or stem-like cells by transplantation of the nucleus of a donor human cell or a human cell into an enucleated human, primate, or non-primate animal oocyte, e.g., an ungulate oocyte, and in a preferred embodiment a bovine enucleated oocyte.
• the embryonic or stem-like cells will be produced by a nuclear transfer process comprising the following steps: (i) obtaining desired human or animal cells to be used as a source of donor nuclei (which may be genetically altered); (ii) obtaining oocytes from a suitable source, e.g.
• Nuclear transfer techniques or nuclear transplantation techniques are known in the literature and are described in many of the references cited in the Background of the Invention.
• Human or animal cells may be obtained and cultured by well known methods.
• Human and animal cells useful in the present invention include, by way of example, epithelial, neural cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), other immune cells, erythrocytes, macrophages, melanocytes, monocytes, mononuclear cells, fibroblasts, cardiac muscle cells, and other muscle cells, etc.
• the human cells used for nuclear transfer may be obtained from different organs, e.g., skin, lung, pancreas, liver, stomach, intestine, heart, reproductive organs, bladder, kidney, urethra and other urinary organs, etc.
• suitable donor cells i.e., cells useful in the subject invention, may be obtained from any cell or organ of the body. This includes all somatic or germ cells.
• the donor cells or nucleus would comprise actively dividing, i.e., non-quiescent, cells as this has been reported to enhance cloning efficacy. Also preferably, such donor cells will be in the Gl cell cycle.
• the resultant blastocytes may be used to obtain embryonic stem cell lines according to the culturing methods reported by Thomson et al., Science 282: 1145-1147 (1998) and Thomson et al, Proc. Natl. Acad. Sci, USA 92:7544- 7848 (1995), incorporated by reference in their entirety herein.
• the cells used as donors for nuclear transfer were epithelial cells derived from the oral cavity of a human donor and adult human keratinocytes.
• the disclosed method is applicable to other human cells or nuclei.
• the cell nuclei may be obtained from both human somatic and germ cells. It is also possible to arrest donor cells at mitosis before nuclear transfer, using a suitable technique known in the art.
• 5-azacytidine can be used to reduce the level of DNA methylation in cells, potentially leading to increased access of transcription factors to DNA regulatory sequences. Accordingly, donor cells may be exposed to 5-azacytidine (5-Aza) previous to fusion, or 5-Aza may be added to the culture medium from the 8 cell stage to blastocyst. Alternatively, other known methods for effecting DNA demethylation may be used.
• the oocytes used for nuclear transfer may be obtained from animals including mammals and amphibians. Suitable mammalian sources for oocytes include sheep, bovines, ovines, pigs, horses, rabbits, goats, guinea pigs, mice, hamsters, rats, primates, humans, etc.
• the oocytes will be obtained from primates or ungulates, e.g., a bovine. Methods for isolation of oocytes are well known in the art. Essentially, this will comprise isolating oocytes from the ovaries or reproductive tract of a mammal or amphibian, e.g., a bovine. A readily available source of bovine oocytes is slaughterhouse materials. For the successful use of techniques such as genetic engineering, nuclear transfer and cloning, oocytes must generally be matured in vitro before these cells may be used as recipient cells for nuclear transfer, and before they can be fertilized by the sperm cell to develop into an embryo.
• This process generally requires collecting immature (prophase I) oocytes from animal ovaries, e.g., bovine ovaries obtained at a slaughterhouse and maturing the oocytes in a maturation medium prior to fertilization or enucleation until the oocyte attains the metaphase II stage, which in the case of bovine oocytes generally occurs about 18-24 hours post-aspiration. For purposes of the present invention, this period of time is known as the "maturation period.” As used herein for calculation of time periods, “aspiration” refers to aspiration of the immature oocyte from ovarian follicles. Additionally, metaphase II stage oocytes, which have been matured in vivo have been successfully used in nuclear transfer techniques.
• mature metaphase II oocytes are collected surgically from either non-superovu- lated or superovulated cows or heifers 35 to 48 hours past the onset of estrus or past the injection of human chorionic gonadotropin (hCG) or similar hormone.
• hCG human chorionic gonadotropin
• the metaphase II stage oocyte as the recipient oocyte because at this stage it is believed that the oocyte can be or is sufficiently "activated" to treat the introduced nucleus as it does a fertilizing sperm.
• the oocyte activation period generally ranges from about 16-52 hours, preferably about 28-42 hours post- aspiration.
• immature oocytes may be washed in HEPES buffered hamster embryo culture medium (HECM) as described in Seshagine et al, Biol.
• HECM HEPES buffered hamster embryo culture medium
• TCM tissue culture medium
• FSH follicle stimulating hormone
• metaphase II oocytes are either placed in HECM, optionally containing 7.5 micrograms per milliliter cytochalasin B, for immediate enucleation, or may be placed in a suitable medium, for example CRlaa, plus 10% estrus cow serum, and then enucleated later, preferably not more than 24 hours later, and more preferably 16-18 hours later. Enucleation may be accomplished microsurgically using a micropipette to remove the polar body and the adjacent cytoplasm. The oocytes may then be screened to identify those of which have been successfully enucleated.
• This screening may be effected by staining the oocytes with 1 microgram per milliliter 33342 Hoechst dye in HECM, and then viewing the oocytes under ultraviolet irradiation for less than 10 seconds.
• the oocytes that have been successfully enucleated can then be placed in a suitable culture medium.
• the recipient oocytes will preferably be enucleated at a time ranging from about 10 hours to about 40 hours after the initiation of in vitro maturation, more preferably from about 16 hours to about 24 hours after initiation of in vitro maturation, and most preferably about 16-18 hours after initiation of in vitro maturation.
• a single animal or human cell or nucleus derived therefrom which is typically heterologous to the enucleated oocyte will then be transferred into the perivitelline space of the enucleated oocyte used to produce the NT unit.
• the animal or human cell or nucleus and the enucleated oocyte will be used to produce NT units according to methods known in the art.
• the cells may be fused by electrofusion. Electrofusion is accomplished by providing a pulse of electricity that is sufficient to cause a transient break down of the plasma membrane. This breakdown of the plasma membrane is very short because the membrane reforms rapidly.
• the human or animal cell and oocyte are electrofused in a 500 ⁇ m chamber by application of an electrical pulse of 90- 120V for about 15 ⁇ sec, about 24 hours after initiation of oocyte maturation. After fusion, the resultant fused NT units are then placed in a suitable medium until activation, e.g., one identified infra.
• the NT unit may be activated by known methods. Such methods include, e.g., culturing the NT unit at sub-physiological temperature, in essence by applying a cold, or actually cool temperature shock to the NT unit. This may be most conveniently done by culturing the NT unit at room temperature, which is cold relative to the physiological temperature conditions to which embryos are normally exposed. Alternatively, activation may be achieved by application of known activation agents. For example, penetration of oocytes by sperm during fertilization has been shown to activate prefusion oocytes to yield greater numbers of viable pregnancies and multiple genetically identical calves after nuclear transfer.
• oocyte activation methods are the subject of U.S. Patent No. 5,496,720, to Susko-Parrish et al, which is herein incorporated by reference.
• oocyte activation may be effected by simultaneously or sequentially: (i) increasing levels of divalent cations in the oocyte, and (ii) reducing phosphorylation of cellular proteins in the oocyte.
• divalent cations into the oocyte cytoplasm, e.g., magnesium, strontium, barium or calcium, e.g., in the form of an ionophore.
• divalent cations include the use of electric shock, treatment with ethanol and treatment with caged chelators.
• Phosphorylation may be reduced by known methods, e.g., by the addition of kinase inhibitors, e.g., serine-threonine kinase inhibitors, such as 6-dimethyl- amino-purine, staurosporine, 2-aminopurine, and sphingosine.
• phosphorylation of cellular proteins may be inhibited by introduction of a phosphatase into the oocyte, e.g., phosphatase 2A and phosphatase 2B.
• Activated NT units may be cultured in a suitable in vitro culture medium until the generation of embryonic or stem-like cells and cell colonies. Culture media suitable for culturing and maturation of embryos are well known in the art.
• Examples of known media which may be used for bovine embryo culture and maintenance, include Ham's F-10 + 10% fetal calf serum (FCS), Tissue Culture Medium- 199 (TCM-199) + 10% fetal calf serum, Tyrodes-Albumin-Lactate- Pyruvate (TALP), Dulbecco's Phosphate Buffered Saline (PBS), Eagle's and Whitten's media.
• FCS fetal calf serum
• TCM-199 Tissue Culture Medium- 199
• TCM-199 Tissue Culture Medium- 199
• TCM-199 Tissue Culture Medium- 199
• TCM-199 Tissue Culture Medium- 199
• TCM-199 Tissue Culture Medium- 199
• PBS Dulbecco's Phosphate Buffered Saline
• Eagle's and Whitten's media One of the most common media used for the collection and maturation of oocytes.
• TCM-199 TCM-199
• serum supplement including fetal
• a preferred maintenance medium includes TCM-199 with Earl salts, 10% fetal calf serum, 0.2 MM Ma pyruvate and 50 ⁇ g/ml gentamicin sulphate. Any of the above may also involve co-culture with a variety of cell types such as granulosa cells, oviduct cells, BRL cells and uterine cells and STO cells.
• human epithelial cells of the endometrium secrete leukemia inhibitory factor (LIF) during the preimplantation and implantation period. Therefore, the addition of LIF to the culture medium could be of importance in enhancing the in vitro development of the reconstructed embryos.
• LIF leukemia inhibitory factor
• Patent 5,712,156 which is herein incorporated by reference.
• Another maintenance medium is described in U.S. Patent 5,096,822 to Rosenkrans, Jr. et al, which is incorporated herein by reference.
• This embryo medium contains the nutritional substances necessary to support an embryo.
• CR1 contains hemicalcium L-lactate in amounts ranging from 1.0 mM to 10 mM, preferably 1.0 mM to 5.0 mM.
• Hemicalcium L-lactate is L-lactate with a hemicalcium salt incorporated thereon.
• suitable culture medium for maintaining human embryonic cells in culture as discussed in Thomson et al. Science 282: 1145- 1147 (1998) and Proc. Natl. Acad.
• the cultured NT unit or units are preferably washed and then placed in a suitable media, e.g., CRIaa medium, Ham's F-10, Tissue Culture Media - 199 (TCM- 199).
• a suitable media e.g., CRIaa medium, Ham's F-10, Tissue Culture Media - 199 (TCM- 199).
• Tyrodes-Albumin-Lactate-Pyrovate (TALP) Dulbecco's Phosphate Buffered Saline (PBS), Eagle's or Whitten's, preferably containing about 10% FCS.
• PBS Phosphate Buffered Saline
• Whitten's preferably containing about 10% FCS.
• Such culturing will preferably be effected in well plates which contain a suitable confluent feeder layer.
• Suitable feeder layers include, by way of example, fibroblasts and epithelial cells, e.g., fibroblasts and uterine epithelial cells derived from ungulates, chicken fibroblasts, murine (e.g., mouse or rat) fibroblasts, STO and SI-m220 feeder cell lines, and BRL cells.
• the feeder cells will comprise mouse embryonic fibroblasts. Means for preparation of a suitable fibroblast feeder layer are described in the example which follows and is well within the skill of the ordinary artisan.
• the NT units are cultured on the feeder layer until the NT units reach a size suitable for obtaining cells which may be used to produce embryonic stem- like cells or cell colonies.
• these NT units will be cultured until they reach a size of at least about 2 to 400 cells, more preferably about 4 to 128 cells, and most preferably at least about 50 cells.
• the culturing will be effected under suitable conditions, i.e., about 38.5 °C and 5% C0 2 , with the culture medium changed in order to optimize growth typically about every 2-5 days, preferably about every 3 days.
• suitable conditions i.e., about 38.5 °C and 5% C0 2
• the culture medium changed in order to optimize growth typically about every 2-5 days, preferably about every 3 days.
• sufficient cells to produce an ES cell colony typically on the order of about 50 cells, will be obtained about 12 days after initiation of oocyte activation.
• culture medium known to be useful for maintaining human cells in tissue culture.
• Examples of a culture media suitable for human embryo culture include the medium reported in Jones et al, Human Reprod., 13(1): 169- 177 ( 1998), the P 1 -catalog #99242 medium, and the P- 1 catalog #99292 medium, both available from Irvine Scientific, Santa Ana, California, and those used by Thomson et al. (1998) and (1995).
• the cells used in the present invention will preferably comprise mammalian somatic cells, most preferably cells derived from an actively proliferating (non-quiescent) mammalian cell culture.
• the donor cell will be genetically modified by the addition, deletion or substitution of a desired DNA sequence.
• the donor cell e.g., a keratinocyte or fibroblast, e.g., of human, primate or bovine origin, may be transfected or transformed with a DNA construct that provides for the expression of a desired gene product, e.g., therapeutic polypeptide.
• the donor cells may be modified prior to nuclear transfer to achieve other desired effects, e.g., impaired cell lineage development, enhanced embryonic development and/or inhibition of apoptosis. Examples of desirable modifications are discussed further below.
• One aspect of the invention will involve genetic modification of the donor cell, e.g., a human cell, such that it is lineage deficient and therefore when used for nuclear transfer it will be unable to give rise to a viable offspring.
• a viable embryo may be an unwanted outcome.
• This can be effected by genetically engineering a human cell such that it is incapable of differentiating into specific cell lineages when used for nuclear transfer.
• cells may be genetically modified such that when used as nuclear transfer donors the resultant "embryos" do not contain or substantially lack at least one of mesoderm, endoderm or ectoderm tissue. It is anticipated that this can be accomplished by knocking out or impairing the expression of one or more mesoderm, endoderm or ectoderm specific genes.
• a desired somatic cell e.g., a human keratinocyte, epithelial cell or fibroblast
• a desired somatic cell will be genetically engineered such that one or more genes specific to particular cell lineages are "knocked out” and/or the expression of such genes significantly impaired. This may be effected by known methods, e.g., homologous recombination.
• a preferred genetic system for effecting "knock- out" of desired genes is disclosed by Capecchi et al, U.S.
• Patents 5,631 , 153 and 5,464,764 which reports positive-negative selection (PNS) vectors that enable targeted modification of DNA sequences in a desired mammalian genome.
• PPS positive-negative selection
• Such genetic modification will result in a cell that is incapable of differentiating into a particular cell lineage when used as a nuclear transfer donor.
• This genetically modified cell will be used to produce a lineage-defective nuclear transfer embryo, i.e., that does not develop at least one of a functional mesoderm, endoderm or ectoderm. Thereby, the resultant embryos, even if implanted, e.g., into a human uterus, would not give rise to a viable offspring.
• the ES cells that result from such nuclear transfer will still be useful in that they will produce cells of the one or two remaining non-impaired lineage.
• an ectoderm deficient human nuclear transfer embryo will still give rise to mesoderm and endoderm derived differentiated cells.
• An ectoderm deficient cell can be produced by deletion and/or impairment of one or both of RNA helicase A or H beta 58 genes.
• These lineage deficient donor cells may also be genetically modified to express another desired DNA sequence.
• the genetically modified donor cell will give rise to a lineage- deficient blastocyst which, when plated, will differentiate into at most two of the embryonic germ layers.
• the donor cell can be modified such that it is "mortal".
• Another preferred embodiment of the present invention is the production of nuclear transfer embryos that grow more efficiently in tissue culture. This is advantageous in that it should reduce the requisite time and necessary fusions to produce ES cells and/or offspring (if the blastocysts are to be implanted into a female surrogate). This is desirable also because it has been observed that blastocysts and ES cells resulting from nuclear transfer may have impaired development potential.
• a DNA construct containing the Ql and/or Q9 gene will be introduced into donor somatic cells prior to nuclear transfer.
• an expression construct can be constructed containing a strong constitutive mammalian promoter operably linked to the Q7 and/or Q9 genes, an IRES, one or more suitable selectable markers, e.g,. neomycin, ADA, DHFR, and a poly-A sequence, e.g, bGH polyA sequence.
• blastocyst development as these genes are highly conserved in different species, e.g, bovines, goats, porcine, dogs, cats, and humans.
• donor cells can be engineered to affect other genes that enhance embryonic development.
• these genetically modified donor cells should produce blastocysts and preimplantation stage embryos more efficiently.
• Still another aspect of the invention involves the construction of donor cells that are resistant to apoptosis, i.e., programmed cell death. It has been reported in the literature that cell death related genes are present in preimplantation stage embryos. (Adams et al, Science, 281(5381):1322-1326 (1998)).
• Genes reported to induce apoptosis include, e.g. Bad, Bok, BH3, Bik, Hrk, BNIP3, Bim L , Bad, Bid, and EGL-1.
• genes that reportedly protect cells from programmed cell death include, by way of example, BcL-XL, Bcl-w, Mcl-1, Al, Nr-13, BHRF-1, LMW5-HL, ORF16, Ks-Bel-2, E1B-19K, and CED-9.
• donor cells can be constructed wherein genes that induce apoptosis are "knocked out” or wherein the expression of genes that protect the cells from apoptosis is enhanced or turned on during embryonic development.
• this can be effected by introducing a DNA construct that provides for regulated expression of such protective genes, e.g, Bcl-2 or related genes during embryonic development.
• the gene can be "turned on” by culturing the embryo under specific growth conditions.
• it can be linked to a constitutive promoter.
• a DNA construct containing a Bcl-2 gene operably linked to a regulatable or constitutive promoter, e.g, PGK, SV40, CMV, ubiquitin, or beta-actin, an IRES, a suitable selectable marker, and a poly-A sequence can be constructed and introduced into a desired donor mammalian cell, e.g, human keratinocyte or fibroblast.
• donor cells when used to produce nuclear transfer embryos, should be resistant to apoptosis and thereby differentiate more efficiently in tissue culture. Thereby, the speed and/or number of suitable preimplantation embryos produced by nuclear transfer can be increased. Another means of accomplishing the same result is to impair the expression of one or more genes that induce apoptosis. This will be effected by knock-out or by the use of antisense or ribozymes against genes that are expressed in and which induce apoptosis early on in embryonic development. Examples thereof are identified above. Still alternatively, donor cells may be constructed containing both modifications, i.e., impairment of apoptosis-inducing genes and enhanced expression of genes that impede or prevent apoptosis.
• a particular cyclin DNA may be operably linked to a regulatory sequence, together with a detectable marker, e.g, green fluorescent protein (GFP), followed by the cyclin destruction box, and optionally insulation sequences to enhance cyclin and marker protein expression.
• a detectable marker e.g, green fluorescent protein (GFP)
• GFP green fluorescent protein
• cyclin Dl gene in order to select for cells that are in Gl .
• any cyclin gene should be suitable for use in the claimed invention. (See, e.g. King et al, Mol. Biol. Cell, Vol. 7(9): 1343-1357 (1996)).
• a less invasive or more efficient method for producing cells of a desired cell cycle stage are needed. It is anticipated that this can be effected by genetically modifying donor cells such that they express specific cyclins under detectable conditions. Thereby, cells of a specific cell cycle can be readily discerned from other cell cycles. Cyclins are proteins that are expressed only during specific stages of the cell cycle.
• cyclin Dl, D2 and D3 in Gl phase include cyclin Bl and B2 in G2/M phase and cyclin E, A and H in S phase.
• These proteins are easily translated and destroyed in the cytogolcytosol. This "transient" expression of such proteins is attributable in part to the presence of a "destruction box", which is a short amino acid sequence that is part of the protein that functions as a tag to direct the prompt destruction of these proteins via the ugiquitin pathway. (Adams et al, Science, 281 (5321): 1322-1326 (1998)).
• donor cells will be constructed that express one or more of such cyclin genes under easily detectable conditions, preferably visualizable, e.g, by the use of a fluorescent label.
• a particular cyclin DNA may be operably linked to a regulatory sequence, together with a detectable marker, e.g, green fluorescent protein (GFP), followed by the cyclin destruction box, and optionally insulation sequences to enhance cyclin and/or marker protein expression.
• GFP green fluorescent protein
• cells of a desired cell cycle can be easily visually detected and selected for use as a nuclear transfer donor.
• An example thereof is the cyclin Dl gene which can be used to select for cells that are in Gl .
• any cyclin gene should be suitable for use in the claimed invention.
• Still another aspect of the invention is a method for enhancing nuclear transfer efficiency, preferably in a cross-species nuclear transfer process. While the present inventors have demonstrated that nuclei or cells of one species when inserted or fused with an enucleated oocyte of a different species can give rise to nuclear transfer embryos that produce blastocysts, which embryos can give rise to ES cell lines, the efficiency of such process is quite low. Therefore, many fusions typically need to be effected to produce a blastocyst the cells of which may be cultured to produce ES cells and ES cell lines.
• Yet another means for enhancing the development of nuclear transfer embryos in vitro is by optimizing culture conditions.
• One means of achieving this result will be to culture NT embryos under conditions impede apoptosis.
• proteases such as capsases can cause oocyte death by apoptosis similar to other cell types.
• blastocyst development will be enhanced by including in culture media used for nuclear transfer and to maintain blastocysts or culture preimplantation stage embryos one or more capsase inhibitors.
• Such inhibitors include by way of example capsase-4 inhibitor I, capsase-3 inhibitor I, capsase-6 inhibitor II, capsase-9 inhibitor II, and capsase- 1 inhibitor I.
• the amount thereof will be an amount effective to inhibit apoptosis, e.g, 0.00001 to 5.0% by weight of medium; more preferably 0.01% to 1.0% by weight of medium.
• the foregoing methods may be used to increase the efficiency of nuclear transfer by enhancing subsequent blastocyst and embryo development in tissue culture. After NT units of the desired size are obtained, the cells are mechanically removed from the zone and are then used to produce embryonic or stem-like cells and cell lines.
• NT unit which typically will contain at least about 50 cells
• washing such cells and plating the cells onto a feeder layer, e.g, irradiated fibro- blast cells.
• the cells used to obtain the stem-like cells or cell colonies will be obtained from the inner most portion of the cultured NT unit which is preferably at least 50 cells in size.
• NT units of smaller or greater cell numbers as well as cells from other portions of the NT unit may also be used to obtain ES-like cells and cell colonies. It may be that a longer exposure of donor cell DNA to the oocyte 's cytosol would facilitate the dedifferentiation process.
• recloning could be accomplished by taking blastomeres from a reconstructed embryo and fusing them with a new enucleated oocyte.
• the donor cell may be fused with an enucleated oocyte and 4 to 6 hours later, without activation, chromosomes may be removed and fused with a younger oocyte. Activation would occur thereafter.
• the cells are maintained in the feeder layer in a suitable growth medium, e.g, alpha MEM supplemented with 10% FCS and 0.1 mM beta- mercaptoethanol (Sigma) and L-glutamine.
• the growth medium is changed as often as necessary to optimize growth, e.g, about every 2-3 days.
• This culturing process results in the formation of embryonic or stem-like cells or cell lines.
• colonies are observed by about the second day of culturing in the alpha MEM medium.
• this time may vary dependent upon the particular nuclear donor cell, specific oocyte and culturing conditions.
• One skilled in the art can vary the culturing conditions as desired to optimize growth of the particular embryonic or stem-like cells.
• the embryonic or stem-like cells and cell colonies obtained will typically exhibit an appearance similar to embryonic or stem-like cells of the species used as the nuclear cell donor rather than the species of the donor oocyte.
• the cells exhibit a morphology more similar to mouse embryonic stem cells than bovine ES-like cells. More specifically, the individual cells of the human ES-line cell colony are not well defined, and the perimeter of the colony is refractive and smooth in appearance. Further, the cell colony has a longer cell doubling time, about twice that of mouse ES cells. Also, unlike bovine and porcine derived ES cells, the colony does not possess an epithelial-like appearance. As discussed above, it has been reported by Thomson, in U.S.
• primate stem cells are SSEA-1 (-), SSEA-4 (+), TRA-1-60 (+), TRA-1-81 (+) and alkaline phosphatase (+). It is anticipated that human and primate ES cells produced according to the present methods will exhibit similar or identical marker expression. Alternatively, that such cells are actual human or primate embryonic stem cells will be confirmed based on their capability of giving rise to all of mesoderm, ectoderm and endoderm tissues. This will be demonstrated by culturing ES cells produced according to the invention under appropriate conditions, e.g, as disclosed by Thomsen, U.S. Patent 5,843,780, incorporated by reference in its entirety herein.
• the resultant embryonic or stem-like cells and cell lines preferably human embryonic or stem-like cells and cell lines, have numerous therapeutic and diagnostic applications. Most especially, such embryonic or stem-like cells may be used for cell transplantation therapies.
• Human embryonic or stem-like cells have application in the treatment of numerous disease conditions.
• mouse embryonic stem (ES) cells are capable of differentiating into almost any cell type, e.g, hematopoietic stem cells. Therefore, human embryonic or stem-like cells produced according to the invention should possess similar differentiation capacity.
• the embryonic or stem-like cells according to the invention will be induced to differentiate to obtain the desired cell types according to known methods.
• the subject human embryonic or stem-like cells may be induced to differentiate into hematopoietic stem cells, muscle cells, cardiac muscle cells, liver cells, cartilage cells, epithelial cells, urinary tract cells, etc, by culturing such cells in differentiation medium and under conditions which provide for cell differentia- tion.
• Medium and methods which result in the differentiation of embryonic stem cells are known in the art as are suitable culturing conditions.
• Palacios et al Proc. Natl Acad.
• hematopoietic stem cells from an embryonic cell line by subjecting stem cells to an induction procedure comprising initially culturing aggregates of such cells in a suspension culture medium lacking retinoic acid followed by culturing in the same medium containing retinoic acid, followed by transferral of cell aggregates to a substrate which provides for cell attachment.
• induction procedure comprising initially culturing aggregates of such cells in a suspension culture medium lacking retinoic acid followed by culturing in the same medium containing retinoic acid, followed by transferral of cell aggregates to a substrate which provides for cell attachment.
• Pedersen, J. Reprod. Fertil Dev., 6:543-552 (1994) is a review article which references numerous articles disclosing methods for in vitro differentiation of embryonic stem cells to produce various differentiated cell types including hematopoietic cells, muscle, cardiac muscle, nerve cells, among others.
• Bcl-2 prevents many, but not all, forms of apoptotic cell death that occur during lymphoid and neural development.
• a thorough discussion of how Bcl-2 expression might be used to inhibit apoptosis of relevent cell lineages following transfection of donor cells is disclosed in U.S. Patent No. 5,646,008, which is herein incorporated by reference.
• the subject embryonic or stem-like cells may be used to obtain any desired differentiated cell type. Therapeutic usages of such differentiated human cells are unparalleled.
• human hematopoietic stem cells may be used in medical treatments requiring bone marrow transplantation.
• Hematopoietic stem cells can be obtained, e.g, by fusing adult somatic cells of a cancer or AIDS patient, e.g, epithelial cells or lympho- cytes with an enucleated oocyte, e.g, bovine oocyte, obtaining embryonic or stem-like cells as described above, and culturing such cells under conditions which favor differentiation, until hematopoietic stem cells are obtained.
• hematopoietic stem cells may be used in the treatment of diseases including cancer and AIDS.
• adult somatic cells from a patient with a neurological disorder may be fused with an enucleated animal oocyte, e.g, a primate or bovine oocyte, human embryonic or stem-like cells obtained therefrom, and such cells cultured under differentiation conditions to produce neural cell lines.
• an enucleated animal oocyte e.g, a primate or bovine oocyte, human embryonic or stem-like cells obtained therefrom, and such cells cultured under differentiation conditions to produce neural cell lines.
• oocyte e.g, a primate or bovine oocyte, human embryonic or stem-like cells obtained therefrom, and such cells cultured under differentiation conditions to produce neural cell lines.
• Specific diseases treatable by transplantation of such human neural cells include, by way of example, Parkinson's disease, Alzheimer's disease, ALS and cerebral palsy, among others.
• Parkinson's disease it has been demonstrated that transplanted fetal brain neural cells make the proper connec- tions with surrounding cells and produce dopamine. This
• donor cells may be transfected with selectable markers expressed via inducible promoters, thereby permitting selection or enrichment of particular cell lineages when differentiation is induced.
• CD34-neo may be used for selection of hematopoietic cells, Pwl-neo for muscle cells, Mash-1-neo for sympathetic neurons, Mal-neo for human CNS heurons of the grey matter of the cerebral cortex, etc.
• the great advantage of the subject invention is that it provides an essentially limitless supply of isogenic or synegenic human cells suitable for transplantation.
• diseases and conditions treatable by isogenic cell therapy include, by way of example, spinal cord injuries, multiple sclerosis, muscular dystrophy, diabetes, liver diseases, i.e., hypercholesterolemia, heart diseases, cartilage replacement, burns, foot ulcers, gastrointestinal diseases, vascular diseases, kidney disease, urinary tract disease, and aging related diseases and conditions.
• human embryonic or stem-like cells produced according to the invention may be used to produce genetically engineered or transgenic human differentiated cells. Essentially, this will be effected by introducing a desired gene or genes, which may be heterologous, or removing all or part of an endogenous gene or genes of human embryonic or stem-like cells produced according to the invention, and allowing such cells to differentiate into the de- sired cell type.
• a preferred method for achieving such modification is by homologous recombination because such technique can be used to insert, delete or modify a gene or genes at a specific cite or cites in the stem-like cell genome.
• This methodology can be used to replace defective genes, e.g, defective immune system genes, cystic fibrosis genes, or to introduce genes which result in the expression of therapeutically beneficial proteins such as growth factors, lymphokines, cytokines, enzymes, etc.
• the gene encoding brain derived growth factor may be introduced into human embryonic or stem-like cells, the cells differentiated into neural cells and the cells transplanted into a Parkinson's patient to retard the loss of neural cells during such disease.
• BDNF BDNF-derived neurotrophic factor
• astrocytes have been transfected with BDNF gene using retroviral vectors, and the cells grafted into a rat model of Parkinson's disease (Yoshimoto et al. Brain Research, 691 :25-36, (1995)). This ex vivo therapy reduced Parkinson's-like symptoms in the rats up to 45% 32 days after transfer.
• the tyrosine hydroxylase gene has been placed into astrocytes with similar results (Lundberg et al. Develop. Neurol, 139:39-53 (1996) and references cited therein).
• cattle and pig embryonic cell lines can be transfected and selected for stable integration of heterologous DNA.
• desired genes may be introduced into the sub- ject human embryonic or stem-like cells, and the cells differentiated into desired cell types, e.g, hematopoietic cells, neural cells, pancreatic cells, cartilage cells, etc.
• Genes which may be introduced into the subject embryonic or stem-like cells include, by way of example, epidermal growth factor, basic fibroblast growth factor, glial derived neurotrophic growth factor, insulin-like growth factor (I and II), neurotrophin-3, neurotrophin-4/5, ciliary neurotrophic factor, AFT-1, cytokine genes (interleukins, interferons, colony stimulating factors, tumor necrosis factors (alpha and beta), etc.), genes encoding therapeutic enzymes, collagen, human serum albumin, etc.
• TK thymidine kinase
• donor cells transfected with the thymidine kinase (TK) gene will lead to the production of embryonic cells containing the TK gene. Differentiation of these cells will lead to the isolation of therapeutic cells of interest which also express the TK gene.
• Such cells may be selectively eliminated at any time from a patient upon gancyclovir administration.
• a negative selection system is described in U.S. Patent No. 5,698,446, and is herein incorporated by reference.
• the subject embryonic or stem-like cells preferably human cells, also may be used as an in vitro model of differentiation, in particular for the study of genes which are involved in the regulation of early development. Also, differentiated cell tissues and organs using the subject embryonic or stem-like cells may be used in drug studies.
• EXAMPLE 1 MATERIALS AND METHODS Donor Cells for Nuclear Transfer Epithelial cells were lightly scraped from the inside of the mouth of a consenting adult with a standard glass slide. The cells were washed off the slide into a petri dish containing phosphate buffered saline without Ca or Mg. The cells were pipetted through a small-bore pipette to break up cell clumps into a single cell suspension.
• TL- HEPES medium containing 10% fetal calf serum (FCS) under oil for nuclear transfer into enucleated cattle oocytes.
• FCS fetal calf serum
• the bovine oocyte cytoplasm and the donor nucleus are fused together using electrofusion techniques.
• One fusion pulse consisting of 90 V for 15 ⁇ sec was applied to the NT unit. This occurred at 24 hours post-initiation of maturation (hpm) of the oocytes.
• the NT units were placed in CRIaa medium until 28 hpm. The procedure used to artificially activate oocytes has been described elsewhere. NT unit activation was at 28 hpm.
• NT units were exposed for four min to ionomycin (5 ⁇ M; CalBiochem, La Jolla, CA) in TL-HEPES supplemented with 1 mg/ml BSA and then washed for five min in TL-HEPES supplemented with 30 mg/ml BSA.
• the NT units were then transferred into a microdrop of CR1 aa culture medium containing 0.2 mM DMAP (Sigma) and cultured at 38.5 °C 5% C0 2 for four to five hours.
• the NT units were washed and then placed in a CRIaa medium plus 10% FCS and 6 mg/ml BSA in four well plates containing a confluent feeder layer of mouse embryonic fibroblasts (described below).
• the NT units were cultured for three more days at 38.5 °C and 5% C0 2 .
• the culture medium was changed every three days until day 12 after the time of activation.
• NT units reaching the desired cell number i.e., about 50 cell number, were mechanically removed from the zona and used to produce embryonic cell lines.
• a photograph of an NT unit obtained as described above is contained in Figure 1.
• Fibroblast feeder layer Primary cultures of embryonic fibroblasts were obtained from 14-16 day old murine fetuses.
• Fibroblast cells were plated in tissue culture flasks and cultured in alpha-MEM medium (BioWhittaker, Walkersville, MD) supple- mented with 10% fetal calf serum (FCS) (Hyclone, Logen, UT), penicillin (100 IU/ml) and streptomycin (50 ⁇ l/ml).
• FCS fetal calf serum
• NT unit cells obtained as described above were washed and plated directly onto irradiated feeder fibroblast cells. These cells included those of the inner portion of the NT unit. The cells were maintained in a growth medium consisting of alpha MEM supplemented with 10% FCS and 0.1 mM befamercaptoethanol (Sigma).
• FIG. 1 Figures 2 through 5 are photographs of ES-like cell colonies obtained as described, supra. Production of Differentiated Human Cells The human embryonic cells obtained are transferred to a differentiation medium and cultured until differentiated human cell types are obtained. RESULTS Table 1. Human cells as donor nuclei in NT unit production and development. TABLE 1
• the one NT unit that developed a structure having greater than 16 cells was plated down onto a fibroblast feeder layer. This structure was attached to the feeder layer and started to propagate forming a colony with a ES cell-like morphology (See, e.g. Figure 2). Moreover, although the 4 to 16 cell stage structures were not used to try and produce an ES cell colony, it has been previously shown that this stage is capable of producing ES or ES-like cell lines (mouse, Eistetter et al, Devel. Growth and Differ., 31 :275-282 (1989); Bovine, Stice et al, 1996)). Therefore, it is expected that 4 - 16 cell stage NT units should also give rise to embryonic or stem-like cells and cell colonies.

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