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  • 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]
• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
  • A61P25/16—Anti-Parkinson drugs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • 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
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  • C12N2517/00—Cells related to new breeds of animals
  • C12N2517/04—Cells produced using nuclear transfer

Definitions

• the present invention relates to production of primate embryonic stem (ES) cells that are MHC-matched to a specific donor individual. More particularly, it relates to ES cells that are designed to be genetically identical for all nuclear genes to the donor.
• ES primate embryonic stem
• transplantation therapies Such cells derived from undifferentiated primate ES cells were in part developed to provide source material for transplantation therapies.
• ES cell-based transplantation therapies a potential problem with ES cell-based transplantation therapies is the possibility of immune rejection of the transplanted cells. While there are a number of drugs that suppress immune rejection responses, they typically have side effects and significant cost. It is preferable to provide transplantation therapies which minimize or eliminate the need for such drugs.
• stem cells e.g. capable of prolonged undifferentiated proliferation, capable of differentiation of the three embryonic germ layers.
• MHC- compatible I mean that the cell has a major histocompatibility complex that matches that of the selected donor.
• the invention provides a purified preparation of primate (preferably human) embryonic stem cells which (i) is capable of proliferation in an in vitro culture, (ii) maintains the potential to differentiate to derivatives of endoderm, mesoderm, and ectoderm tissues in the culture, (iii) is inhibited from differentiation when cultured on a fibroblast feeder layer, and (iv) has a major histocompatibility complex from a foreign source (e.g. one derived from a donor) .
• a foreign source e.g. one derived from a donor
• the cell is genetically identical with respect to all nuclear genes to a specified nuclear donor .
• ES cell are viable with changes in the status of imprinted genes.
• the derivation of ES cells by the transfer of differentiated cell nuclei to an enucleated oocytes should allow the formation of histocompatible ES cells even when the nucleus is from a cell that would not allow the cloning of an intact adult primate.
• a method for creating the aforesaid cells wherein the cells are derived by transplantation of cell nucleus from a differentiated cell of the donor to an enucleated primate (preferably human) oocyte.
• an enucleated primate preferably human
• ES cells By reprogramming the nucleus of a differentiated cell from a specific individual to an ES cell nuclear state, it is possible to make ES cells that are MHC- matched to the specific individual. So, for example, one can take a skin fibroblast or other cell from a patient with juvenile onset diabetes, reprogram the differentiated nucleus by transfer to an enucleated oocyte, culture the nuclear transfer product to the blastocyst stage, derive undifferentiated ES cells from the inner cell mass, differentiate the ES cells to pancreatic ⁇ -cells, and use those ⁇ -cells for transplantation therapy to treat the diabetes in the same way that whole pancreas transplants are currently used for this purpose (albeit without the need for immunosuppressive drugs) .
• the reprogrammed cells would be genetically matched for all nuclear genes of the nuclear donor including genes of the major histocompatibility complex (MHO . Immune rejection of these cells by the individual that donated the nucleus is therefore much less likely.
• the MHC-matched ES cells can be formed by fusion between differentiated cell karyoplasts from a donor and primate ES or primate embryonal carcinoma (EC) cell cytoplasts.
• Human embryonal carcinoma (EC) cells are the stem cells of teratocarcinomas .
• EC cells are essentially the malignant equivalent to ES cells, and some human EC cell lines maintain the potential to form all three embryonic germ layers. Because human EC cells are highly aneuploid (lack a normal complement of chromosomes) , they have a significantly more restricted developmental potential than ES cells.
• the objects of the present invention therefore include providing :
• Primate oocytes can then be activated by known osmotic (J. Levron et al . , 3 Zygote 157-161 (1995)), electrical (V. Marshall et al . , 9 Abstr. Serv. 98 J. Reprod. Fertil. (1992)), chemical (6-DMAP/ionomycin, SR 2+ or cyclohexamide - G. Wu, 55 Biol. Reprod. 260-270 (1996); L. Meng et al . , 57 Biol. Reprod. 454-459 (1997); T. Wakayama et al. , 394 Nature 369 (1998)), or biological (injection of sperm factor - H. Wu e_t al .
• Fibroblasts or other donor cell types can be prepared from fetal or postnatal tissues (e.g. skin biopsy) by mechanical disruption followed by trypsinization and cultured in DMEM-10% Fetal Calf Serum (FCS) . See generally E. Robertson, Teratocarcinoma And Embryonic Stem Cells : A Practical Approach, IRL Press, Washington, D.C. 71-112 (1987) .
• Mil oocytes are proposed to be incubated in transfer medium containing the cytoskeletal inhibitor cytochalasin B (7.5 ⁇ g/ml) . See generally L. Meng et al . , 57 Biol. Reprod. 454-459 (1997) .
• Enucleation is then proposed to be accomplished with a beveled micropipette (J. McGrath et al . , 228 Exper. Zoo . 355-362 (1983) and J. McGrath et al. 220 Science 1300-1302 (1983) , and enucleation confirmed by staining of the removed karyoplast with the dye Hoechst 33342 (3 ⁇ g/ml) and direct observation under epifluorescence . See generally S. Stice et al . , 54 Biol. Reprod. 100-110 (1996) . A single donor cell is then proposed to be selected with the micropipette and injected under the zona pellucida. Fusion
• Fusion of the donor cell and oocyte cytoplast are proposed to be accomplished by viral (J. McGrath et al., 228 Exper. Zoo . 355-362 (1983) and J. McGrath et al. 220 Science 1300-1302 (1983), chemical (M. Sims et al., 91 P.N.A.S. USA 6143-6173 (1994), or electrical methods (L. Meng et al., 57 Biol. Reprod. 454-459 (1997).
• nuclei are proposed to be injected directly into the cytoplasm using techniques analogous to the injection techniques described in T. Wakayama et al . , 394 Nature 369 (1998)
• the nuclear transfer products are proposed to be cultured to the blastocyst stage in standard medium used in human IVF clinics, such as S1/S2 or G1.2/G2.2 medium (Scandinavian IVF Science AB, Gothenburg, Sweden) .
• the inner cell mass of the resulting blastocyst should then be removed by immunosurgery (D. Solter et al . , 72 P.N.A.S. USA 5099-5102 (1975)), and cultured on mitotically inactivated fibroblasts. After approximately 9-15 days, the resulting mass of cells should then be dissociated, replated on fibroblasts, and ES cells selected by their characteristic morphology and expanded. See J. Thomson et al . , 38 Curr. Top.
• ES cells or preferably ES cell cytoplasts
• rhesus ES cells XX karyotype
• primary embryonic fibroblasts an XY karyotype
• the rhesus ES cells and fibroblasts containing these markers are then proposed to be fused by electrofusion or by other chemical fusion methods. See generally N. Duzgunes 220 Methods Enzymol . (1993), and heterokaryons selected for hygromycin/neomycin resistance.
• the resulting clones are proposed to be expanded, karyotyped, injected into SCID mice, and analyzed for contributions to derivatives of all three embryonic germ layers. This will determine whether undifferentiated ES cells can reprogram differentiated nuclei using this technique to create tetraploid heterokaryons.
• rhesus ES cell cytoplasts and neomycin resistant fibroblast karyoplasts be prepared and fused by similar methods. See generally N. Duzgunes, 220 Methods Enzymol. (1993) . The fusion products will be plated on neomycin resistant primary fibroblasts under
• ES cells can be characterized by their ability to differentiate to derivatives of all three embryonic germ layers, endoderm, mesoderm, and ectoderm even after prolonged culture. This is demonstrated either by letting the cells over-grow and pile up in culture, when spontaneous differentiation occurs, or by injecting into immunocompromised mice, where teratomas form with cells representing all three germ layers. D. Application
• a cell e.g. a skin fibroblast or other cell
• a disease e.g. juvenile onset diabetes
• the invention is designed to provide precursors for developing transplantable cells. These cells are intended to be useful for therapeutic and other purposes.

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• 1999
  • 1999-05-05 AU AU38814/99A patent/AU3881499A/en not_active Abandoned
  • 1999-05-05 WO PCT/US1999/009784 patent/WO2000012682A1/en not_active Application Discontinuation
  • 1999-05-05 JP JP2000567669A patent/JP2002523084A/ja active Pending
  • 1999-05-05 IL IL14169999A patent/IL141699A0/xx unknown
  • 1999-05-05 CA CA002342205A patent/CA2342205A1/en not_active Abandoned
  • 1999-05-05 EP EP99921665A patent/EP1117764A1/en not_active Withdrawn

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