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  • 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
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  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
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  • A61P25/00—Drugs for disorders of the nervous system
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• • A—HUMAN NECESSITIES
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  • A61P25/00—Drugs for disorders of the nervous system
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  • A61P25/16—Anti-Parkinson drugs
• • A—HUMAN NECESSITIES
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  • 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
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  • 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
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV
• • 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
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • 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
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  • 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
• 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
• a bovine enucleated oocyte a bovine enucleated oocyte.
• the present invention further relates to the use of the resultant embryonic or stem ⁇
• embryonic or stem-like cells differentiated cells, cell lines, tissues and organs. Also, the embryonic or stem-like cells
• obtained according to the present invention may themselves be used as nuclear donors in
• clones preferably transgenic cloned or chimeric animals.
• preimplantation mouse embryos are well known. (See, e.g., Evans et al., Nature, 29: 154-
• ES cells can be
• Mouse ES cells can give rise to germline chimeras when introduced into
• nuclei from like preimplantation livestock embryos support the
• endoderm-like cells but that after prolonged culture only endoderm-like cells are evident.
• STO mouse fibroblast
• pluripotent embryonic stem cells derived from porcine and bovine species which
• WO 94/26884 published November 24, 1994, disclosed embryonic stem cells which are
• the reference disclosed culturing of embryos in vitro for seven days to
• bovine ICM cells as donor nuclei in nuclear transfer procedures
• bovine ICM cells into enucleated mature oocytes bovine ICM cells into enucleated mature oocytes.
• bovine pluripotent embryonic cells in nuclear transfer
• bovine embryonic cells were fused with bison oocytes to produce some cross species NT units possibly having an inner
• the dogma has been that embryonic cells are more easily
• Collas et al taught the use of granulosa cells (adult somatic cells) to
• marker express alkaline phosphatase activity, are pluripotent, and have karyotypes which
• Parkinson's disease is caused by degenera ⁇
• neurons from a variety of brain regions can be incorporated into the adult brain.
• tissue are subject to graft rejection. Moreover, in some cases, it would be beneficial to
• animal or human oocyte e.g., an ungulate, human or primate enucleated oocyte.
• mitochondrial DNA derived from the same species preferably
• an enucleated somatic cell e.g., an enucleated human somatic
• species NT unit which may be cleaved or uncleaved.
• 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
• genes that enhance embryonic development e.g., genes that enhance embryonic development
• embryos e.g., cross-species nuclear transfer embryos, by the introduction of transgenes
• a cell death gene such as BAX, Apaf-1, or capsase, or a portion thereof, or
• apoptosis e.g., Bcl-2 or Bcl-2 family members and/or by the expression of antisense
• cyclin linked to a detectable marker e.g., one that encodes a visualizable (e.g.,
• inhibitors preferably one or more capsase inhibitors, thereby inhibiting apoptosis.
• oocyte of a different species oocyte of a different species.
• embryonic or stem-like cells produced by transplantation of the nucleus of a primate or
• human cell into an enucleated animal oocyte e.g., a human, primate or ungulate enucle-
• stem-like cells for treatment or diagnosis of any disease wherein cell, tissue or organ
• transplantation is therapeutically or diagnostically beneficial. It is another specific object of the invention to use the embryonic or stem-like cells
• stem-like cells produced accordmg to the invention for the production of differentiated
• onic or stem-like cells which cells may be used to produce genetically engineered or
• transgenic differentiated human cells, tissues or organs e.g., having use in gene therapies.
• transplantation therapy comprising the usage of isogenic or synegenic cells, tissues or
• Such therapies include by way of example treatment of diseases and injuries
• Parkinson's Huntington's, Alzheimer's, ALS, spinal cord injuries, multiple
• embryonic or stem-like cells produced according to the invention for gene therapy in
• transgenic animals e.g., non-transgenic animals
• transgenic animals can be used to produce,
• polypeptides e.g., therapeutics or nutripharmaceuticals.
• Figure 1 is a photograph of a nuclear transfer (NT) unit produced by transfer of an
• FIGS. 2 to 5 are photographs of embryonic stem-like cells derived from a NT
• the present invention provides a novel method for producing embryonic or stem-
• bovine-goat and bovine-bison have been reported, e.g., bovine-goat and bovine-bison, they did
• 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
• NT nuclear transfer
• transplantation involving the introduction of a differentiated donor cell or nucleus into an
• enucleated oocyte of a genetically dissimilar species e.g., the transplantation of cell
• nuclei from a differentiated animal or human cell e.g., adult cell, into the enucleated egg
• the 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
• embryonic or stem-like cells refer to cells produced
• the present application refers to such cells as stem ⁇
• stem- like cells may possess the mitochondria of the oocytes used for nuclear transfer, and thus not
• bovine oocyte resulted in the formation of nuclear transfer units, the cells of which upon
• bovine oocytes and human oocytes and likely mammals in general
• oocytes in general comprise factors
• RNAs and/or telomerase may comprise material RNAs and/or telomerase.
• 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 amphibians, rodents, rabbits, guinea pigs, etc. Further, using similar methods, it should
• blastocysts to produce human ES cells.
• the present invention involves the
• NT unit containing cells which may be used to obtain embryonic
• nucleus may be effected before or after nuclear transfer.
• the invention may be effected before or after nuclear transfer.
• the invention may be effected before or after nuclear transfer.
• the invention may be effected before or after nuclear transfer.
• the invention may be effected before or after nuclear transfer.
• the invention may be effected before or after nuclear transfer.
• the invention may be effected before or after nuclear transfer.
• the invention may be effected before or after nuclear transfer.
• the invention may
• oocyte of another species e.g., another ungulate or non-ungulate, by injection or fusion
• the cells of such NT units may be used to produce embryonic or stem-like cells or cell
• 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
• the embryonic or stem-like cells will be produced by a nuclear transfer
• oocytes obtained from a suitable source, e.g. a mammal and most preferably
• a primate or an ungulate source e.g. bovine
• steps (iii) and (iv) may be effected in either order;
• Human or animal cells preferably mammalian cells, may be obtained and cultured
• Human and animal cells useful in the present invention include,
• fibroblasts fibroblasts, cardiac muscle cells, and other muscle cells, etc.
• human cells fibroblasts, cardiac muscle cells, and other muscle cells, etc.
• used for nuclear transfer may be obtained from different organs, e.g., skin, lung, pancreas,
• Suitable donor cells include Suitable donor cells,
• cells useful in the subject invention may be obtained from any cell or organ of the
• the body This includes all somatic or germ cells.
• the donor cells or nucleus are selected from the body.
• such donor cells will be in the Gl cell cycle.
• the resultant blastocysts may be used to obtain embryonic stem cell lines
• epithelial cells derived from the oral cavity of a human donor and adult human
• cell nuclei may be obtained from both human somatic and
• Zygote gene activation is associated with hyperacetylation of Histone H4.
• Trichostatin-A has been shown to inhibit histone deacetylase in a reversible manner
• butyrate is also believed to cause hyper-acetylations of histones by
• donor cells may be exposed to Trichostatin-A or another
• 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
• the oocytes used for nuclear transfer may be obtained from animals including
• Suitable mammalian sources for oocytes include sheep,
• the oocytes will be obtained from primates
• oocytes e.g., a bovine.
• Methods for isolation of oocytes are well known in the art. Essentially, this will
• 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
• oocytes must generally be matured in vitro before these cells may be used
• recipient cells for nuclear transfer, and before they can be fertilized by the sperm cell
• prophase I oocytes from animal ovaries, e.g., bovine ovaries obtained at a
• oocytes generally occurs about 18-24 hours post-aspiration.
• this period of time is known as the "maturation period.”
• the “maturation period” As used herein for
• metaphase II stage oocytes which have been matured in vivo have
• oocytes are collected surgically from either non-superovulated or superovulated cows or
• gonadotropin hCG
• hCG gonadotropin
• the oocyte can be or is sufficiently "activated" to treat the intro ⁇
• the oocyte activation period generally ranges from about 16-52 hours, preferably about
• immature oocytes may be washed in HEPES buffered hamster
• HECM embryo culture medium
• TCM tissue culture medium
• gonadotropins such as luteinizing hormone (LH) and follicle stimulating
• FSH fluorescence hormone
• estradiol estradiol
• the oocytes will typically be enucleated.
• the oocytes Prior to enucleation the oocytes will preferably be removed and placed in HECM
• the stripped oocytes are then screened for polar bodies, and the selected
• metaphase II oocytes as determined by the presence of polar bodies, are then used for nuclear transfer. Enucleation follows. As noted above, enucleation may be effected
• Enucleation may be effected by known methods, such as described in U.S. Patent
• 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
• Enucleation may be accomplished microsurgically using a micropipette to remove
• the oocytes may then be screened to identify
• This screening may be effected by
• oocytes that have been successfully enucleated can then be placed in a suitable culture
• the recipient oocytes will typically be enucleated at a time
• Enucleation may be effected before, simultaneous or after nuclear transfer. Also,
• enucleation may be effected before, after or simultaneous to activation.
• a single animal or human cell or nucleus derived therefrom which is typically
• endogenous nucleus may alternatively be effected after nuclear transfer.
• the cells may be fused by electro-
• Electrofusion is accomplished by providing a pulse of electricity that is sufficient
• media can be used including e.g., sucrose, mannitol, sorbitol and phosphate buffered
• Fusion can also be accomplished using Sendai virus as a fusogenic agent
• the human or animal cell and oocyte are electrofused in a 500 ⁇ m
• a suitable medium e.g., one identified infra. -
• oocyte before or proximate (simultaneous) to nuclear transfer, which may or may not be
• activation may be effected from about twelve hours prior to
• activation is effected simultaneous or shortly after nuclear transfer, e.g., about four to nine
• the NT unit may be activated by known methods. Such methods include, e.g.,
• activation may be achieved by application of known activation
• oocyte activation may be effected by simultaneously or sequentially:
• cytoplasm e.g., magnesium, strontium, barium or calcium, e.g., in the form of an iono-
• Phosphorylation may be reduced by known methods, e.g., by the addition of kinase
• inhibitors e.g., serine-threonine kinase inhibitors, such as 6-dimethylamino-purine,
• staurosporine 2-arninopurine, and sphingosine.
• phosphorylation of cellular proteins may be inhibited by
• a phosphatase into the oocyte, e.g., phosphatase 2A and phosphatase 2B.
• nucleus is of ungulate origin, e.g., a sheep, buffalo, horse, goat, bovine, pig and/or
• oocyte is of ungulate origin, e.g., sheet, pig, buffalo, horse, goat, bovine, etc.,
• activation may be effected before, simultaneous, or after nuclear
• Activation is preferably effected after or proximate to in vitro or in
• vivo maturation of the oocyte e.g., approximately simultaneous or within about 40 hours
• Activated NT units may be cultured in a suitable in vitro culture medium until the
• FCS fetal calf serum
• TCM-199 Tissue Culture Medium- 199 (TCM-199) + 10% fetal calf serum
• TCM-199 Tissue Culture Medium- 199 (TCM-199) + 10% fetal calf serum
• TCM-199 Tissue Culture Medium- 199 (TCM-199) + 10% fetal calf serum
• TCM-199 Tissue Culture Medium- 199 (TCM-199) + 10% fetal calf serum
• TCM-199 10% fetal calf serum
• TCM-199 TCM-199, and 1 to 20% serum supplement
• fetal calf serum including fetal calf serum, newborn serum, estrual cow serum, lamb serum or steer
• a preferred maintenance medium includes TCM-199 with Earl salts, 10% fetal
• BRL cells and uterine cells and STO cells BRL cells and uterine cells and STO cells.
• human epithelial cells of the endometrium secrete leukemia
• LIF inhibitory factor
• CR1 contains the nutritional substances necessary to support an embryo.
• hemicalcium L-lactate in amounts ranging from 1.0 mM to 10 mM, preferably 1.0 mM
• Hemicalcium L-lactate is L-lactate with a hemicalcium salt incorporated
• 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-
• Suitable feeder layers include, by way of example, fibroblasts and epithelial cells, e.g.,
• fibroblasts e.g., mouse or rat
• STO and SI-m220 feeder cell lines e.g., BRL cells
• the feeder cells will comprise mouse embryonic
• fibroblasts Means for preparation of a suitable fibroblast feeder layer are described in
• the NT units are cultured on the feeder layer until the NT units reach a size
• these NT units will be cultured until they reach a size of at least
• the culturing will be effected under suitable conditions, i.e., about 38.5 °C
• Examples of a culture media suitable for human embryo culture include the medium
• Another preferred medium comprises ACM + uridine + glucose + 1000 IU of LIF.
• the cells used in the present invention will preferably be
• mammalian somatic cells most preferably cells derived from an actively
• the donor cell will be genetically modified by the addition, deletion or
• the donor cell e.g., a keratinocyte or fibroblast, e.g., of human, primate or bovine origin, may be transfected or transformed
• DNA construct that provides for the expression of a desired gene product, e.g.,
• lymphokines e.g., IGF-I, IGF-II,
• interferons such as colony stimulating factors, connective tissue polypeptides such as collagens,
• the donor cells may be modified prior to nuclear transfer
• One aspect of the invention will involve genetic modification of the donor cell
• a human cell e.g., a human cell, such that it is lineage deficient and therefore when used for nuclear
• cells when used for nuclear transfer.
• cells may be genetically modified
• mesoderm, endoderm or ectoderm specific genes examples thereof include:
• Mesoderm SRF, MESP- 1 , HNF-4, beta-I integrin, MSD ; Endoderm: GATA-6, GATA-4;
• Ectoderm RNA helicase A, H beta 58.
• a desired somatic cell e.g., a human keratinocyte, epithelial cell or
• fibroblast will be genetically engineered such that one or more genes specific to
• a preferred genetic system for effecting "knock-out" of desired genes is
• PPS positive-negative selection
• 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
• An ectoderm deficient cell can be produced by deletion and/or impairment of one
• RNA helicase A or H beta 58 genes are or both of RNA helicase A or H beta 58 genes.
• These lineage deficient donor cells may also be genetically modified to express
• 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
• the donor cell can be modified such that it is "mortal". This can be
• Another preferred embodiment of the present invention is the production of
• ES cells and/or offspring if the blastocysts are to be implanted into a female surrogate.
• an expression construct can be constructed containing a strong constitutive mammalian promoter
• markers e.g,. neomycin, ADA, DHFR, and a poly-A sequence, e.g., bGH polyA
• insulates by the inclusion of insulates. It is anticipated that these genes will be expressed early on
• genetically modified donor cells should produce blastocysts and preimplantation stage
• Still another aspect of the invention involves the construction of donor cells that
• genes that reportedly protect cells from programmed cell death include, by way of
• donor cells can be constructed wherein genes that induce apoptosis are
• this can be effected by introducing a DNA construct that provides
• the gene can be "turned on” by culturing the embryo
• regulatable or constitutive promoter e.g., PGK, SV40, CMV, ubiquitin, or beta-actin, an
• IRES a suitable selectable marker
• poly-A sequence can be constructed and
• a desired donor mammalian cell e.g., human keratinocyte or fibroblast.
• genes that may be expressed in the antisense orientation include BAX, Apaf-1, and
• transgene may be introduced that encodes for methylase or
• donor cells may be constructed containing both modifications, i.e., impairment of apoptosis-inducing genes
• Another means of enhancing cloning efficiency is to select cells of a particular cell
• a particular cyclin DNA may be operably linked
• a detectable marker e.g., green fluorescent protein
• GFP GFP
• cyclin gene should be suitable for use in the claimed invention. (See, e.g., King et al,
• Cyclins are proteins that are expressed only during specific stages of the cell cycle.
• donor cells will be constructed that express one or more
• a particular cyclin DNA may be operably
• telomere linked to a regulatory sequence, together with a detectable marker, e.g., green fluorescent
• GFP GFP protein
• cyclin Dl gene which can be used to select for cells that
• the present invention provides different methods for enhancing
• nuclear transfer efficiency preferably a cross-species nuclear transfer process.
• nuclei or cells of one species when inserted or
• proteases such as capsases can cause
• capsase inhibitors include, but not limited to
• capsase-4 inhibitor I 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
• the foregoing methods may be used to increase the efficiency of nuclear transfer by enhancing subsequent blastocyst and
• This is preferably effected by taking the clump of cells which comprise the NT unit,
• a feeder layer e.g., irradiated fibroblast cells.
• a feeder layer e.g., irradiated fibroblast cells.
• the cultured NT unit which is preferably at least 50 cells in size.
• cytosol may facilitate the dedifferentiation process. This can be accomplished by re-
• the donor cell may be fused with an enucleated oocyte
• the cells are maintained in the feeder layer in a suitable growth medium, e.g.,
• the growth medium is changed as often as necessary to optimize growth,
• 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
• the embryonic or stem-like cells and cell colonies obtained will typically exhibit
• the cells exhibit a morphology more similar to mouse
• the individual cells of the human ES-line cell colony are not
• the cell colony has a longer cell doubling time, about twice that of mouse ES
• 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
• Such cells are actual human or primate embryonic stem cells
• mesoderm a differentiated tissue
• 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 tasks
• embryonic or stem-like cells may be used for cell
• Still another object of the present invention is to improve the efficacy of nuclear
• human mitochondrial DNA will be derived from cells of the
• donor e.g., liver cells and tissue.
• tissue culture or from tissue.
• tissue or from tissue. The particular cells or tissue will be isolated from cells in tissue culture, or from tissue. The particular cells or tissue will be isolated from cells in tissue culture, or from tissue. The particular cells or tissue will be isolated from cells in tissue culture, or from tissue. The particular cells or tissue will be isolated from cells in tissue culture, or from tissue. The particular cells or tissue will be isolated from cells in tissue culture, or from tissue. The particular cells or tissue will be isolated from cells in tissue culture, or from tissue. The particular cells or tissue will be isolated from cells in tissue culture, or from tissue. The particular cells or tissue will be isolated from cells in tissue culture, or from tissue. The particular cells or tissue will be isolated from cells in tissue culture, or from tissue. The particular cells or tissue will be isolated from cells in tissue culture, or from tissue. The particular cells or tissue will be isolated from cells in tissue culture, or from tissue. The particular cells or tissue will be isolated from cells in tissue culture, or from tissue. The particular cells or tissue will be isolated from cells in tissue culture, or from tissue. The particular cells or tissue will
• mitochondria may be used as sources of mitochondria include fibroblasts, epithelium, liver, lung,
• keratinocyte stomach, heart, bladder, pancreas, esophageal, lymphocytes, monocytes,
• hematopoietic cells and tissues containing such cells.
• mitochondria can be isolated from tissue culture cells and rat liver.
• preferred source of mitochondria comprises
• the isolated DNA can also be further purified, if desired,
• mouse embryonic stem (ES) cells are capable of ES cells
• embryonic or stem-like cells produced according to the invention should possess similar
• the embryonic or stem-like cells according to the invention will have a differentiation capacity.
• the embryonic or stem-like cells according to the invention will have a differentiation capacity.
• the subject human embryonic or stem-like cells may 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
• cartilage cells epithelial cells, urinary tract cells, etc., by culturing such cells in
• stem cells subjecting stem cells to an induction procedure comprising initially culturing aggregates
• references are exemplary of reported methods for obtaining differentiated cells from
• the subject embryonic or stem-like cells to obtain desired differentiated cell types, e.g.,
• Bcl-xl might be useful for enhancing in vitro development of specific cell lineages.
• Bcl-2 prevents many, but not all, forms of apoptotic cell death that occur during
• the subject embryonic or stem-like cells may be used to obtain any desired
• human hematopoietic stem cells may be used in medical
• epithelial cells or lymphocytes with an enucleated oocyte, e.g., bovine oocyte, obtaining
• hematopoietic 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 obtained from a patient with a neurological disorder.
• oocyte e.g., a primate or bovine oocyte, human
• embryonic or stem-like cells obtained therefrom, and such cells cultured under
• transplantation of such human neural cells include, by way of example, Parkinson's
• donor cells may be any suitable cells.
• CD34-neo may be used for selection of hematopoietic cells, Pwl-neo for
• rejection i.e., rejection of the transplanted tissue which may occur because of host-vs- graft or graft-vs-host rejection. Conventionally, rejection is prevented or reduced by the
• anti-rejection drugs such as cyclosporin.
• the present invention should eliminate, or at least greatly
• anti-rejection drugs such as cyclosporine, imulan, FK-506,
• glucocorticoids and rapamycin, and derivatives thereof.
• diseases i.e., hypercholesterolemia, heart diseases, cartilage replacement, burns, foot
• ulcers ulcers, gastrointestinal diseases, vascular diseases, kidney disease, urinary tract disease,
• This methodology can be used to replace defective genes, e.g., defective immune
• cystic fibrosis genes or to infroduce genes which result in the expression
• therapeutically beneficial proteins such as growth factors, lymphokines, cytokines,
• the gene encoding brain derived growth factor may be any suitable enzymes, etc.
• the gene encoding brain derived growth factor may be any suitable enzymes, etc.
• the gene encoding brain derived growth factor may be any suitable enzymes, etc.
• immortalized cell lines either neural or non-neural (myoblast and fibroblast) derived
• asfrocytes have been transfected with BDNF gene using retro viral
• cattle and pig embryonic cell lines can be transfected and
• desired genes may be introduced into the subject human embryonic
• stem-like cells and the cells differentiated into desired cell types, e.g., hematopoietic
• epidermal growth factor include, by way of example, epidermal growth factor, basic fibroblast growth factor, glial
• neurotrophin-4/5 ciliary neurotrophic factor
• AFT-1 ciliary neurotrophic factor
• cytokine genes interleukins
• genes encoding therapeutic enzymes collagen, human serum albumin, etc.
• TK thymidine kinase
• cells may be selectively eliminated at any time from a patient upon gancyclovir
• the subject embryonic or stem-like cells preferably human cells, also may be used.

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