EP1282685A2 - Pluripotente zellen mit allogenem zellkern und mitochondrien - Google Patents

Pluripotente zellen mit allogenem zellkern und mitochondrien

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Publication number
EP1282685A2
EP1282685A2 EP01930525A EP01930525A EP1282685A2 EP 1282685 A2 EP1282685 A2 EP 1282685A2 EP 01930525 A EP01930525 A EP 01930525A EP 01930525 A EP01930525 A EP 01930525A EP 1282685 A2 EP1282685 A2 EP 1282685A2
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Prior art keywords
cells
allochimeric
human
chimeric
nucleus
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English (en)
French (fr)
Inventor
Michael D. West
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Astellas Institute for Regenerative Medicine
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Advanced Cell Technology Inc
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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
    • 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
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C12N5/166—Animal cells resulting from interspecies fusion
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/04—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from germ 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

Definitions

  • the field of the invention is the manipulation of cells to provide non-human oocytes for nucler transfer procedures wherein such nuclear transfer units possess allogenic nucleus and mitochondria from two different cellular sources of the same species.
  • Embryonic stem (“ES”) cells offer opportunities to produce differentiated cells, organs and complete individuals upon demand, so that one may clone a particular genotype.
  • livestock animals e.g.
  • modified oocytes and ES cells capable of being expanded in culture and differentiating to different cell types, engineered ES cells, differentiated cells derived therefrom, organs or portions thereof and organisms derived thereby.
  • the methods comprise employing an allogenic egg cell, where allogenic intends the ultimate species. intended for the cell, which is enucleated and into which is introduced a xenogenic nucleus, to provide a chimeric cell having cytoplasm and mitochondria from one species and a nucleus from a different species.
  • the ES cells After expansion of the chimeric cell to provide a nuclear transfer (NT) unit from which ES cells may be derived, the ES cells may be used for further expansion and scientific investigation, or the NT unit may be transferred into an allogenic, syngenic or other compatible xenogenic host uterus (based on the original oocyte) for pregnancy and birth.
  • Oocytes from this Fl animal may then be harvested, enucleated and an allogenic nucleus introduced to provide an egg cell which can provide cells having proteins of a single species, be immunologically competent and which can be accepted with a reduced risk of rejection when implanted in an allogenic host.
  • these cells and their progeny will be referred to as "allochimeric.”
  • Allochimeric cells comprising mitochondria from a first individual of a first species and a nucleus from a second individual from the same first species.
  • pluripotent cells capable of development into a fetus or into a plurality of different cell types will be produced comprising mitochondria from an allogenic species, where allogenic species intends the species of interest, and a xenogenic nucleus, where xenogenic intends the species in which the cells will be implanted or a different species compatible with implantation of the cells and the cells differentiating into a fetus.
  • the developed or matured fetus will then be used for harvesting oocytes, enucleating the oocytes, and introducing a nucleus from the same species as the mitochondria.
  • the resulting allochimeric cells may be used in vitro and in vivo for studying differentiation, for producing cells along different differentiation paths, e.g. ectoderm, endoderm or mesoderm, and cells within these classifications, such as neuronal cells, neurons, astrocytes, glial cells, ganglions, etc., hematopoietic cells, e.g. lymphocytes, macrophages, NK cells, erythrocytes, megakaryocytes, etc., fibroblasts, myoblasts, etc.
  • the subject methodology involves the following primary stages: •
  • Egg cells may be harvested from any convenient host of interest, for which one wishes mitochondria from a species of interest.
  • any mammalian species of interest may serve as the source of the egg cells, particularly domestic animals, more particularly large domestic animals, exemplified by equine, bovine, ovine, porcine, feline, canine, lagomorpha, murine, etc., and primate, exemplified by human, monkey and ape.
  • the oocytes may be harvested in any convenient way, depending upon whether there are sources of follicles from slaughtered animals or a surgical procedure is required.
  • primate cells more particularly human cells, which may serve for production of differentiated cells.
  • the cells may come from any member of the species, as the nucleus which will be subsequently employed will determine the species and the histocompatibility of the cells.
  • the subject invention finds particular application with human cells, although there will be instances with other species where allochimeric cells can play a role, e.g. rare and valuable animals, animals for which in vitro fertilization is not reasonably feasible, and the like.
  • the egg cells may be matured in vitro in accordance with known techniques, using appropriate maturation medium.
  • Oocytes having polar bodies may be employed as the host cells. See, for example, Prather et al., Differentiation 48:1-8 (1991) and Seshagine et al., Biol. Reprod. 40:544-606 (1989))
  • Enucleation is achieved in accordance with conventional ways, conveniently using a micropipette to remove polar bodies and surrounding cytoplasm. The oocytes are then screened to establish the successful enucleation.
  • the somatic nuclei may be engineered by the introduction of genes important for mitochondrial function in the type of mitochondria present in the egg cell.
  • the subject invention finds particular application in the introduction of human nuclear mitochondrial genes into animal, particularly bovine somatic cells to facilitate cellular function of an animal cell with human mitochondria.
  • a wide variety of mammalian hosts may be employed as the source of the nuclei, where the issue will be primarily one of convenience. Besides the necessity for growing the chimeric NT unit to term, other considerations in the selection of the host source will be ease of harvesting the oocytes from the host, ease of manipulation and growth in culture, maturity of the technology, prior success rate in achieving progeny, ease of growth of the host, viability of the resulting chimeric oocytes, number of available oocytes, compatibility of the mitochondria with the host nucleus, as well as other practical considerations. While any mammalian host may be employed, of particular interest are domestic animals, more particularly, large domestic animals or primates other than humans.
  • Exemplary mammals include ungulates, bovine and ovine, porcine, feline, equine, lagomorpha, murine, canine, etc.
  • the isolated oocytes are matured in vitro and selected for the presence of polar bodies.
  • the nuclei may come from any convenient source, germ or somatic cell, which can provide for the production of progeny. Therefore, the nuclei may come from fetal cells, neonate cells, mature cells, cells produced in a culture medium, or the like. Generally, neoplastic cell nuclei will not be used, but may have application depending on the nature of the neoplasm.
  • the source may ne differentiated cells, which may be quiescent, G 0 or in an active state e.g. G, or G 2 .
  • Cell types include one cell embryos (zygotes) or pronuclei, egg cells, blastomeres, epithelial cells, endothelial cells, muscle cells, keratinocytes, skin cells, alveolar cells, hepatocytes, renal cells, neuronal cells, hematopoietic cells, fibroblasts, endothelial cells, parenchymal cells, adipose cells, glial cells, follicular cells, etc., where the choice may be determined by a number of factors associated with the end purpose.
  • Fusion and injection has been shown to be efficient, where the cell, which is the source of the nucleus, is placed into the perivitelline space of the enucleated oocyte and fused in a fusion chamber by means of an electrical pulse or using a fine glass needle, the donor nucleus or pronuclei from a fertilized oocyte can be isolated and delivered into the cytoplasm of the recipient egg.
  • pronuclei may be from animals whose genomes have been modified to include nuclear mitochondrial genes of the same species as the recipient oocyte.
  • Fusion can also be accomplished by using electrical pulses or by using various fusogenic reagents, e.g. Sendai virus. (See, for example, Graham, Wister Inot. Symp. Monogr. 9:19 (1969) and Collas and Barnes, Mol. Reprod. Dev. 38:264-267 (1994).)
  • NT fused nuclear transfer
  • a suitable medium e.g. CRIaa medium.
  • activation will be effected shortly thereafter, usually within 24h, more usually 4 - 9h later.
  • Activation may be accomplished by methods known for mammalian cells, such as culturing the NT unit at sub-physiological temperature, e.g. room temperature, penetration of oocytes by sperm, electrical and chemical shock, etc. See, for example, Susko-Parrish et al., U.S. Patent No. 5, 496, 720.
  • Other methods include effecting simultaneous or sequential increasing of levels of divalent cations in the oocyte, e.g.
  • kinase inhibitors e.g 6-diniemylaminopurine, staurosporine, roscovitine, butyrolactone, 2-aminopurine, and sphingosine, or introducing one or more phosphatases into the oocyte, e.g. phosphatase 2A or 2B.
  • a technique of interest for activation is: activation of NT units within 18 to 30 hours after the start of culture in maturation medium.
  • NT units are then removed from the culture media and placed in activation media (2mL TL Hepes with 2 mg of bovine serum albumin, 5mM of Ionomycin and 2mM of 6-DMAP for 4 minutes on a slide warmer. At the end of 4 minutes, the NT units are rinsed in TL Hepes and placed in culture media with 2mM of 6-DMAP at 38.5 ° and 5% CO 2 for 2 to 5 hours. At the end of this period, NT units are rinsed 4 times in TL Hepes and placed in culture. When the desired developmental stage is reached, ES cells will be derived or NT units will be transferred to recipient females.
  • activation media 2mL TL Hepes with 2 mg of bovine serum albumin, 5mM of Ionomycin and 2mM of 6-DMAP for 4 minutes on a slide warmer. At the end of 4 minutes, the NT units are rinsed in TL Hepes and placed in culture media with 2mM of 6-DMAP at 38.5 ° and 5% CO 2 for 2
  • the activated NT units of chimeric cells may then be cultured in a suitable in vitro or in vivo culture medium until the generation of ES cells and cell colonies developing embryos, such as blastocysts.
  • Culture media suitable for culturing and maturation of ES cells and embryos are well known in the art.
  • the chimeric cells may be treated as cells from the species of the nucleus, having the surface membrane proteins, cytoplasmic and nuclear proteins defined by the nuclear genes.
  • Examples of known media include Ham's F-10 + 10% fetal calf serum (“FCS”), Tissue culture medium-199 (“TCM-199”) + 10% fetal calf serum, TyrodespAlbumin-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
  • 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
  • TCM-199 Tissue culture medium-199
  • TPM-199 10% fetal
  • An exemplary media is TCM-199 with Earl salts, 10% fetal calf serum, 0.2mM Na pyruvate and 50 ⁇ g/ml gentamycin. These media may be used with cell lines to provide for conditioned media, using granulosa cells, oviduct cells, BRL cells, uterine cells and STO cells. Alternatively, the media described by Rosenkrans, jr., U.S. Patent No. 5, 096, 822 may be used.
  • CR1 contains hemicalcium L-lactate in from about 1 to lOmM, usually 1 to 5mM, sodium chloride, potassium chloride, sodium bicarbonate, and a minor amount of fatty-acid free bovine serum albumin, and when essential and non-essential amino acids are added, the medium is referred to as CRlaa.
  • An exemplary CR1 media comprises 114.7mM NaCl, 3.1 mM KC1, 26.2mM Na 2 CO 3 , 5mM hemicalcium L-lactate and 3mg/ml of fatty acid-free bovine serum albumin.
  • the activated NT unit chimeric ES cells are placed in CRlaa media containing 1.9mM DMAP for about 4h, followed by a wash with HECM and then cultured in CRlaa containing BSA, at about 38.5°C, 5% CO 2 , for about 4 - 5 h.
  • the cultured NT units are usually washed and placed in a suitable conditioned media for growth.
  • An effective media is CRlaa media containing 10% FCS and 6 mg/ml BSA.
  • Suitable feeder layers include fibroblasts and epithelial cells, particularly uterine epithelial cells, form sources such as ungulates, chicken, murine, STO, SI-m220 and BRL cells. Mouse embryonic fibroblasts have been found to be particularly useful. After sufficient time, which will vary with the species of the nucleus, chimeric ES cells are obtained which may now serve for msemination in an appropriate host.
  • NT units are transferred to the uterus.
  • the host is monitored to ensure that the NT units have been successfully implanted.
  • the host may be restrained to prevent a spontaneous abortion.
  • the delivery of the fetus or neonate may be spontaneous or induced abortion, natural delivery or cesarean section.
  • the above stages will depend on the nature of the host. Normal maintenance of the host is employed to permit the neonate to grow as a healthy host. The harvesting of the oocytes may be performed as described above.
  • the manner of enucleation and introduction of nuclei has already been discussed.
  • the selection of the species for supplying the nucleus will depend on the purpose for which the cells are to be used.
  • the cells may be used for cloning a particular species, particularly where oocytes are only difficultly grown in the species, such as humans or rare species, or as a source of differentiated cells, where the cells have both a nucleus and mitochondria from the same species.
  • Other than human, other species which find application with the subject invention include the cloning of rare and endangered animals.
  • the NT units are cultured on the feeder layer until the NT units reach a size suitable for the isolation of ES cells, generally requiring at least 4 cells, preferably at least 50 cells, and usually not exceeding 400 cells. Culturing will usually employ conditions of 38.5°C and 5% CO 2 , with the culture medium changed about every 1 - 5 days.
  • the final stage involves mechanically removing the NT units from the culture, isolating cells from the inner portion of the NT unit, although cells from other portions of. the NT unit may find application, washing the cells from the NT unit and plating the cells on a feeder layer, e.g. irradiated fibroblast cells, from the same or different species from the nucleus.
  • the cells are maintained on the feeder layer in a suitable growth media, e.g alpha- MEM supplemented with 10% FCS, O.lmM ⁇ -mercaptoethanol and L-glutamine.
  • the growth media is conveniently changed about every 1 - 3 days.
  • the individual cells are not well defined and the perimeter of the colony is refractive and smooth in appearance.
  • the colony does not possess an epithelial-like appearance.
  • the NT unit cells may use the NT unit cells to provide inner cell mass (ICM) cells of blastocysts.
  • ICM inner cell mass
  • the ICM cells can be grown in culture using feeder layers, e.g STO (mouse fibroblasts) with charcoal stripped serum
  • the ES cells may be modified with DNA in accordance with conventional techniques. Bare DNA, recA coated DNA, viruses, plasmids, YACs, extrachromosomal DNA, chromosomal fragments, cDNA or other source of the desired gene, regulating sequence or the like may be introduced into the ES cells, using appropriate media, liposomes, transporting sequences, permeabilization, electrofusion or the like. See, for example, Schneike et al., Science 278:2130-3 (1997).
  • the modifications can provide for the constitutive or inducible expression of a particular product, e.g. insulin angiogenesis factors, cytokines, e.g. interleukins, interferons and colony stimulating factors, blood factors, e.g. serum albumin, thrombin, fibrinogen, thrombopoietin, erythropoietin, tissue plasminogen activator,, etc. hormones, e.g. growth hormones, growth factors, e.g. epidermal growth factor, basic fibroblast growth factor, glial derived neurofrophic growth factor, etc., enzymes, enzyme inhibitors, e.g. ⁇ - antitrypsin, telomere associated proteins, e.g.
  • a particular product e.g. insulin angiogenesis factors, cytokines, e.g. interleukins, interferons and colony stimulating factors
  • blood factors e.g. serum albumin, thrombin, fibrinogen, thrombo
  • Sir, telomerase, etc. neuronal proteins, e.g. neurotrophin-3,4/5, ciliary neurotrophic factor, etc., pancreatic proteins, basal proteins, taste bud proteins, eye proteins, hemoglobin, transcription factors, lipoproteins, immunoglobulins, surface membrane receptors, e.g. insulin receptor, oncogene repressors, L-dopamine, and angiogenesis inhibitors.
  • neuronal proteins e.g. neurotrophin-3,4/5, ciliary neurotrophic factor, etc.
  • pancreatic proteins basal proteins
  • taste bud proteins eye proteins
  • hemoglobin transcription factors
  • lipoproteins e.g. insulin receptor
  • immunoglobulins e.g. insulin receptor, oncogene repressors, L-dopamine, and angiogenesis inhibitors.
  • surface membrane receptors e.g. insulin receptor, oncogene repressors, L-dopamine, and angiogenesis inhibitors.
  • nuclei from subjects suffering from sickle cell anemia may be genetically modified to provide cells which provide a native hemoglobin; subjects suffering from hemophilia may have the mutant gene for the blood factor corrected, e.g. Factor NHIc or NHIvw, Christmas factor; subjects susceptible to diseases because of genetic factors, may have their nuclei modified to provide alleles less susceptible to such diseases, e.g. AIDS, juvenile onset diabetes, muscular dystrophy, Alzheimer's disease, Parkinson's disease, and so on.
  • blood factor corrected e.g. Factor NHIc or NHIvw, Christmas factor
  • subjects susceptible to diseases because of genetic factors may have their nuclei modified to provide alleles less susceptible to such diseases, e.g. AIDS, juvenile onset diabetes, muscular dystrophy, Alzheimer's disease, Parkinson's disease, and so on.
  • genes which may be knocked out include oncogenes, histocompatibility proteins, blood type proteins and other genes whose dominant expression leads to pathology.
  • Cells of interest include hematopoietic cells, neuronal cells, skeletal and cardiac muscle cells, skin cells, epithelial cells, endothelial cells, structural cells, osteoclasts and osteoblasts, follicle cells, eye cells, cells associated with sensing, such as taste buds inner ear cells, bone cells, renal cells, hepatocytes, pancreatic cells, e.g. ⁇ -islet cells, fibroblasts, and chondrocytes.
  • the differentiated ES cells can find application in a wide variety of venues.
  • the cells having allogenic mitochondria and nucleus provide cells which represent normal cells comprised of D ⁇ A from a single species.
  • the proteins which are present in the cytoplasm are all of the same species, the interaction between the nucleus and the mitochondria, e.g. transport of proteins is natural, and the immunodominant sequences present on the surface of the cell will all be from the same species.
  • ce ⁇ lular processes which depend on the interaction between the mitochondria and other infracellular compartments would also be natural.
  • the ES cells and differentiated cells offer many opportunities for dissection of cellular processes, response to exogenous agents, e.g. genes, drugs, factors, etc., being used for screening the effect of these exogenous agents in the development of drugs, identifying specific alleles or mutations associates with the response to these outside agents, and investigating variations in transcription and expression in response to various agents.
  • exogenous agents e.g. genes, drugs, factors, etc.
  • the ES cells may be maintained in culture and expanded, one can repetitively produce the differentiated cells, where the cells will be based on the same genotype.
  • the subject invention also offers the capability to use the nucleus from an individual for investigation and diagnosis. Thus, one may determine the susceptibility of a particular individual to outside agents, such as carcinogens, allergens, toxins, and mutagens where there is a complete cell of a single species having the native metabolism of such species. One can also investigate the cellular response to a particular regimen, e.g. drug regimen, to determine the effect on the normal cells of a particular patient.
  • a particular regimen e.g. drug regimen
  • ES cells which can be directed to produce various differentiated cells at different stages of differentiation, which can be subjected to the regimen and changes in the character of the cells determined.
  • the subject invention allows for studying processes of mitochondria in conjunction with a particular nucleus.
  • the subject methodology allows for cloning of such cells and seeing the effect of differentiation on the mitochondrial processes and the effect on growth patterns, phenotype, etc.
  • the use of the native mitochondria avoids incompatibility when mating the resulting individual with other individuals of the same species.
  • compositions which find use in the subject invention are oocytes comprising mitochondria from one species and a nucleus from a different species, isolated and in culture.
  • the nucleus and mitochondria will be from species which substantially differ, generally being from a different genus and even different families.
  • the nucleus will be chosen to provide for a useful source of the enucleated ovum, such as domestic animals (ungulates), e.g. bovine, ovine and porcine, and laboratory animals, e.g. murine.
  • ova comprising mitochondria from one individual and a nucleus from a different individual of the same species, ES cells derived therefrom, cultures comprising such ES cells, differentiated cells derived therefrom, and cultures comprising such differentiated cells.
  • compositions include allochimeric oocytes and ES cells in growth cultures for expansion of the cells.
  • the compositions also include mixtures of allochimeric cells and differentiated cells of inner cell masses or concomitant growth or maintenance of ES cells and differentiated cells in culture media for cell differentiation.
  • rIL-3 interleukin-3
  • F final concentration 10%v/v
  • FLS4.1 supernatant contains FLT3 ligand, steel factor and a new factor that supports growth of hematopoietic stem cells in 2 - 2.5 ml of culture medium [Iscove's Dulbecco's modified medium/50 ⁇ M 2-mercaptoethanol/2mM L-glutamine/gentamycin at 50 ⁇ g/ml /7.5% v/v FCS] at 37°C in a 7.5%CO 2 /92.5% air atmosphere. Every 5 - 7 days the cells are harvested and subcultured in new Costar 6-well plates containing mitomycin C-treated RP.0.10 stromal cells and freshly prepared cytokine-supplemented culture medium. (Palacios, et al., Proc. Natl.
  • the ES cells are subject to an 8-day induction procedure which consists of 4 days of culture as aggregates without retinoic acid (RA) followed by 4 days of culture in the presence of RA.
  • the medium is DMEM (high glucose with L-glutamine, without pyruvate; GIBCO 11965-043), 10% fetal bovine serum, 10% newborn calf serum and nucleosides stock.
  • Other media may be employed for directing differentiation into other cell types, using factors which exist naturally and affect the differentiation in vivo, such as hematopoietic factors, e.g. G-CSF, M-CSF, GM-CSF, interleukins, interferons and other cytokines and growth factors.
  • factors which exist naturally and affect the differentiation in vivo such as hematopoietic factors, e.g. G-CSF, M-CSF, GM-CSF, interleukins, interferons and other cytokines and growth factors.
  • the enucleated oocytes may be stored frozen in a suitable medium for extended periods and then carefully thawed before use.
  • the differentiated cells find use in therapy, where healthy cells can be introduced into the appropriate compartment to perform a desired function.
  • myocytes may be used for grafting to myocardial tissue or other muscle tissue, where natural or genetically modified cells may provide an advantage, e.g. correction of a mutant protein, as in the case of muscular dystrophy. See, for example, U.S. Patent No. 5,602,301.
  • Hematopoietic cells may be infused into a patient, such as lymphocytes, natural killer cells, megakaryocytes, eosinophils, basophils, mononuclear cells, or the precursors thereof, where the precursors may be dedicated to a specific cell type or may be multipotent and will differentiate to a number of different type cells, ⁇ -islet cells may be grafted onto the pancreas or provided in a protected container which may be innervated by a blood supply for treatment of diabetes.
  • Neuronal cells may be introduced into the brain cavity to provide therapies for Parkinson's disease, Alzheimer disease, cerebral palsy, and the like, by providing a source of L-dopamine, NGE or other factors or other composition which can affect brain function.
  • ES cells or their differentiated progeny include spinal cord injuries, multiple sclerosis, liver diseases, vascular diseases, bums cartilage replacement, heart diseases, kidney diseases, urinary tract diseases, prostate diseases, and diseases resulting from aging.
  • the subject cells may be used to provide for enhanced supplies of particular factors, consfitutively or inducibly.
  • Pruschy et al., Chem. Biol. 1: 163-72 (1997) provide modifications which allow for inducing production of a product by orally taking a pill, which activates a transcription factor.
  • tissue plasminogen activator in the case of a heart attack, production of insulin for diabetics, activation of lymphocytes in the case of infection, through the introduction of non-human receptors such as theecdysone receptor, and the like.
  • non-human receptors such as theecdysone receptor, and the like.
  • Oocytes from human fetuses Fetal ovaries are obtained from human fetuses of 16- 20 weeks gestation following elective abortion. Ovarian tissues are minced into ⁇ lmm size and cultured in Waymouth media supplemented with 15%(v/v) fetal bovine serum, 0.03 IU/ml FSH and 35 ng/ml insulin. The tissue is cultured at 37°C in 5%CO 2 air for 5 - 25 days in Falcon dishes and 3 - 40 days in Costar Transwell-COL membranes prior to induction of final maturation in the presence of LH and human follicular fluid. Patches of monolayer consisting of fibroblasts form within 2 - 3 days of the culture of the fresh tissues.
  • follicles After 1 week of culture, follicles separate out from the ovarian tissue, but remain attached to the monolayer. The maximal number of follicles separating out from the tissue appear about 1 week after initiating culture. After 40 days of culture in Costar dishes, a significant fraction of the ova reach a diameter of more than 80 ⁇ , of which about one-third are surrounded by the zona pellucida. Following induction of final maturation, extrusion of the first polar body is noted in ova grown in Costar dishes for 40 days. The mature ova may be collected and used directly for enucleation. Oocytes from mature human females. Human ova are obtained using the technique of Trotnow et al., Arch. Gynecol. 236: 211-7 (1985). A steering attachment is employed with the transducer of the Diasonics DS 1 sector scanner employing an aspiration needle gliding through a trocar, with continuous sonographic imaging of the aspiration needle. The patient is given epidural anaesthesia
  • the oocytes may then be matured in vitro as follows.
  • the immature oocytes are washed in TL-HEPES buffered medium containing 3 mg ml bovine serum bumin
  • Fraction N The oocyte cumulus complexes are placed in TCM-199 containing 10% fetal calf serum, LH and/or FSH and estradiol at 39 °C. After about 20h in maturation medium, the oocytes are removed and placed in TL-HEPES with 1 mg/ml hyaluronidase and the cumulus cells are removed by repeated pipetting through a fine-bore pipet. The stripped oocytes are screened for polar bodies and those containing polar bodies (metaphase II oocytes) are selected for further use.
  • Heifers are superovulated and artificially inseminated . Embryos are recovered from the reproductive tract on day 5 or 6 after standing estrus. The embryos are flushed from the reproductive tract with PBS and collected. In vitro matured, fertilized and cultured embryos are used as donor embryos on day 5 after fertilization. The fertilization process is described in Keefer et al., Mol. Reprod. Dev. 36: 469-74 (1993).
  • Enucleation is performed using a beveled micropipette at approximately 18h post- initiation of maturation (hpm). Enucleation is confirmed in TL-HEPES medium plus bis- benzimide (Hoechst 33342, 3 ⁇ g/ml). Nuclear transfer
  • NT unit bovine donor nucleus
  • NT units are exposed for 4 min to ionomycin-6-DMAP (2mM) followed by 4 hrs in DMAP only (2mM) in TL-HEPES supplemented with lmg/ml serum albumin and then washed for 5min in TL-HEPES supplemented with 30mg/ml serum albumin.
  • the NT units are then transferred into a microdrop of CRlaa culture media plus 10% FCS and 6mg/ml serum albumin in 4 well plates containing a confluent feeder layer of mouse embryonic fibroblasts.
  • the NT units are cultured for 3 more days at 38.5 °C and 5% CO 2 .
  • the culture medium is changed every 3 days until day 12 after the initiation of activation. At this time about 50 cells should be formed, which cells are mechanically removed from the zona and used to produce embryonic cell lines.
  • mice embryonic fibroblasts Primary cultures of mouse embryonic fibroblasts are obtained from 14 - 16 day old murine fetuses. After the head, liver, heart and alimentary tract are aseptically removed, the embryos are minced and incubated for 30min at 37°C in prewarmed trypsin EDTA solution (0.05% try ⁇ sin/0.02% EDTA; GTBCO, Grand Island, NY). Fibroblast cells are plated in in tisssue culture flasks and cultured in alpha-MEM medium (BioWhittaker, Walkersville, MD) supplemented with 10% FCS, penicillin (lOORJ/ml) and streptomycin (50 ⁇ l/ml).
  • alpha-MEM medium BioWhittaker, Walkersville, MD
  • embryonic fibroblasts in 35 x 10 Nunc culture dishes (Baxter Scientific, McGaw Park, IL) are irradiated.
  • the irradiated fibroblasts are grown and maintained in a humidified atmosphere with 5% CO 2 in air at 37 °C.
  • Culture plates having a uniform monolayer of cells are used to culture embryonic cell lines. Production of embryonic cell lines:
  • NT unit cells obtained as described above were washed and plated directly onto irradiated feeder fibroblast cells (see above). These cells included those of the inner portion of the NT unit.
  • the cells are maintained in a growth medium consisting of alpha- MEM supplemented with 10% FCS and O.lmM ⁇ -mercaptoethanol. Growth medium is exchanged every 2 to 3 days. The initial colony is observed by the second or third day of culture. The colony is propagated and exhibits a similar morphology to previously disclosed mouse and bovine embryonic stem (ES) cells. Individual cells within the colony are not well defined and the perimeter of the colony is refractile and smooth in appearance. The cells have an epithelial appearance.
  • ES mouse and bovine embryonic stem
  • Chimeric NT units are selected and implanted in cows according to the following procedure: 1-5 NT units are implaned in the uterus. The resulting calves are grown to the appropriate stage for oocyte development, at which time the ova are aspirated and isolated. Alternatively, oocytes are obtained from adult females by superovulation and ultrasound- guided oocyte retrieval or from slaughtered cow ovaries.
  • the chimeric ova obtained as described above are treated as described above for growth and enucleation, and are then ready to be used for receipt of a human nucleus.
  • Human epithelial cells are lightly scraped from the inside of the mouth from a consenting human adult with a standard glass slide. The cells are washed off the slide into a Petri dish containing PBS with Ca or Mg. The cells are pipetted through a small-bore pipette to break up cell clumps into a single cell suspension. The cells are then transferred into a microdrop of TL-HEPES media containing 10% FCS under oil for nuclear transfer to enucleated chimeric bovine oocytes comprising human mitochondria using the procedure described above.
  • the allochimeric cells may emplpy nuclei from substantially any convenient human cell source, such a fibroblasts, epithelial cells, keratinocytes, blood lymphocyte or bladder epithelial cell.
  • the allochimeric cells are grown in culture and directed into various differentiation patterns, as described by Stice et al., (1998), supra, including preparation of myocytes.
  • allochimeric human cells having a human nucleus and human mitochondria from different individuals. These cells find extensive use in that they provide a simulated natural human cell for studying cellular processes in the development of embryos and differentiation to differentiated cell types. The cells can also be used for producing differentiated cells for cell therapy or production of human factors.
  • the subject cells provide cells which produce only human proteins, so as to be less likely to induce an immune response, permit genetic manipulation of the nucleus, to provide human cells which may be introduced into an individual who is the source of the nucleus, but where the nucleus is modified for therapeutic or other purposes, and provide a source of genotypically identical cells, which may be used for cloning of a particular genotype.

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