WO2008104065A1 - Préparations de cellules neuronales et procédés de production et d'utilisation correspondants - Google Patents

Préparations de cellules neuronales et procédés de production et d'utilisation correspondants Download PDF

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WO2008104065A1
WO2008104065A1 PCT/CA2008/000365 CA2008000365W WO2008104065A1 WO 2008104065 A1 WO2008104065 A1 WO 2008104065A1 CA 2008000365 W CA2008000365 W CA 2008000365W WO 2008104065 A1 WO2008104065 A1 WO 2008104065A1
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cells
neural
multipotent
cell
lin
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PCT/CA2008/000365
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Ian Rogers
Robert Casper
Shawn J. CHUA
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Mount Sinai Hospital
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • 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
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • 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
    • A61K35/44—Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
    • 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
    • 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
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • 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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
    • 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
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/119—Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/26—Flt-3 ligand (CD135L, flk-2 ligand)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/90—Polysaccharides
    • C12N2501/91—Heparin

Definitions

  • the invention relates to methods for producing neural cells or precursors thereof, in particular neural cell preparations comprising oligodendrocytes or precursors thereof, pharmaceutical compositions comprising the neural cells or preparations, and the use of the neural cells, preparations and compositions in research or commercial applications.
  • SCI spinal cord injury
  • oligodendrocyte development from pluripotent neuroepithelial cells into oligodendrocyte precursor cells is dependent on positive and negative signals (Wada, Kagawa et al, 2000).
  • One positive signal involved is Sonic hedgehog (Shh), which can induce OPC development (Roelink, Porter et al. 1995; Pringle, Yu et al. 1996).
  • OPCs at this stage express the markers OHgI (Lu, Yuk et al. 2000; Zhou, Wang et al. 2000) and NG2 proteoglycan (Levine and Nishiyama 1996).
  • OPCs proliferate in response to PDGF and differentiate into oligodendrocytes (Fruttiger, Karlsson et al, 1999).
  • PDGF Rostiger, Karlsson et al. 1988
  • TH thyroid hormone
  • Candidate cells for remyelination include neural progenitor cells (Park, Liu et al. 1999), bone marrow stem cells (Sanchez-Ramos 2002), embryonic stem cells (Liu, Qu et al. 2000), olfactory ensheathing cells (Chuah, Choi-Lundberg et al. 2004) and umbilical cord blood cells (Sanchez-Ramos, Song et al. 2001).
  • neural progenitor cells Park, Liu et al. 1999
  • bone marrow stem cells Sanchez-Ramos 2002
  • embryonic stem cells Liu, Qu et al. 2000
  • olfactory ensheathing cells Chouah, Choi-Lundberg et al. 2004
  • umbilical cord blood cells Sanchez-Ramos, Song et al. 2001
  • Retinoic acid (RA) and neural growth factor (NGF) can induce UCB mononuclear cells to express neural (Musachi, Tujl) and astrocyte (GFAP) markers (Sanchez-Ramos, Song et al. 2001 ).
  • UCB mononuclear cells were injected into a neonatal rat brain and 20% of the cells were found to engraft and express the glial marker GFAP and the neural markers Tuj 1 (Zigova, Song et al.
  • a mouse model of amyotrophic lateral sclerosis was used to demonstrate that UCB cells have the ability to delay the onset of the disease and cells were positive for neural (nestin, Tuj 1) and glial (GFAP) markers (Garbuzova-Davis, Willing et al. 2003).
  • GFAP glial
  • a mouse model of spinal cord injury was injected with mononuclear UCB cells and found to increase locomotor activity (Saporta, Kim et al. 2003).
  • the invention provides neural cell preparations comprising neural cells differentiated from multipotent cells having properties of multipotential mesenchymal cells.
  • Neural cells may have properties of glial cells, neurons or oligodendrocytes.
  • the neural cells are characterized by one or more of the following properties: an oligodendrocyte morphology, Lin neg , and expressing the oligodendrocyte markers 2',3'-cyclic nucleotide 3'- phosphohydrolase (CNPase), oligodendrocyte marker 04, myelin and/or galactocerebroside (GaIC).
  • Neural cells can be isolated and purified from a neural cell preparation of the invention.
  • the invention provides cell preparations isolated and cultured in vitro enriched for characteristics of oligodendrocytes.
  • the invention provides neural cell preparations comprising neural cells differentiated in vitro from multipotent cells having properties of multipotential mesenchymal cells and having an oligodendrocyte morphology and expressing CNPase, 04, myelin and/or 5 GaIC.
  • the neural cells can have functional features of oligodendrocytes including one or more of the following: a) the ability to myelinate ganglia in a coculture assay, b) the ability to restore myelin to demyelinated axons in vivo, and, c) the ability to improve neurological function in subjects.
  • the invention provides neural cell preparations comprising neural cells l o differentiated in vitro from multipotent cells having properties of multipotential mesenchymal cells wherein the neural cells having characteristics of glial or neuronal cells
  • the invention also relates to a system or method for production of purified neural cell preparations of the invention comprising culturing multipotent cells having properties of multipotential mesenchymal cells in the presence of or media comprising one or more
  • the multipotent cells are cultured in neural differentiation media for at least one, two, three, or four weeks. In another embodiment of the
  • the multipotent cells are cultured in the presence of or media comprising retinoic acid.
  • the invention also relates to a system or method for production of purified neural cell preparations of the invention comprising culturing multipotent cells having properties of multipotential mesenchymal cells in the presence of or media comprising one or more
  • the multipotent cells may be produced by culturing Lin neg stem and progenitor cells, preferably isolated from umbilical cord blood, under proliferation conditions, in particular in the presence of or media comprising positive
  • the multipotent cells are CD45 + HL A-ABC + cells, more particularly CD45 + HL A- ABC + Lm ' cells.
  • the multipotent cells may comprise neural precursor cells, in particular oligodendrocyte precursor cells.
  • the multipotent cells may comprise cells that express nestin and/or neurofilament, more preferably nestin and neurofilament.
  • Another aspect of the invention is an enriched or purified neural cell preparation, including multipotent cells comprising neural precursor cells, produced by a method of the invention.
  • the invention provides a purified neural cell preparation consisting essentially of neural cells differentiated in vitro from multipotent CD45 + HLA-ABC + Lin " cells expressing nestin and neurofilament, and characterized by the following properties: an oligodendrocyte morphology , Lin neg , and expressing the oligodendrocyte markers 2',3'-cylid nucleotide 3'-phosphohydrolase (CNPase), oligodendrocyte marker 04, myelin and/or galactocerebroside (GaIC).
  • the multipotent cells and neural cell preparations may be used for the preparation of pharmaceutical compositions.
  • the invention also relates to a pharmaceutical composition, in particular a purified pharmaceutical composition, comprising multipotent cells, a neural cell preparation of the invention or neural cells or precursors thereof isolated therefrom, and a pharmaceutically acceptable carrier, excipient or diluent.
  • a pharmaceutical composition may include a targeting agent to target cells to particular tissues or organs.
  • the invention also contemplates a cell line comprising neural cells or precursors thereof derived from a cell preparation of the invention.
  • the invention also contemplates multipotent cells, neural cell preparations and pharmaceutical compositions of the invention in combination with a substrate or matrix, preferably a substrate or matrix adapted for transplantation into a patient.
  • the substrate may be an engineered biomaterial or porous tissue culture insert.
  • the multipotent cells, neural cell preparations and pharmaceutical compositions may be used in research or in medical applications.
  • the multipotent cells, neural cell preparations and compositions of the invention and cells therefrom can be used in a variety of methods (e.g. transplantation or grafting) and they have numerous uses in the field of medicine.
  • the multipotent cells and neural cell preparations may be used for the replacement of body tissues, organs, components or structures which are missing or damaged due to trauma, age, metabolic or toxic injury, disease, idiopathic loss, or any other cause.
  • the multipotent cells, neural cell preparations and pharmaceutical compositions comprising the neural cells, and cells therefrom can be used for neuronal transplantation to improve neurological deficit and to effect repair of neural/neuronal tissue, and to treat neurodegenerative diseases.
  • the invention provides use of multipotent cells, cell preparations or compositions described herein or neural cells obtained therefrom for treating neural diseases or in the preparation of a medicament for treating neural diseases.
  • the multipotent cells, cell preparations or compositions of the invention or cells therefrom are used to treat conditions associated with defects in myelination of exons.
  • the multipotent cells, cell preparations or compositions of the invention or cells therefrom are used to treat spinal cord injury.
  • the multipotent cell preparations, neural cell preparations and pharmaceutical compositions, or cells therefrom can be used in cell therapies and gene therapies aimed at alleviating disorders and diseases involving neural cells, progressive demyelination, and/or trauma of the central nervous system.
  • the invention obviates or reduces the need for human tissue to be used in various medical and research applications.
  • the invention thus provides a method of treating a patient with a condition involving neural cells, in particular a defect in neural cells, comprising transferring or administering an effective amount of multipotent cells, a neural cell preparation or pharmaceutical composition or cells therefrom, optionally with a substrate into the patient.
  • the invention provides a method of treating a patient with a condition involving neural cells comprising administering to the patient multipotent CD45 + HLA-
  • ABC + Lin cells that differentiate into neural cells or neural cells differentiated in vitro from the multipotent CD45 + HLA-ABC + Lin ' cells, wherein the neural cells have characteristics of glial cells or neurons or the neural cells are characterized by the following: an oligodendrocyte morphology, Lin neg , and expressing one or more of 2',3'-cyclic nucleotide 3'- phosphohydrolase (CNPase), oligodendrocyte marker 04, myelin and/or galactocerebroside
  • CNPase 2',3'-cyclic nucleotide 3'- phosphohydrolase
  • oligodendrocyte marker 04 myelin and/or galactocerebroside
  • the invention provides a method of treating a patient with a condition involving neural cells comprising:
  • the invention provides a method of treating a patient with a condition involving neural cells comprising: (a) culturing Lin neg stem and progenitor cells under proliferation conditions to provide multipotent cells wherein the multipotent cells are CD45+HLA-ABC+ cells that express nestin and/or neurofilament; and (b) administering multipotent cells of (a) in an effective amount to the patient to treat the condition.
  • the invention also provides a method of treating a mammalian individual suffering from a disease associated with demyelination of central nervous system axons, comprising: ( 1 ) using a method of the invention to obtain multipotent cells or a cell preparation comprising or consisting essentially of neural cells; (2) introducing the multipotent cells or neural cells from the cell preparation to the mammalian individual, in an amount effective to treat the disease.
  • the mammalian individual is a human.
  • the multipotent cells, cell preparation or cells therefrom are administered to the mammalian individual by cell transplantation.
  • the invention further provides a method for regenerating nerve tissue comprising administering to a patient in need thereof, a therapeutically effective amount of multipotent cells, a cell preparation or composition or neural cells therefrom.
  • Methods of the invention can further comprise co-administering to the mammalian individual a second pharmaceutical composition effective for treating the disease.
  • an immunosuppressive agent is co-administered with the multipotent cells, cell preparations, cell compositions or cells therefrom.
  • multipotent cells, neural cell preparations and pharmaceutical compositions of the invention or cells therefrom are used for autografting, i.e., cells from an individual are used in the same individual.
  • the multipotent cells, neural cell preparations and pharmaceutical compositions or cells therefrom are used in allografting, i.e., cells from one individual are used in another individual.
  • the multipotent cells, neural cell preparations and pharmaceutical compositions or cells therefrom are used for xenografting, i.e., transplantation from one species to another species.
  • the invention provides a method for obtaining compositions for autologous transplantation from a subject's own hematopoietic cells comprising (a) obtaining hematopoietic cells, in particular hematopoietic cells from fresh or cryopreserved umbilical cord blood or bone marrow, from a subject; (b) separating out an enriched cell preparation comprising hematopoietic stem and progenitor cells, in particular Lin ' stem and progenitor cells; (c) culturing the cells under proliferation conditions, in particular in the presence of or media comprising FGF4, SCF, and FLT-3 ligand to produce multipotent cells, more particularly CD45 + HLA-ABC + Lin " cells; and (d) culturing the multipotent cells in the presence of or in media comprising one
  • Neural cell preparations and neural compositions may be used to screen for potential therapeutics that modulate development or activity of neural cells.
  • neural cell preparations and neural compositions may be used to screen compounds for an effect on neural cells, in particular oligodendrocytes, in which the presence of the compound is correlated with cell maintenance, toxicity, or the ability to function as an oligodendrocyte.
  • the neural cell preparations and pharmaceutical compositions of the invention and cells therefrom may be used as immunogens that are administered to a heterologous recipient.
  • the neural cell preparations and pharmaceutical compositions of the invention and cells therefrom may be used to prepare model systems of disease, or to produce growth factors, hormones, etc.
  • the invention also relates to a method for conducting a regenerative medicine business. Still further the invention relates to a method for conducting a stem cell business involving identifying agents that affect the proliferation, differentiation, function, or survival of neural cells.
  • An identified agent(s) can be formulated as a pharmaceutical preparation, and manufactured, marketed, and distributed for sale.
  • the invention contemplates methods for influencing the proliferation, differentiation, or survival of neural cells by contacting a neural cell preparation or pharmaceutical composition of the invention or cells therefrom with an agent or agents identified by a method of the invention.
  • the invention also contemplates a method of treating a patient comprising administering an effective amount of an agent identified in accordance with a method of the invention to a patient with a disorder affecting the proliferation, differentiation, function, or survival of neural cells.
  • the invention also contemplates a method for conducting a drug discovery business comprising identifying factors or agents that influence the proliferation, differentiation, function, or survival of neural cells, and licensing the rights for further development.
  • the invention further contemplates a method of providing drug development wherein a neural cell preparation of the invention or progeny of neural cells in the preparation are used as a source of biological components of neural cells in which one or more of these biological components are the targets of the drugs that are being developed.
  • the invention also relates to methods of providing a bioassay.
  • the invention also features a kit including multipotent cells, neural cell preparations or pharmaceutical compositions of the invention.
  • the invention is also directed to a kit for transplantation of neural cells comprising a flask with medium and multipotent cells, a neural cell preparation or a pharmaceutical composition of the invention.
  • the invention also relates to a method of using the neural cell preparations or compositions of the invention or cells therefrom in rational drug design.
  • the invention relates to a kit for rational drug design comprising a neural cell preparation obtained by a method of the invention.
  • Figure 1 is an outline of the differentiation protocol used to produce oligodendrocyte cells from multipotent cells from umbilical cord blood Lin neg cells.
  • Figure 3 shows CNPase (oligodendrocyte marker) mRNA expression by RT-PCR. Results from 2 individual experiments show positive expression of CNPase. Primers were designed to span 2 introns. Total RNA from all Lin neg UCB cells that underwent differentiation culture conditions was isolated, and treated with DNAse.
  • Figure 4 shows that oligodendrocyte-like Lin neg UCB cells stained positive for
  • FIG. 5 shows that human umbilical cord cells in rat spinal cord tissue stained positive for myelin basic protein after six weeks. Rats underwent spinal cord injury with a 35g clip and were directly inj ected with 8 x 10 5 d8 F/S/F HUBC cells.
  • FIG. 6 shows that Lin neg cells grown in FSF medium for 8 days could be differentiated into multiple neural cell types, (a) glial, neuron and oligodendrocyte cells can be found in a single culture, (b) Cells are positive for the neurofilament protein (green). Nuclei-DAPI (Blue).
  • Figure 7 shows changes in morphology of Lin neg cells.
  • Lin neg cells grown in FSF media for 8 days are round and non-adherent (a) DSCN1434. After exposure to 5 days of differentiation media, colonies form (b) DSCN1487. After 30 days, cells exhibit egg shaped (c) DSCN0720 and spindle shaped morphologies (d) DSCN0925.
  • Figure 8 shows the following: (a). The human oligodendrocyte cell line MO3.13 is positive for GalC(green) and MBP (red); (b) MO3.13 is also positive for GaIC (green) and 04(red); (c) Oligodendrocyte-like Lin neg UCB cells stained positive for CNPase, GaI-C,
  • FIG. 9 shows differentiation of a multipotent cell preparation into neural cells.
  • the culture of UCB-derived CD45+/lin- cells in a medium containing exogenous SCF, FL and FGF results in the expansion of CD34+ and CD45+ cells.
  • the expanded cell product is capable of differentiation into neural cells, as determined by in vitro differentiation assays.
  • Prolonged culture duration resulted in 100% of the surviving cells developing neural morphology and expressing neurofilament (a).
  • Differentiation regimens to induce oligodendrocytes resulted in ⁇ 1% of multipotent cells surviving and developing oligodendrocyte morphology (b).
  • Immunocytochemistry demonstrated that the multipotent cells, after culture in neural differentiation medium, express the neural protein ⁇ -III tubulin (c).
  • the multipotent cells could also be differentiated into highly specialized tyrosine hydroxylase expressing cells after a 6-week, 3-stage differentiation regime (d).
  • Tissue culture reagents and materials are commercially available from companies such as Gibco/BRL, Nalgene-Nunc International, Sigma Chemical Co., StemCell Technologies and ICN Biomedicals. For convenience, certain terms employed in the specification and claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
  • “Patient”, “subject” or “individual” refers to an animal, preferably a human, to whom treatment, including prophylactic treatment, with the cells, preparations, and compositions of the present invention, is provided.
  • treatment including prophylactic treatment, with the cells, preparations, and compositions of the present invention.
  • the term refers to that specific animal.
  • the terms refer to a human.
  • the terms also include domestic animals bred for food, sport, or as pets, including horses, cows, sheep, poultry, fish, pigs, cats, dogs, and zoo animals.
  • a “donor” refers to an individual (animal, including a human) who or which donates cells, in particular hematopoietic cells, more particularly umbilical cord blood for use in a patient.
  • Effective amount refers to concentrations of components such as growth factors, cells, preparations, or compositions effective for producing an intended result including production of neural cell preparations, or treating a disease or condition with multipotent cells, neural cell preparations, and pharmaceutical compositions of the invention, or for effecting a transplantation of such cells, neural cell preparations or pharmaceutical compositions within a patient to be treated.
  • an effective amount can provide a dosage which is sufficient in order for prevention and/or treatment of a condition or disease in the patient compared with no treatment or another treatment.
  • administering refers to the process by which multipotent cells, neural cells, neural cell preparations, or compositions of the invention are delivered to a patient for treatment purposes.
  • Cells, preparations, or compositions may be administered a number of ways including parenteral (e.g. intravenous and intraarterial as well as other appropriate parenteral routes), intrathecal, intraventricular, intraparenchymal (including into the spinal cord, brainstem, or motor cortex), intracisternal, intracranial, intrastriatal, oral, subcutaneous, inhalation, transdermal, or intranigral among others.
  • parenteral e.g. intravenous and intraarterial as well as other appropriate parenteral routes
  • intrathecal e.g. intravenous and intraarterial as well as other appropriate parenteral routes
  • intraventricular e.g. intraparenchymal (including into the spinal cord, brainstem, or motor cortex)
  • intracisternal e.g. intracranial, intrastriatal, oral, subcutaneous, in
  • Cells, preparations, and compositions of the invention are administered in accordance with good medical practices taking into account the patient's clinical condition, the site and method of administration, dosage, patient age, sex, body weight, and other factors known to physicians.
  • administration is typically via a parenteral route, for example intravenously, by administration into the cerebral spinal fluid or by direct administration into the affected tissue in the brain.
  • a parenteral route for example intravenously, by administration into the cerebral spinal fluid or by direct administration into the affected tissue in the brain.
  • the route of administration can be a transplant directly into the striatum
  • Parkinson's disease the route of administration can be atransplant directly into the substantia nigra.
  • Cerebrospinal fluid is the preferred route of administration for amyotrophic lateral sclerosis and multiple sclerosis.
  • Intravenous administration or administration through the cerebrospinal fluid are the preferred routes of administration in lysosomal storage disease.
  • the preferred route of administration in the case of a stroke will depend on the affected tissue which can be determined using imaging techniques such as MRI.
  • Transplanting “transplantation”, “grafting” and “graft” are used to describe the process by which cells, preparations, and compositions of the invention are delivered to the site within the patient where the cells are intended to exhibit a favorable effect, such as repairing damage to a patient's tissues, treating a disease, injury or trauma, or genetic damage or environmental insult to an organ or tissue caused by, for example an accident or other activity.
  • Cells, preparations, and compositions may also be delivered in a remote area of the body by any mode of administration relying on cellular migration to the appropriate area in the body to effect transplantation.
  • pharmaceutically acceptable carrier, excipient or vehicle refers to a medium which does not interfere with the function or activity of the multipotent cells or neural cells and which is not toxic to the hosts to which it is administered.
  • a carrier, excipient or vehicle includes diluents, binders, adhesives, lubricants, disintegrates, bulking agents, wetting or emulsifying agents, pH buffering agents, and miscellaneous materials that may be needed in order to prepare a particular composition.
  • treating refers to reversing, alleviating, or inhibiting the progress of a neural disease, or one or more symptoms of such disease, to which such term applies.
  • the term also refers to preventing a neural disease, and includes preventing the onset of a neural disease, or preventing the symptoms associated with a neural disease.
  • a treatment may be either performed in an acute or chronic way.
  • the term also refers to reducing the severity of a neural disease or symptoms associated with such disease prior to affliction with the disease.
  • prevention or reduction of the severity of a disease prior to affliction refers to administration of multipotent cells or a cell preparation or pharmaceutical composition of the present invention or cells therefrom to a subject that is not at the time of administration afflicted with the disease.
  • Preventing also refers to preventing the recurrence of a disease or of one or more symptoms associated with such disease.
  • Treatment and “therapeutically,” refer to the act of treating, as “treating” is defined above.
  • Essentially refers to a population of cells or a method which is at least 20+%, 30+%,
  • a method that enriches for a given cell population enriches at least about 20+%, 30+%, 40+%, 50+%, 60+%, 70+%, 80%, 85%,
  • the targeted cell population most preferably at least about 98% of the cell population, most preferably about 99% of the cell population.
  • Isolated or purified refers to altered “by the hand of man” from the natural state i.e. anything that occurs in nature is defined as isolated when it has been removed from its original environment, or both.
  • a preparation, population or composition of cells is substantially free of cells and materials with which it may be associated in nature.
  • substantially free or substantially purified is meant at least 50% of the population are the target cells, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90%, 95% or 99% are free of other cells. Purity of a population or composition of cells can be assessed by appropriate methods that are well known in the art.
  • Gene therapy refers to the transfer and stable insertion of new genetic information into cells for the therapeutic treatment of diseases or disorders.
  • a foreign gene is transferred into a cell that proliferates to introduce the transferred gene throughout the cell population. Therefore, multipotent cells, neural cells, preparations and compositions of the invention may be the target of gene transfer, since they will produce various lineages which will potentially express the foreign gene.
  • hematopoietic cells refers to cells that are related to the production of blood cells, including cells of the lymphoid, myeloid and erythroid lineages.
  • exemplary hematopoietic cells include hematopoietic stem cells, primordial stem cells, early progenitor cells, CD34 + cells, early lineage cells of the mesenchymal, myeloid, lymphoid and erythroid lineages, bone marrow cells, blood cells, umbilical cord blood cells, stromal cells, and other hematopoietic precursor cells that are known to those of ordinary skill in the art.
  • the hematopoietic cells may be obtained from fresh blood, reconstituted cryoperserved blood, or fresh or reconstituted fractions thereof.
  • the hematopoietic cells may also be obtained from bone marrow cells.
  • the hematopoietic cells are preferably mammalian cells, more preferably the cells are primate, pig, rabbit, dog, or rodent (e.g. rat or mouse) in origin. Most preferably, the cells are human in origin.
  • the hematopoietic cells may be obtained from a fetus, a child, an adolescent, or an adult.
  • umbilical cord blood generally refers to blood obtained from a neonate or fetus. In a preferred embodiment, umbilical cord blood refers to blood obtained form the umbilical cord or placenta of newborns. Hematopoietic cells obtained from UCB offer several advantages including less invasive collection and less severe graft versus host (GVH) reaction [Gluckman et al, N. Eng. J. Med 337:373-81, 1993]. The use of umbilical cord blood also eliminates the use of human embryos as a source of embryonic stem cells. Cord blood may be obtained by direct drainage from the cord and/or by needle aspiration from the delivered placenta at the root and at distended veins.
  • VH graft versus host
  • Multipotent cells refers to cells that show at least one phenotypic characteristic of an early stage non-hematopoietic cell (e.g. stem, precursor, or progenitor non- hematopoietic cells), and preferably at least one phenotypic characteristic of an embryonic stem cell.
  • phenotypic characteristics can include expression of one or more proteins specific for early stage non-hematopoietic cells, or a physiological, morphological, immunological, or functional characteristic specific for an early stage non-hematopoietic cell or embryonic stem cell [e.g. Oct4, Nanog, Stage Specific Embryonic Antigen-3 (SSEA3), and/or Stage Specific Embryonic Antigen-4 (SSEA4)].
  • Multipotent cells can be produced by first obtaining hematopoietic cells and enriching the cells for hematopoietic stem cells and progenitor cells (sometimes referred to herein as "enriched hematopoietic cell preparation").
  • stem cells refers to undifferentiated cells that are capable of essentially unlimited propagation either in vitro, in vivo or ex vivo and capable of differentiation to other cell types.
  • Progenitor cells are cells that are derived from stem cells by differentiation and are capable of further differentiation to more mature cell types. Negative and positive selection methods known in the art can be used for enrichment of the hematopoietic cells.
  • cells can be sorted based on cell surface antigens using a fluorescence activated cell sorter, or magnetic beads which bind cells with certain cell surface antigens, in particular lineage specific cell surface antigens (e.g. CD2, CD3, CD14, CD16, CD 19, CD24, CD56, CD66b, glycophorin A and/or dextran).
  • Negative selection columns can be used to remove cells expressing lineage specific surface antigens.
  • the mature blood cells are removed.
  • the enriched hematopoietic cell preparation essentially comprises or consists essentially of Lin " stem and progenitor cells.
  • An enriched hematopoietic cell preparation can be cultured under proliferation conditions (e.g. in the presence of or media comprising positive growth factors, in particular FGF4, SCF, Flt-3 ligand) to produce multipotent cells.
  • multipotent cells are characterized as follows:
  • CD45 + HLA-ABC + cells more particularly CD45 + HLA-ABC + Lin " cells.
  • the multipotent cells are CD45 + HLA-ABC + cells that express nestin and/or neurofilament, preferably nestin and neurofilament.
  • a multipotent cell preparation may be enriched or purified and comprise cells that are at least 70%, 80%, 90%, 95%, 98%, or 99% CD45 + HLA- ABC + Lin " cells.
  • a “neural differentiation media” or “neural differentiation medium” generally refers to any cellular media which provides appropriate elements to enable efficient differentiation of multipotent cells to neural cells or preparations of the invention.
  • a differentiation medium generally comprises a minimum essential medium plus optional agents such as growth factors, non-essential amino acids, and other agents known in the art. In aspects of the invention a minimum essential medium is used which is supplemented with non-essential amino acids, glutamine, mercaptoethanol, fetal bovine serum, and one or more differentiation factors.
  • An example of a neural differentiation medium is a mixture of DMEM and F 12, optionally supplemented with N2 and/or neurobasal medium supplemented with B27.
  • a neural differentiation medium may contain serum (FCS) or be serum free.
  • a “differentiation factor” refers to an agent which can be added to neural differentiation media which induces multipotent cells to neural cells or preparations of the invention.
  • differentiation factors include antioxidants including retinoic acid, fetal or mature neuronal cells including mesencephalic or striatal cells or a growth factor or cytokine including brain derived neurotrophic factor (BDNF); glial derived neurotrophic factor (GDNF); nerve growth factor (NGF); fibroblast growth factor (FGF); transforming growth factors (TGF); ciliary neurotrophic factor (CNTF); bone-morphogenetic proteins
  • BMP leukemia inhibitory factor
  • GGF glial growth factor
  • TNF tumor necrosis factors
  • IGF insulin-like growth factors
  • CSF colony stimulating factors
  • KIT-SCF KIT receptor stem cell factor
  • SHH sonic hedge hog
  • neurotrophins such as neurotrophin 3 (NT3); triiodothyronine
  • antioxidants such as selenium, Vitamin E, and factors that increase activity of enzymes for which selenium is a co-factor such as thioredoxin reductase, and the family of iodothyronine deiodinases; thyroxine; erythropoietin; thrombopoietin; silencers (including glial-cell missing, neuron restrictive silencer factor); Src- homology-2 domain transforming protein; neuroproteins; proteoglycans; glycoproteins; neural adhesion molecules; and other cell-signaling molecules and mixtures
  • the differentiation factor is selenium, an antioxidant that is believed to participate in the upregulation of myelin genes in differentiating oligodendrocytes.
  • the differentiation factors employed are retinoic acid, FGF -2, NT3 and/or Sonic Hedgehog.
  • the differentiation factors are ligands and antibodies that bind thyroid hormone receptors on the cell surface or nucleus, exemplified by T3 (3,5,3 '-triiodo-L- thyronine) and T4 (L-thyroxin).
  • immunosuppressive agent refers to any agent which inhibits or prevents an immune response.
  • immunosuppressive agents are drugs, for example, a rapamycin; a corticosteroid; an azathioprine; mycophenolate mofetil; a cyclosporine; a cyclophosphamide; a methotrexate; a 6-mercaptopurine; FK506; 15-deoxyspergualin; an FTY 720; a mitoxantrone; a 2-amino-l,3-propanediol; a 2-amino-2[2-(4- octylphenyl)ethyl]; propane-l,3-diol hydrochloride; a 6-(3 dimethyl-aminopropionyl) forskolin; interferon and a demethimmunomycin.
  • an immunosuppressive agent is an antibody including without limitation hut 124; BTI-3
  • CBL CBL; 0KT3; ATGAM; basiliximab; daclizumab; thymoglobulin; ISAtx247; Medi-500; Medi- 507; Alefacept; efalizumab; or infliximab.
  • the immunosuppressive agent is one or more of dexamethasone, cyclosporin A, azathioprine, brequinar, gusperimus, 6-mercaptopurine, mizoribine, rapamycin, tacrolimus (FK-506), folic acid analogs (e.g., denopterin, edatrexate, methotrexate, piritrexim, pteropterin, Tomudex®, trimetrexate), purine analogs (e.g., cladribine, fludarabine, 6-mercaptopurine, thiamiprine, thiaguanine), pyrimidine analogs (e.g.
  • ancitabine azacitidine, 6-azauridine, carmofur, cytarabine, doxifluridine, emitefur, enocitabine, floxuridine, fluorouracil, gemcitabine, tegafur), fluocinolone, triaminolone, anecortave acetate, flurometholone, medrysone and prednislone.
  • a “neural disease” refers to a condition which can be treated and/or prevented using a neural cell preparation and pharmaceutical composition of the invention.
  • a neural disease includes a condition involving progressive demyelination, and trauma of the central nervous system where the ability to maintain or produce myelin may either contribute to healing or help to prevent further deterioration.
  • a neural disease may be a chronic or acute condition.
  • a neural disease includes a neurodegenerative disease.
  • the term “neurodegenerative disease” refers to a disease caused by damage to the central nervous system, in particular a disease associated with demyelination.
  • a neurodegenerative disease is a disease where damage can be reduced or alleviated by transplantation of a neural cell preparation or cells thereof to damaged areas of the brain and/or spinal cord of a subject.
  • neurodegenerative diseases include Alzheimer's disease (AD) Creutzfeldt-Jakob disease,
  • Huntington's disease Huntington's disease, Lewy body disease, Pick's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Rett Syndrome, lysosomal storage diseases including Sanfilippo, Tay Sachs disease, other genetic diseases, multiple sclerosis, neurofibromatosis, and diseases without a necessary genetic component such as multiple infarct dementia, stroke, and brain injury or trauma caused by ischemia, accidents, environmental insult such as spinal cord damage, ataxia, and alcoholism.
  • ALS amyotrophic lateral sclerosis
  • neural disease includes stroke or myocardial infarction caused by lack of blood flow or ischemia to a site in the brain or which has occurred from physical injury to the brain and/or spinal cord.
  • the term also includes neurodevelopmental disorders such as autism and neurological diseases such as schizophrenia.
  • brain tumors such as oligodendrogliomas and glioblastoma multiforme.
  • the neural disease is a demyelinating disease i.e., diseases in which myelin is the primary target.
  • demyelinating diseases include acquired demyelinating diseases including Multiple sclerosis (MS) and its alternating relapsing/remitting phases, and hereditary metabolic disorders including the leukodystrophies such as metachromatic leukodystrophy, Refsum's disease, adrenoleukodystrophy, Krabbe's disease, phenylketonuria, Canavan disease, Pelizaeus-Merzbacher disease and Alexander's disease.
  • MS Multiple sclerosis
  • hereditary metabolic disorders including the leukodystrophies such as metachromatic leukodystrophy, Refsum's disease, adrenoleukodystrophy, Krabbe's disease, phenylketonuria, Canavan disease, Pelizaeus-Merzbacher disease and Alexander's disease.
  • the neural disease is a characterized by myelin destruction, such as in multiple sclerosis (MS), after viral infection, and other trauma or chemical insults-induced demyelination.
  • MS multiple sclerosis
  • the disease is damaged neural tissue and the methods of the invention provide a relatively non-invasive treatment.
  • the neural disease is multiple sclerosis, acute disseminated encephalomyelitis, transverse myelitis, demyelinating genetic disease, spinal cord injury, virus-induced demyelination, Progressive Multifocal Leucoencephalopathy,
  • HBVI Human Lymphotrophic T-cell Virus I
  • the neural disease is multiple sclerosis, a slowly progressive disease characterized by disseminated patches of demyelination in the brain and spinal cord.
  • the neural disease is acute disseminated encephalomyelitis (postinfectious encephalomyelitis) characterized by perivascular CNS demyelination that can occur spontaneously or following a viral infection or vaccination.
  • the neural disease is a congenital metabolic disorder affecting the myelin sheath in the CNS, including without limitation phenylketonuria and other aminoacidurias, Tay-Sachs, Niemann-Pick, Gaucher's diseases, Hurler's syndrome, Krabbe's disease and other leukodystrophies, adrenoleukodystrophy, adrenomyeloneuropathy,
  • the neural disease is an acute or long-term abnormality caused by trauma to the CNS or a condition related to loss of myelin through anoxia and ischemia, including without limitation stroke, periventricular leukomalacia (PVL), cerebral palsy, or traumatic brain injury.
  • trauma to the CNS or a condition related to loss of myelin through anoxia and ischemia, including without limitation stroke, periventricular leukomalacia (PVL), cerebral palsy, or traumatic brain injury.
  • PVL periventricular leukomalacia
  • cerebral palsy or traumatic brain injury.
  • the neural disease is a spinal cord injury, in particular a spinal cord injury that causes paraplegia or incomplete motor function.
  • injuries to the cervical, lumbar, thoracic and sacral spine derive a benefit or stabilization of the condition.
  • Acute spinal cord injuries can be treated simultaneously or soon after decompression surgery.
  • Chronic spinal cord injuries can be treated or retreated as desired.
  • the neural disease is a traumatic brain injury (TBI) i.e, an injury which results in damage to the brain.
  • TBI traumatic brain injury
  • a head injury may be a closed head injury or penetrating head injury.
  • a closed head injury may occur when the head is hit by a blunt object causing the brain to interact with the hard bony surface inside the skull.
  • a closed head injury may also occur without direct external trauma to the head if the brain undergoes a rapid forward or backward movement, (e.g. whiplash).
  • a penetrating head injury may occur when a fast moving object such as a bullet pierces the skull.
  • a closed or penetrating head injury may result in localized and widespread, or diffuse, damage to the brain which may manifest as memory loss, emotional disturbances, motor difficulties, including paralysis, damage to the senses, and death.
  • the term also includes secondary damage that follows an injury including swelling and fluid buildup and the accumulation of substances toxic to surrounding neurons such as the neurotransmitter glutamate.
  • Multipotent cells may be produced by culturing an enriched hematopoietic cell preparation, preferably derived from umbilical cord blood, under proliferation conditions, in particular in the presence of or media comprising one or more positive growth factors and isolating the multipotent cells in the culture.
  • the enriched hematopoietic cell preparation essentially comprises Lin neg cells.
  • An enriched hematopoietic cell preparation may be prepared using positive or negative selection techniques known in the art. For example, a source of hematopoietic cells (e.g., umbilical cord blood) can be treated to remove mature myeloid cells and lymphocytes using antibodies specific to the mature cells (e.g., CD2,
  • a source of hematopoietic cells generally contains a minimum total nucleated cell count of about 50-1000 million cells, 500-1000 million cells, 500 to 700 million cells, 600 to 700 million cells, in particular 650 million cells, to ensure a sufficient cell dose in the final multipotent cell preparation.
  • Proliferation conditions are those conditions that give rise to multipotent cells.
  • the proliferation conditions preferably involve culturing the enriched hematopoietic cell preparation in the presence of or media comprising one or more positive growth factors for a sufficient time, in particular a sufficient time to enable the cells to complete sufficient cell cycles, to form multipotent cells.
  • Positive growth factors are growth factors that promote and maintain cell proliferation.
  • a positive growth factor may be human in origin, or may be derived from other mammalian species when active on human cells.
  • FGF fibroblast growth factor
  • GM-CSF GM-CSF
  • HGF HGF
  • Hox family HGF
  • Notch HGF
  • the positive growth factors or combination of growth factors used to produce the multipotent cells are fibroblast growth factor (FGF) (e.g. FGF-4 and FGF-2), IL- 3, stem cell factor (SCF), Flt-3 ligand, thrombopoietin (TPO), granulocyte macrophage- colony stimulating factor (GM-CSF), and neural growth factor (NGF).
  • FGF fibroblast growth factor
  • SCF stem cell factor
  • Flt-3 ligand Flt-3 ligand
  • TPO thrombopoietin
  • GM-CSF granulocyte macrophage- colony stimulating factor
  • NGF neural growth factor
  • the proliferation conditions involve using FGF-4 or FGF- 2, SCF and Flt-3 ligand, in particular FGF-4, SCF and Flt-3 ligand, to prepare multipotent cells.
  • the proliferation conditions involve using TPO, SCF and FLT-3 ligand to prepare multipotent cells.
  • the proliferation conditions involve using NGF, SCF, and Flt-3 to prepare multipotent cells.
  • the growth factors may be used in combination with equal molar or greater amounts of a glycosaminoglycan such as heparin sulfate. Growth factors may be commercially available or can be produced by recombinant
  • growth factors are commercially available from several vendors such as, for example, Genzyme (Framingham, Mass.), Genentech (South San Francisco, Calif), Amgen (Thousand Oaks, Calif), R&D Systems (Minneapolis, Minn.) and Immunex (Seattle, Wash.).
  • Some growth factors may be purified from culture media of cell lines by standard biochemical techniques.
  • molecules having similar biological activity as wild-type or purified growth factors are intended to be used within the spirit and scope of the invention.
  • an effective amount of a positive growth factor(s) is used in the culture medium.
  • concentration of a positive growth factor in the culture medium is between 10 and 150 ng/ml, preferably 20 to 100ng/ml or25 to 100 ng/ml, more preferably 20 to 50 ng/m, 20 to 60 ng/ml, 20 to 55 ng/ml, 25 to 55 ng/ml, most preferably 25 to 50 ng/ml.
  • the growth factors are typically applied at sufficient intervals to maintain high proliferation levels. In an embodiment, the growth factors are applied about 2-4 times per week, preferably 2-3 times per week.
  • the culture medium may comprise conditioned medium, non-conditioned medium, or embryonic stem cell medium.
  • suitable conditioned medium include IMDM,
  • DMEM fetal bovine serum
  • calf bovine serum horse serum
  • human serum or an artificial serum substitute [e.g.
  • bovine serum albumin 10 ⁇ g/ml bovine pancreatic insulin, 200 ⁇ g/ml human transferrin, 10 "4 M ⁇ -mercaptoethanol, 2 mM L- glutamine and 40 ⁇ g/ml LDL (Low Density Lipoproteins)], or it may be serum free.
  • the culture medium is serum free to provide multipotent cells that are free of serum proteins or biomolecules that may bind to the surface of the cells.
  • the culture medium comprises FGF-4, SCF and Flt-3 ligand in a serum free medium, in particular BIT or STI (sometimes referred to herein as "FSFl medium”).
  • concentration of FGF-4, SCF and Flt-3 ligand in the culture medium can be between about 10 to 75 ng/ml, 15 to 60 ng/ml, 20 to 60 ng/ml, 30 to 60 ng/ml, 20-55 ng/ml, 25-55 ng/ml, 25-50 ng/ml, 40 to 55 ng/ml, 45 to 55 ng/ml, or 45 to 50 ng/ml preferably
  • the enriched hematopoietic cell preparation may be seeded into the culture medium at a concentration of about 1 x 10 3 cells/ml to 5 x 10 7 cells/ml, Ix 10 4 cell/ml to 1 x 10 5 cells/ml, or 1 xlO 4 cells/ml to 5 x 10 4 cells/ml.
  • the proliferation conditions entail culturing the enriched hematopoietic cell preparation for a sufficient period of time to produce multipotent cells.
  • the enriched hematopoietic cells are generally maintained so that the cells complete about 1-100 cell cycles, preferably 5-75 cell cycles, more preferably 2-50, 2-40 or 2 -20, most preferably at least about 2-10 or 4-5 cell cycles.
  • the enriched hematopoietic cells are typically maintained in culture for about 4 to 40 days, preferably about 2-20 days, more preferably at least or about 2-15 days, 2-12 days, 4-10 days, or 8-12 days, and most preferably at least about 4-8 days, 8- 12 days, 8-10 days or 8 days.
  • the frequency of feeding hematopoietic cells is selected to promote the survival and growth of multipotent cells.
  • the hematopoietic cells are fed once, twice, three times or four times a week.
  • the cells may be fed by replacing the entirety of the culture media with new media.
  • the cells in culture may be selected for hematopoietic stem and progenitor cells (e.g.
  • CD45 + HLA-ABC + cells at a frequency to promote the survival and growth of multipotent cells.
  • cells enriched for hematopoietic stem and progenitor cells e.g. CD45 + HLA-ABC + cells
  • Multipotent cells may be produced on a large-scale, for example multipotent cells may be isolated and/or expanded in a bioreactors.
  • the multipotent cells are characterized by one or more of the following:
  • h express embryonic stem cell proteins such as Oct4, Stage Specific Embryonic Antigen-3 (SSEA3), and/or Stage Specific Embryonic Antigen-4 (SSEA4);
  • SSEA3 Stage Specific Embryonic Antigen-3
  • SSEA4 Stage Specific Embryonic Antigen-4
  • Multipotent cells may comprise cells with the characteristics (a) and (c); (a), (b), and (c); (a), (b), (C) and (d); (a), (b), (c), (d) and (e); (a), (b), (c), (d), (e), (f), and (g); (a) through (e) inclusive; (a) through (f) inclusive; (a) through (g) inclusive; (a) through (h) inclusive; (a) through (i) inclusive; (a) through (j) inclusive; (a) through (k) inclusive; (a) through (1)
  • the multipotent cells are CD45 + HLA-ABC + Lin " and express nestin, neurofilament, and/or 04.
  • the multipotent cells have the phenotypic characteristics shown in Table 1 for post-culture cells.
  • l o Multipotent cells may be expanded using proliferation conditions described herein or known in the art (e.g., using one or more positive growth factors).
  • a multipotent cell preparation comprises at least 60%, 70%, 80% or 85% CD45+ cells. In aspects of the invention, a multipotent cell preparation comprises about 1-5 x 10 7 cells, preferably 2 x 10 7 cells.
  • the multipotent cells may be induced to differentiate into neural cells and neural tissues in vitro or in vivo.
  • the multipotent cells may be induced to differentiate into neural cells, in particular cells that exhibit morphological, physiological, functional, and/or immunological features of oligodendrocytes.
  • Neural cells obtained by a method of the invention are characterized by one or more of the following properties:
  • neural cells may comprise cells with the characteristics (a) and (c); (a), (b), and (c); (a), (c) and (f); (a), (d), and (e); (a) and (i), or (a) and (h).
  • a neural cell population of the invention comprises at least about 60%, 70%, 80%, 90%, 95%, or 98% oligodendrocyte cells identified as being positive for one, two, or three of any of the phenotypic markers CNPase, 04, GaIC.
  • Neural cells in neural cell preparations of the invention may also be characterized by expression of one or more of the following markers: myelin basic protein (MBP); amarker of mature myelin and myelin-producing cells; PDGFR ⁇ , a membrane receptor for PDGF, expressed by oligodendrocytes and other cells types; TR ⁇ l, a nuclear receptor for thyroid hormone, expressed by oligoprogenitors, oligodendrocytes, and neurons; myelin proteolipid protein, a component of myelin that is expressed on oligodendrocytes and glial precursors, the epitope recognized by RIP antibody, which stains oligodendrocytes and their processes, and coincides with myelinated axons in both the spinal cord and the cerebellum; SoxlO, a Sox family transcription factor expressed by oligoprogenitors, oligodendrocytes, Schwann cells, neural rest, cochlea, prostate and melanocytes
  • Markers can be detected using any suitable immunological technique such as flow immunocytochemistry for cell-surface markers or immunohistochemistry of, for example, fixed cells or tissues for intracellular or cell-surface markers.
  • a cell is positive for a marker if it shows substantially higher staining using a specific antibody in an immunocytochemistry, flow cytometry assay or immunohistochemisty technique compared with a control.
  • Tissue- specific gene products can be detected at the mRNA level by Northern blot analysis, dot-blot hybridization analysis, or by reverse transcriptase initiated polymerase chain reaction (RT-
  • Sequence information for markers may be obtained from public databases such as GenBank.
  • Neural cells in neural cell preparations of the invention can be characterized by morphological features of oligodendrocyte precursors or mature oligodendrocytes.
  • the cells can take a bipolar shape, having two processes extending off opposite poles from the central body.
  • the cells can also be relatively flat cells expressing many of the same markers and other characteristics of oligodendrocytes.
  • the cells can also be characterized by relatively slow growth rates and dependence on extracellular matrix and soluble factors.
  • Mature oligodendrocytes can be characterized by increased processes which appear to have myelin webbing in between and which are capable of wrapping around individual axons to form myelin sheath that promotes neural transmission along the axon.
  • a neural cell preparation of the invention may comprise at least about 20%, 40%, 60%, 80% or 90% or more of the bipolar or the flat cell phenotype.
  • Neural cells of preparations of the invention can also be characterized by functional criteria.
  • the neural cells may be assessed for their ability to remyelinate neuronal tissue in tissue culture or in vivo, to repair sites of demyelination in vivo, or restore neurological function in a subject.
  • in vitro myelination in coculture can be assessed in adult dorsal root ganglion (DRG) cultures (see Wood PM and Bunge RP, J Neurol Sci. 1986 Jul;74(2-3): 153-69; Rosen, CL. et al, J Neurosci. 1989 Oct;9(10):3371-9; and Wang Z, et al., Glia. 2007 Apr l;55(5):537-45).
  • DRG dorsal root ganglion
  • Neural cells of this invention can be obtained by culturing multipotent cells in a special growth environment that enriches and/or expands cells with the desired neural phenotype.
  • the growth environment may specifically direct differentiation into the oligodendrocyte lineage, promote outgrowth of the desired cells, inhibit growth of other cell types or perform any combination of these activities.
  • the growth environment generally provides tissue culture conditions for differentiating neural cells, including without limitation the use of neural differentiation media including differentiation factors.
  • the invention provides a method for producing an isolated or purified cell preparation comprising neural cells disclosed herein comprising culturing multipotent cells disclosed herein in the presence of or in media comprising one or more differentiation factors or in neural differentiation media comprising such factors.
  • the differentiation factors are one or more of retinoic acid,
  • Neural cells may be obtained by growing multipotent cells on media that induces differentiation of the cells to neural cells (e.g. DMEM medium supplemented with differentiation factors). Neural cells may be identified based on expression of neural specific markers. For example, oligodendrocyte type cells can be identified based on the expression of CNPase, 04, myelin and GaIC.
  • a purified neural cell preparation consisting essentially of neural cells, in particular, at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99%, preferably at least 80% or 90% neural cells characterized by the following properties: an oligodendrocyte morphology, Lin neg , and expressing the oligodendrocyte markers 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase), oligodendrocyte marker 04, myelin and galactocerebroside (GalC).
  • the purified preparation can be produced by culturing multipotent CD45 + HLA-ABC + Lin " cells expressing nestin and neurofilament in neural differentiation media for at least one, two, three, four, five or six weeks, preferably about 3 weeks.
  • the purified preparation can be produced by culturing multipotent CD45 + HLA-ABC + Lin ' cells expressing nestin and neurofilament in neural differentiation media comprising retinoic acid.
  • the neural cells may be separated to obtain a population of cells largely or essentially consisting of the neural cells. This may be accomplished using various separation procedures such as antibody or lectin mediated adherence or sorting for cell surface markers.
  • positive selection of neural cells may be carried out using antibodies to identify tissue specific cell surface markers or negative selection may be carried out using neural cell specific markers (e.g., CNPase, 04, myelin and GaIC).
  • Neural cells with oligodendrocyte properties can also be separated from other cells by adhering the cells to a suitable substrate. Oligodendrocytes possess cell-specific carbohydrates and cell-surface receptors and they will preferentially adhere to a conjugate ligand. In aspects of the invention, neural cells with oligodendrocyte properties can be separated by adherence to certain basement membrane components such as laminin, gelatin, or Matrigel®. After oligodendrocytes adhere to the matrix, other cell types can be washed away and the adherent cells recovered, for example by enzymatic digestion (e.g., trypsin digestion).
  • enzymatic digestion e.g., trypsin digestion
  • the neural cells can be used to prepare a cDNA library relatively uncontaminated with cDNA preferentially expressed in cells from other lineages, and they can be used to prepare antibodies that are specific for particular markers of neural cells.
  • the number of neural cells in the preparation can be increased by causing them to proliferate further in culture. This can be accomplished by culturing the neural cells in the presence of or media comprising one or more positive growth factors.
  • positive growth factors which can be used for proliferation of neural cells are fibroblast growth factors (e.g., FGF-2 and FGF -4), epidermal growth factor (EGF), functional homologs, and other factors that bind the EGF receptor; platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), and factors that elevate cyclic AMP levels such as forskolin. It may be beneficial to include differentiation factors in the medium to maintain preferential growth of neural cells. Expansion of the number of neural cells allows large populations of neural cells to be produced. Modification of Cells
  • a neural cell preparation or neural cell composition of the invention may be derived from or comprised of cells that have been genetically modified (transduced or transfected) either in nature or by genetic engineering techniques in vivo or in vitro.
  • Cells in cell preparations and compositions of the invention can be modified by introducing mutations into genes in the cells (or the cells from which they are obtained) or by introducing transgenes into the cells. Insertion or deletion mutations may be introduced in a cell using standard techniques.
  • a transgene may be introduced into cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran- mediated transfection, lipofection, electroporation, or microinjection. Suitable methods for transforming and transfecting cells can be found in Sambrook et al. [Sambrook, Fritsch, &
  • a transgene may be introduced into cells using an appropriate expression vector including but not limited to cosmids, plasmids, or modified viruses (e.g. replication defective retroviruses, adenoviruses and adeno-associated viruses). Transfection is easily and efficiently obtained using standard methods including culturing the cells on a monolayer of virus- producing cells (see Van der Putten, 1985, ProcNatl Acad Sci U S A.;82:6148-52; Stewart et al. 1987, EMBO J. 6:383-388).
  • a gene encoding a selectable marker may be integrated into cells of a cell preparation or composition of the invention.
  • a gene which encodes a protein such as ⁇ - galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or a fluorescent protein marker may be integrated into the cells.
  • fluorescent protein markers are the Green Fluorescent Protein (GFP) from the jellyfish ⁇ , victoria, or a variant thereof that retains its fluorescent properties when expressed in vertebrate cells. (For example, the GFP variants described in Heim et al, 1994, Proc. Natl. Acad. Sci. 91 : 12501; M. Zernicka-Goetz et al, 1997,
  • Another aspect of the present invention relates to genetically engineering the cells in the cell preparations and compositions of the invention in such a manner that they or cells derived therefrom produce, in vitro or in vivo, polypeptides, hormones and proteins not normally produced in the cells in biologically significant amounts, or produced in small amounts but in situations in which regulatory expression would lead to a therapeutic benefit.
  • the cells could be modified such that a protein normally expressed will be expressed at much lower levels. These products would then be secreted into the surrounding media or purified from the cells.
  • the cells formed in this way can serve as continuous short term or long term production systems of the expressed substance.
  • neural cells can be genetically engineered to produce L- dihydroxy phenylalanine (L-DOPA) by transfection with genes for tyrosine hydroxylase and guanosine triphosphate cylohydrolase-1.
  • L-DOPA L- dihydroxy phenylalanine
  • Multipotent cells used to produce neural cell preparations can be modified with genetic material of interest.
  • the modified cells can be cultured in vitro under suitable conditions as disclosed herein so that they differentiate into neural cells.
  • the neural cells are able to express the product of the gene expression or secrete the expression product.
  • These modified neural cells can be administered to a target tissue where the expressed product will have a beneficial effect.
  • the transduced multipotent cells can be induced in vivo to differentiate into neural cells that will express the gene product.
  • the transduced multipotent cells may be administered to induce production of neural cells having the transduced gene.
  • the cells may be administered in admixture with each other or separately and may be delivered to a targeted area.
  • the cells can be introduced intravenously and home to the targeted area.
  • the cells may be used alone and caused to differentiate in vivo.
  • genes can be introduced into cells which are then injected into arecipient where the expression of the gene will have a therapeutic effect.
  • the technology may also be used to produce additional copies of essential genes to allow augmented expression by neural cells of certain gene products in vivo.
  • These genes can be, for example, matrix proteins, cell membrane proteins, cytokines, adhesion molecules, or "rebuilding" proteins important in tissue repair.
  • the multipotent cells, cell preparations and compositions of the invention and cells therefrom can be used in a variety of methods (e.g. transplantation) and they have numerous uses in the field of medicine. They may be used for the replacement of body tissues, organs, components or structures which are missing or damaged due to trauma, age, metabolic or toxic injury, disease, idiopathic loss, or any other cause.
  • Transplantation or grafting can include the steps of isolating multipotent cells, or a neural cell preparation according to the invention and transferring the multipotent cells or cells in the preparation into a mammal or a patient.
  • Transplantation can involve transferring the cells into a mammal or a patient by inj ection of a cell suspension into the mammal or patient, surgical implantation of a cell mass into a tissue or organ of the mammal or patient, or perfusion of a tissue or organ with a cell suspension.
  • the route of transferring the cells may be determined by the requirement for the neural cells to reside in a particular tissue or organ and by the ability of the neural cells to find and be retained by the desired target tissue or organ. Where the transplanted neural cells are to reside in a particular location, they can be surgically placed into a tissue or organ or simply injected into the bloodstream if the cells have the capability to migrate to the desired target organ.
  • the invention may be used for autografting (cells from an individual are used in the same individual), allografting cells (cells from one individual are used in another individual) and xenografting (transplantation from one species to another).
  • the multipotent cells, neural cell preparations and pharmaceutical compositions of the invention and cells therefrom may be used in autologous or allogenic transplantation procedures to improve a neural cell deficit or to repair tissue.
  • the multipotent cells and/or newly created neural cell preparations can be used in both cell therapies and gene therapies aimed at alleviating disorders and diseases involving neural cells.
  • the invention obviates the need for human tissue to be used in various medical and research applications.
  • the cell therapy approach involves the use of transplantation of the multipotent cells and/or newly created neural cell preparations comprising neural cells as a treatment for injuries and diseases.
  • the steps in this application include: (a) producing multipotent cells or a neural cell preparation as described herein; and (b) allowing the neural cells to form functional connections either before or after a step involving transplantation of the cells or preparation.
  • the gene therapy approach also involves multipotent cells and neural cell preparations, however, following the culturing step in proliferation conditions, the newly created cells are transfected with an appropriate vector containing a cDNA for a desired protein and the cells are optionally differentiated, followed by a step where the modified cells are transplanted.
  • multipotent cells or cell preparations of the invention or cells therefrom can be transplanted in, or grafted to, a patient in need.
  • the multipotent cells or cell preparations or cells therefrom can be used to replace neural cells in a patient in a cell therapy approach, useful in the treatment of tissue injury, and diseases.
  • These cells can be also used as vehicles for the delivery of specific gene products to a patient.
  • One example of how these newly created cells or cell differentiated therefrom can be used in a gene therapy method is in treating the effects of Parkinson's disease.
  • tyrosine hydrolase a key enzyme in dopamine synthesis
  • tyrosine hydrolase may be delivered to a patient via the transplantation of multipotent cells or a neural cell preparation or composition of the invention where the cells therein have been transfected with a vector suitable for the expression of tyrosine hydrolase.
  • the invention also provides a method of treating a patient with a neural disease comprising transferring multipotent cells or a neural cell preparation of the invention or cells therefrom into the patient.
  • a method of the invention may involve producing or obtaining cells for autologous transplantation from the patient's own hematopoietic cells comprising (a) obtaining a sample comprising hematopoietic cells from the patient, preferably from umbilical cord blood, more preferably fresh or cryopreserved umbilical cord blood; (b) separating out an enriched cell preparation comprising Lin neg stem cells and progenitor cells; (c) culturing the cells under proliferation conditions to produce multipotent cells, preferably CD45 + HLA-ABC + cells, in particular expressing nestin, neurofilament and/or 04; (d) culturing the multipotent cells in the presence of or media comprising one or more differentiation factors to produce a neural cell preparation; and (e) transferring the multipotent cells of (c) or the neural cell preparation of (d) to the patient.
  • a method of the invention may involve producing or obtaining cells for allogeneic transplantation comprising (a) obtaining a sample comprising hematopoietic cells from a donor subject, preferably from umbilical cord blood; (b) separating out an enriched cell preparation comprising comprising Lin neg stem cells and progenitor cells; (c) culturing the cells under proliferation conditions to produce multipotent cells preferably CD45 + HLA-ABC + cells, in particular expressing nestin, neurofilament and/or 04; (d) culturing the multipotent cells in the presence of or in media comprising one or more differentiation factors to produce a neural cell preparation; and (e) transferring the multipotent cells of (c) or the neural cell 5 preparation of (d) to another subject to treat a neural disease.
  • the invention also contemplates a pharmaceutical composition
  • a pharmaceutical composition comprising multipotent cells or a neural cell preparation or composition or neural cells therefrom and a pharmaceutically acceptable carrier, excipient, or diluent.
  • the pharmaceutical compositions herein can be prepared by per se known methods for the preparation of pharmaceutically l o acceptable compositions which can be administered to subj ects, such that an effective amount of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in the standard texts Remington: The Science and Practice of Pharmacy (21 st Edition.2005, University of the Sciences in Philadelphia (Editor), Mack Publishing Company), and in The United States Pharmacopeia: The National
  • compositions include, albeit not exclusively, solutions of the multipotent cells, neural cell preparations or neuronal cells therefrom in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
  • the treatment methods of the invention may be used with one or more other treatment methods effective for the same neural disease.
  • multiple sclerosis may be additionally treated with a tetracycline derivative such as minocycline and doxycycline.
  • a treatment or treatment method may be used prior to or at the same time as the patient receives a transplant of multipotent cells, a cell preparation or composition of the invention. Examples
  • a cell preparation composition, medicament, or treatment of the invention may comprise a single unit dosage of multipotent cells or neural cells.
  • a "unit dosage” refers to a unitary i.e. a single dose which is capable of being administered to a patient, and which may be readily handled and packed, remaining as a physically and chemically stable unit dose comprising either the cells, cell preparations or compositions as such or a mixture with one or more pharmaceutical excipients, carriers, or vehicles.
  • a cell preparation, composition or unit dose may comprise a cell dose of greater than 1 x 10 5 to 5 x 10 8 , 1 x 10 6 to 1 x 10 8 ,or 1 x 10 7 to 5 x 10 7 , in particular greater than 2.O x IO 7 cells.
  • the invention contemplates a kit for producing neural cell preparations of the invention comprising multipotent cells capable of differentiating into neural cells both in vitro and in vivo.
  • the kit includes the reagents for a method of the present invention for producing a neural cell preparation.
  • This kit preferably would include at least one differentiation factor, and instructions for use.
  • the invention contemplates a kit comprising multipotent cells or a neural cell preparation of the invention in kit form.
  • a kit may comprise a package which houses a container which contains a preparation or composition of the invention and also houses instructions for administering the preparation or composition to a subject.
  • kits can also comprise cell preparations of the invention or cells therefrom for conducting the screening and testing methods disclosed herein
  • neural cell preparations and pharmaceutical compositions disclosed herein can be used for toxicity testing for drug development testing.
  • Toxicity testing may be conducted by culturing neural cell preparations or pharmaceutical compositions or cells obtained or derived therefrom in a suitable medium and introducing a substance, such as a pharmaceutical or chemical, to the culture. The neural cells are examined to determine if the substance has had an adverse effect on the culture.
  • Drug development testing may be done by developing derivative cell lines which may be used to test the efficacy of new drugs. Affinity assays for new drugs may also be developed from the neural cell preparations or cell lines. Using a method of the invention it is possible to identify drugs that are potentially toxic to neural cells.
  • the neural cell preparations of the invention may be used to screen for potential therapeutics that modulate development or activity of neural cells.
  • the cells of a neural cell preparation of the invention may be subjected to a test substance, and the effect of the test substance may be compared to a control (e.g. in the absence of the substance) to determine if the test substance modulates development or activity of the neural cells
  • a method for using neural cell preparations to screen a potential new drug to treat a disorder involving neural cells comp ⁇ sing the steps of
  • hematopoietic cells obtained from a sample from a patient with a disorder involving neural cells, (b) preparing from the hematopoietic cells an en ⁇ ched hematopoietic cell preparation comp ⁇ sing hematopoietic stem cells and progenitor cells (e g , Lin ne8 cells), (c) cultu ⁇ ng the en ⁇ ched hematopoietic cell preparation under proliferation conditions to obtain multipotent cells (e g , CD45 + HLA-ABC + ), (d) cultu ⁇ ng the multipotent cells in the presence of or in media comp ⁇ sing one or more differentiation factors under suitable conditions in vitro,
  • multipotent cells e g , CD45 + HLA-ABC +
  • the invention also relates to the use of neural cell preparations and pharmaceutical compositions of the invention in drug discovery.
  • the invention provides methods for drug development using the neural cell preparations and pharmaceutical compositions of the invention.
  • Neural cell preparations and pharmaceutical compositions of the invention may comprise neural cells that secrete novel or known biological molecules or components. In particular, culturing in the absence of serum may provide cells that have minimal interference from serum molecules and thus, may be more physiologically and topologically accurate. Therefore, proteins secreted by neural cells described herein may be used as targets for drug development. Drugs can also be made to target specific proteins on neural cells described herein. In addition, drugs specific for regulatory proteins of neural cells may be used to arrest growth of cells. Any of the proteins can be used as targets to develop antibody, protein, antisense, aptamer, ribozymes, or small molecule drugs.
  • Agents, test substances, or drugs identified in accordance with a method of the invention or used in a method of the invention include but are not limited to proteins, peptides such as soluble peptides including Ig-tailed fusion peptides, members of random peptide libraries and combinatorial chemistry-derived molecular libraries made of D- and/or L- configuration amino acids, phosphopeptides (including members of random or partially degenerate, directed phosphopeptide libraries), antibodies [e.g. polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, single chain antibodies, fragments, (e.g. Fab, F(ab) 2 , and
  • the neural cell preparations and pharmaceutical compositions of the invention can be used in various bioassays.
  • the neural cell preparations are used to determine which biological factors are required for proliferation or differentiation of neural cells.
  • multipotent cells or neural cell preparations in a stepwise fashion in combination with different biological compounds (such as hormones, specific growth factors, etc.)
  • one or more specific biological compounds can be found to induce differentiation to neural cells or proliferation of neural cells.
  • Other uses in a bioassay for the cells are differential display (i.e.
  • Protein-protein interactions can be determined with techniques such as a yeast two-hybrid system. Proteins from neural cell preparations and pharmaceutical compositions of the invention can be used to identify other unknown proteins or other cell types that interact with the cells. These unknown proteins may be one or more of the following: growth factors, hormones, enzymes, transcription factors, translational factors, and tumor suppressors. Bioassays involving neural cell preparations and pharmaceutical compositions of the invention, and the protein-protein interactions these cells form and the effects of protein-protein or cell-cell contact may be used to determine how surrounding tissue contributes to proliferation or differentiation of neural cells.
  • neural cell preparations comprising, produced or derived from multipotent cells obtained after culturing a preparation from cord blood stem cells may be used to repair cell or tissue injury. They may also be used in the treatment of genetic defects that result in nonfunctional cells. Neural cells obtained from differentiating multipotent cells derived from umbilical cord blood may be used for treating a neural disease, in particular neurodegenerative disorders, a brain or spinal cord inj ury, or neurological deficit.
  • neurodegenerative disorders which can be treated include for example, Parkinson's disease, Huntington's disease, multiple sclerosis, Alzheimer's disease, Tay Sach's disease, lysosomal storage disease, brain and/or spinal cord injury due to ischemia, stroke, head injury, cerebral palsy, spinal cord and brain damage/injury, depression, epilepsy, schizophrenia, and ataxia and alcoholism.
  • Neural cells generated in accordance with a method of the invention may be transfected with a vector that can express a desired protein such as growth factors, growth factor receptors, and peptide neurotransmitters, or express enzymes involved in the synthesis of neurotransmitters. These transfected cells may be transplanted into regions of neurodegeneration.
  • a desired protein such as growth factors, growth factor receptors, and peptide neurotransmitters, or express enzymes involved in the synthesis of neurotransmitters.
  • the multipotent cells, neural cell preparations, pharmaceutical compositions and neural cells isolated or derived therefrom may be used as immunogens that are administered to a heterologous recipient.
  • Administration of neural cells obtained in accordance with the invention may be accomplished by various methods. Methods of administering neural cells as immunogens to a heterologous recipient include without limitation immunization, administration to a membrane by direct contact (e.g. by swabbing or scratch apparatus), administration to mucous membranes (e.g. by aerosol), and oral administration.
  • Immunization may be passive or active and may occur via different routes including intraperitoneal injection, intradermal injection, and local injection.
  • the route and schedule of immunization are in accordance with generally established conventional methods for antibody stimulation and production. Mammalian subjects, particularly mice, and antibody producing cells therefrom may be manipulated to serve as the basis for production of mammalian hybidoma cell lines.
  • the cell preparations of the invention may be used to prepare model systems of disease.
  • the cell preparations and compositions of the invention can also be used to produce growth factors, hormones, etc.
  • the invention provides a culture system from which genes, proteins, and other metabolites involved in proliferation or differentiation of neural cells can be identified and isolated.
  • the cells in a culture system of the invention may be compared with other cells (e.g. differentiated cells) to determine the mechanisms and compounds that stimulate production of neural cells.
  • the neural cell preparations of the invention can be used to screen for genes expressed in or essential for differentiation of neural cells. Screening methods that can be used include
  • RDA Representational Difference Analysis
  • SA-lacZ D.P. Hill and W. Wurst, 1993, Methods in Enzymology, 225: 664.
  • Gene trapping can be used to induce dominant mutations (e.g. by deleting particular domains of the gene product) that affect differentiation or activity of neural cells and allow the identification of genes expressed in or essential for differentiation of these cells.
  • the invention also relates to a method for conducting a regenerative medicine business, comprising: (a) a service for accepting and logging in samples from a client comprising hematopoietic cells capable of forming multipotent cells; (b) a system for culturing cells dissociated from the samples, which system provides conditions for producing multipotent cells and neural cell preparations therefrom; and/or (c) a cell preservation system for preserving multipotent cells and neural cell preparations generated by the system in (b) for later retrieval on behalf of the client or a third party.
  • the method may further comprise a billing system for billing the client or a medical insurance provider thereof.
  • the invention features a method for conducting a neural cell business comprising identifying agents which influence the proliferation, differentiation, or survival of neural cells.
  • agents are small molecules, antibodies, and extracellular proteins. Identified agents can be profiled and assessed for safety and efficacy in animals. In another aspect, the invention contemplates methods for influencing the proliferation, differentiation, or survival of neural cells by contacting the cells with an agent or agents identified by the foregoing method.
  • the identified agents can be formulated as a pharmaceutical preparation, and manufactured, marketed, and distributed for sale.
  • the invention provides a method for conducting a neural cell business comprising (a) identifying one or more agents which affect the proliferation, differentiation, function, or survival of neural cells from a neural cell preparation of the invention; (b) conducting therapeutic profiling of agents identified in (a); or analogs thereof for efficacy and toxicity in animals; and (c) formulating a pharmaceutical composition including one or more agents identified in (b) as having an acceptable therapeutic profile.
  • the method may further comprise the step of establishing a distribution system for distributing the pharmaceutical preparation for sale.
  • the method may also comprise establishing a sales group for marketing the pharmaceutical preparation.
  • the invention also contemplates a method for conducting a drug discovery business comprising identifying factors that influence the proliferation, differentiation, function, or survival of neural cells from neural cell preparations of the invention, and licensing the rights for further development.
  • the therapeutic efficacy of the multipotent cells, neural cell preparations, compositions and agents identified using the methods of the invention can be confirmed in animal disease models.
  • the therapeutic efficacy of a neural cell preparation of the invention can be tested in disease models such as models for spinal cord injury, neurodegenerative diseases, lysosomal storage diseases, stroke, brain tumors, Parkinson disease, Alzheimer disease, Huntington disease or stroke (see Kim SU, Neuropathology, 2004, 24(3): 159-71 Kim, SU, Brain Dev. Feb 13, 2007).
  • the ability of multipotent cells or neural cell preparations of the invention and neural cells therefrom to survive and enhance myelination or axonal regrowth in vivo can be demonstrated using suitable animal models.
  • a model for chronic demyelination involving inducing regions of chronic inflammation in the adult rat dorsal column can be utilized (see Keirstead et al., GHa. 1998 Feb;22(2): 161-70; KeirsteadHS andBlakemore WF, Adv Exp Med Biol. 1999;468: 183-97).
  • the therapeutic efficacy of the neural cells can also be tested in congenital models of dysmyelination.
  • Models involving a mutation or defect in myelin basic protein include the shiverer mutant mouse (Roach A et al, Cell. 1985, 42(1): 149- 55; Popko, B. et al., Cell.
  • Remyelination of axons by oligodendrocytes can be an effective treatment strategy to treat spinal cord injury (SCI).
  • Candidate cells for remyelination include human umbilical cord blood (UCB) cells.
  • Lineage negative cells (Lin neg : stem/progenitor cells) from UCB develop into multipotent cells and have properties like those of multipotential mesenchymal cells found in the bone marrow.
  • the objective of this study is to determine if Lin" 68 UCB cells can differentiate into oligodendrocytes in vitro. In vitro studies show that lin neg human umbilical cord blood cells exhibit characteristics of oligodendrocyte cells.
  • UCB was collected from informed consensual donors at Mount Sinai Hospital (Toronto, Ontario) and Joseph Brant Memorial Hospital (Burlington, Ontario), by qualified hospital personnel following protocols approved by the human ethics committees of those hospitals. Briefly, the umbilical cord blood was collected in a 250 ml single blood pack collection bag containing a citrate phosphate dextrose solution as an anticoagulant (Baxter, Fenwal #4R-08-37Q). All blood samples were processed within 24 hours of collection. The mononuclear fraction of the cord blood was isolated by adding Pentaspan (Bristol-Myers Squibb #4745) starch solution at a ratio of 1 :5 (blood:starch).
  • Pentaspan Bristol-Myers Squibb #4745
  • the cells were centrifuged at 50 g for 10 minutes at 1O 0 C to sediment the red blood cells. The supernatant was removed and the resulting pellet was resuspended in RPMI 1640 (Gibco #31800-022) and centrifuged at 400 g for 10 minutes. The resulting pellet was resuspended in a solution of 45% RPMI 1640, 45% human serum and 10% dimethyl sulphoxide (Edwards Lifesciences #CRY-VB01). Cells were cryopreserved by first placing them in a controlled freezing chamber down to -20 0 C (descending by l°/min) and then storing them in liquid nitrogen. Enrichment and proliferation of UCB cells
  • a negative selection column was used to remove mature cells according to the manufacturer's instructions (Stem Cell Technologies #14056). Briefly, UCB cells were quickly thawed in a 37°C water bath. RPMI with penicillin streptomycin (Gibco # 15070-063) was added drop wise at a ratio of 5:1 ml of cells. Cells were centrifuged at 400 g for 10 minutes and resuspended in HBSS/2% serum for a concentration of 50 x 10 6 cells/ml.
  • An antibody cocktail was added to the cells containing monoclonal antibodies (CD2, CD3, CD14, CD 16, CD 19, CD24, CD56, CD66b, glycophorin A and dextran) directed against mature human hematopoietic cells (mature myeloid and lymphocytes) and coupled to colloidal magnetic iron particles.
  • the magnetically labelled mature blood cells were then separated from unlabeled cells by passing them through a magnetic separation column.
  • the resulting enriched hematopoietic progenitor cells that passed through the column were counted and centrifuged at 400 g for 10 mins.
  • the cells were then resuspended and plated in a 12 well plate (100,000 cells/ well) in StemSpan media supplemented with FGF4 (25 ng/ml), SCF (25 ng/ml), Flt-3 ligand (25 ng/ml), heparin (1 ⁇ g/ml), penicillin/streptomycin and low density lipoprotein (Sigma L7914, 0.5 mg/ml) (FSFl medium). All cultures were incubated at 37° in
  • the protocol is a modification of one developed for the differentiation of embryonic stem cells into oligodendrocytes. Briefly, day 8 FSFl-grown Lin neg cells were centrifuged (400 g for 10 mins.) and resuspended in a basal media supplemented with retinoic acid (Sigma
  • the basal media contained a 50:50 mixture of DMEM/F 12 supplemented with N2 and Neurobasal medium supplemented with B27 (Gibco #12400024, #17502048, #21103, #17504 respectively).
  • the cells were plated on poly-D- lysine (2 ⁇ g/cm 2 )/Laminin (0.2 ⁇ g/cm 2 ) coated 12 well plates (BD Falcon) or CC2 coated 8 well chamber slides (Nunc #154941). The media was changed after 48 hours.
  • the media was replaced with the basal media, except the retinoic acid was removed and FGF-2 (R&D #234-FSE-025) was added at a concentration of 20 ng/ml.
  • FGF-2 R&D #234-FSE-025
  • the media was changed every 48 hours.
  • the media was additionally supplemented withNT3 (R&D #267- N3-005) at a concentration of 5ng/ml, forskolin (Sigma #F-6886) at a concentration of 5 ⁇ M, and BSA (Sigma #A9647) at a concentration of lOO ⁇ g/ml.
  • PDGF-AA R&D #211 -AA-010
  • TH Sigma #T5516
  • the media was changed every 48 hours.
  • the cells were allowed to differentiate for another 8 days. All media was supplemented with penicillin/streptomycin. All cultures were incubated at 37° in 5% CO 2 , 95% humidity.
  • the human oligodendrocyte cell line MO3-13 was obtained from the University of Toronto, Ontario Canada.
  • This cell line was generated from the fusion of a 6-thioguanine-resistant mutant of the human rhabdomyosarcoma RD with adult human oligodendrocytes cultured from a surgical specimen (McLaurin, Trudel et al. 1995). It expresses immunoreactivity for the immature oligodendrocyte markers CNPase GaIC, and for the mature markers myelin oligodendrocyte glycoprotein and MBP (Buntinx, Vanderlocht et al. 2003). RT-PCR Cells were harvested and total RNA was collected using a Qiagen Rneasy Mini kit
  • RNA concentration was then determined using a ⁇ Quant plate reader (Biotek Instruments).
  • cDNAs were synthesised from 0.5 ⁇ g of RNA using Omniscript RT (Qiagen #205111) with oligo(dT) 12-18 primers (Invitrogen #18418-012) and RNAse inhibitor (Roche #3335399) according to manufacturers instructions. The resulting cDNAs were then used for the PCR reaction using Taq polymerase (Qiagen).
  • reaction were carried out in a total volume of 50 ⁇ l containing a final concentration of 0.3 ⁇ M of each primer, 200 ⁇ M of each dNTP, 2.5 units Taq and 2 ⁇ l of Qiagen PCR buffer. Reactions were then placed in a PCR machine. The reaction mixture was initially denatured for 3 min. at 94°C. Then the mixture was again denatured at 94°C for 1 min., annealed at 60 0 C for 1 min., denatured at 72 0 C for 1 min. for 30 cycles. The mixture then underwent a final extension at 72°C for 10 min.
  • Glyceraldehyde-3-phosphate dehydrogenase G3PDH
  • the 5' primer was 5'-CCATGTTCGTCATGGGTGTGAAC CA-3' [SEQ ID NO.: 1] and the 3' primer was 5'-GCCAGTAGAGGCAGGGATGATGTTC-S' [SEQ ID NO. : 2].
  • 2',3'-cyclic nucleotide 3'-phosphohydrolase CNP
  • the CNPase primers span 2 introns to avoid genomic amplification.
  • Cells for immunocytochemistry were grown on 8-well chamber slides as described above. Following the differentiation protocol, the cells were rinsed twice with PBS and fixed for 10 minutes with 4% paraformaldehyde. The cells were then washed four times with PBS and allowed to air dry for 30 minutes. For staining, the slides were immersed in PBS for 5 minutes, blocked in 10% goat serum/0.1% tritonx-100/PBS for 3 hours. They were rinsed with PBS (3 x 5 minutes). They were then incubated at 4 0 C overnight in the primary antibody. The slides were washed with PBS (5 x 15 minutes) and then incubated at room temperature with the secondary antibody for 1 hour.
  • the slides were then washed (5 x 15 minutes) and then placed in a DAPI solution (2 ⁇ g/ml) for 2 minutes.
  • the slides were washed again in PBS for 5 minutes and then mounted using a 10% Dabco solution in 50% glycerol/PBS.
  • the following primary antibodies were used after dilution in 1% goat serum/0.1% tritonX-100: monoclonal mouse anti-2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) diluted lO ⁇ g/ml (Chemicon MAB326R); monoclonal mouse anti-oligodendrocyte marker 04, diluted 20 ⁇ g/ml (Chemicon MAB345); monoclonal mouse anti-myelin oligodendrocyte specific protein (MOSP) diluted 5 ⁇ l/ml (Chemicon MAB328); monoclonal mouse anti- galactocerebroside (GaIC) diluted 10 ⁇ g/ml (Chemicon MAB342); myelin basic protein
  • MBP diluted 5 ⁇ l/ml
  • Chemicon MAB386 diluted 5 ⁇ l/ml
  • the following secondary antibodies were used after dilution in 1% goat serum/0.1% tritonx-100: Oregon Green 488 goat anti-mouse IgG diluted 2 ⁇ g/ml (Molecular Probes 06380); Alexa Fluor 594 goat anti-mouse IgM diluted 2 ⁇ g/ml (Molecular Probes A21044); Alexa Fluor 594 goat anti-rat IgG diluted 2 ⁇ g/ml (Molecular Probes Al 1007).
  • RA retinoic acid
  • Lin neg cells were first propagated for 8 days in media containing FGF4, SCF, Flt-31 to expand the stem/progenitor cell numbers, followed by differentiation in media designed to promote oligodendrocyte formation.
  • the differentiation protocol is similar to one used for the differentiation of embryonic stem cells into oligodendrocytes and mimics the normal timing of oligodendrocyte development (Billon, Jolicoeur et al.2002; Nistor, Totoiu et al. 2005). Cells were grown on laminin coated chamber slides. This protocol mimics the normal timing of oligodendrocyte development ( Figure 1). At day 0, the cells are round and non-adherent.
  • Oligodendrocytes-like cells derived from Lin ne8 UCB cells express CNPase, O4, GaIC and MOSP
  • the cells that were obtained after the differentiation culture were morphologically similar to brain derived oligodendrocytes or cell lines (Sperber and McMorris 2001; Paez, l o Garcia et al. 2005). Their ability to express oligodendrocyte specific proteins CNPase, GaIC,
  • CNPase is an early marker of oligodendrocyte development. It is a myelin-associated enzyme found in oligodendrocyte and Schwann cells and localized in the cytoplasm of the cell body and major growth areas of oligodendrocytes (Yin, Peterson et al. 1997; Buntinx, Vanderlocht et al. 2003). GaIC binds specifically to oligodendrocytes and
  • oligodendrocytes 15 Schwann cells and is also an external marker found on the cell surfaces and processes.
  • 04 is a sulfatide found on the surface of oligodendrocytes of the central nervous system and is a marker for cell bodies and processes of oligodendrocytes. Its expression appears early and continues to be expressed in mature oligodendrocytes.
  • GaIC binds specifically to oligodendrocytes and Schwann cells and is also an external marker.
  • CNPase, 04 and GaIC Positive expression of CNPase, 04 and GaIC suggests the Lin neg cells are undergoing differentiation in a pattern similar to oligodendrocyte progenitor cells found in the embryo. With each subsequent differentiation stage, cells presumed not to be capable of differentiating further died leaving behind a population of cells with strong oligodendrocyte morphology. These cells were tested for expression of a mature marker associated with myelin and the cultures were found to be positive for the expression of myelin oligodendrocyte specific protein (MOSP). This myelin antibody is specific to oligodendrocytes and not Schwann cells or fibroblasts. The predicted MOSP amino acid sequence and protein structure suggest that it is the CNS homologue of PMP-22 (Bronstein, Kozak et al. 1996). Discussion and conclusion
  • Lin neg cells were isolated from UCB samples and expanded in FGF-4, SCF and Flt-3 ligand supplemented medium. At the end of the 8-day culture period the cells express the stem cell markers Oct-4 and Nanog as well as nestin and neurofilament (Rogers, Yamanaka et al. 2007).
  • culture conditions were used specifically designed to recapitulate the normal timing of oligodendrocyte differentiation in vivo (Billon, Jolicoeur et al. 2002).
  • the cells After 21- 30 days in culture, the cells exhibited morphological characteristics of oligodendrocytes. They exhibited irregular multiple long branched projections resembling what others have observed culturing oligodendrocytes from neural origins (Osterhout, Wolven et al. 1999; Sperber and McMorris 2001 ; Paez, Garcia et al. 2005). With each subsequent differentiation phase, cell loss occurred suggesting that neural fate is determined early on. Freshly isolated Lin ncg cells are negative for nestin and neurofilament by PCR but become positive for both these markers as well as 04 at day 8 of culture. Therefore prior to placement into specific neural differentiation medium, the cells already demonstrate some neural characteristics.
  • neural progenitor cell markers neural progenitor cell markers
  • MOSP tyrosine hydroxylase
  • the immature cells have retained the capacity for proliferation proliferation of the nestin positive cells can be utilized to increase the over all yield of oligodendrocytes from a single UCB sample.
  • the expression of myelin associated proteins from the Lin neg cells is not a random event due to tissue culture.
  • the cells sequentially express multiple neural and oligodendrocyte proteins in a manner similar to embryo development.
  • Four key oligodendrocyte specific markers CNPase, 04, GaIC and MOSP are expressed from cells with strong oligodendrocyte morphology.
  • ES cells (Brustle, Jones et al. 1999; Billon, Jolicoeur et al. 2002; Glaser, Perez-Bouza et al. 2005) BM cells (Eglitis and Mezey 1997; Sanchez- Ramos, Song et al. 2000; Sanchez-Ramos 2002; Zhao, Duan et al. 2002; Ortiz-Gonzalez, Keene et al.2004; Bonilla, Silva et al. 2005) and UCB (Ha, Choi et al. 2001; Sanchez-Ramos, Song et al. 2001; Buzanska, Machaj et al. 2002; Sanchez-Ramos 2002) can be differentiated into neural cells.
  • the method described herein can be utilized to produce cell preparations with clinically relevant populations of cells positive for oligodendrocyte-like characteristics.
  • One umbilical cord yields 2 x 10 6 Lin neg cells and after 8 days, the cells expand 10 fold in the FGF/SCF/Flt-31 media to 2 x 10 7 . Therefore, one cord could yield 2 x 10 5 oligodendrocyte- like cells. This is clinically relevant as one oligodendrocyte can be responsible for myelinating
  • neural progenitor cells should provide an advantage over mature cells.
  • isolation and in vitro culture of mature oligodendrocytes is difficult.
  • the efficiency of differentiation varies inversely with cell maturation and the isolation of a pure oligodendrocyte population from a donor source is inefficient.
  • embryonic neural cells that are cultured over a longer period have a lower chance of survival and engraftment, probably due to profound changes in gene expression leading to an abnormal cell type (Zietlow, Pekarik et al. 2005).
  • the cultured cells could simply be too old to survive transplantation (Walczak, Chen et al.2007).
  • cell-cell contact with axons within the spinal cord is important (Barres and Raff 1999).
  • the UCB cells can be differentiated into oligodendrocyte-like cells in vitro, to be used in therapy for CNS diseases. Studies were conducted to determine if the cells are functional oligodendrocytes and able to form compact myelin sheaths around axons. In vitro co-culture experiments with neurons as well as transplantation studies into an animal model of spinal cord injury will be conducted. The results of one study are shown in Figure 5.
  • Human umbilical cord cells in rat spinal cord tissue stained positive for myelin basic protein after 6 weeks. Rats underwent spinal cord injury with a 35g clip and were directly injected with 8 x 10 3 d8 F/SF HUBC cells. After 6 weeks, the rats were perfused and spinal cords double labelled with human mitochondria (green) and myelin basic protein (red). Cell nuclei were counterstained with
  • This example describes methods for the preparation of a cellular product with an expanded population of CD45+ multipotential cells from human UCB. These cells are non- adherent at the time of isolation. After 8 days of culture in a defined medium, the cellular product can be differentiated into mesenchymal and neural cells.
  • UCB-derived CD45-positve/lineage-negative (CD45+/lin-) cells are expanded in a medium designed to promote stem cell proliferation without differentiation and the resulting cell population and its in vitro differentiation potential is characterized.
  • UCB- derived lin- cells were cultured in a serum-free medium supplemented with stem cell factor
  • SCF stromal growth factor-4
  • FL Flt-3 ligand
  • FGF-4 fibroblast growth factor-4
  • Cells were maintained at 37 0 C in a humidified atmosphere of 5% CO 2 in air for 8 days. Fifty percent medium replacement occurred every 48 hours. The phenotype and cell expansion was assessed at culture termination. The final cell product was also assessed for its ability to differentiate into a variety of cell lineages in vitro.
  • Nestin an early neural differentiation marker, is detectable in the multipotent cell product, as determined by RT-PCR. Transfer of the multipotent cell product into neural differentiation medium resulted in the cells becoming adherent after 7-14 days. Prolonged culture duration resulted in 100% of the surviving cells developing neural morphology and expressing neurofilament ( Figure 9a). Differentiation regimens to induce oligodendrocytes resulted in ⁇ 1% of multipotent cells surviving and developing oligodendrocyte morphology ( Figure 9b). Immunocytochemistry demonstrated that the multipotent cells, after culture in neural differentiation medium, express the neural protein ⁇ -IH tubulin (Figure 9c).
  • the multipotent cells could also be differentiated into highly specialized tyrosine hydroxylase expressing cells after a 6-week, 3-stage differentiation regime (Figure 9d). Again, uncultured UCB-derived lin- cells failed to survive if cultured directly into neural differentiation medium.
  • the culture of UCB-derived CD45+/lin- cells in amedium containing exogenous SCF, FL and FGF results in the expansion of CD34+ and CD45+ cells.
  • the expanded cell product is capable of differentiation into neural cells.

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Abstract

La présente invention concerne des procédés de production de cellules neuronales, en particulier de préparations de cellules neuronales comprenant des oligodendrocytes, des compositions pharmaceutiques contenant les cellules neuronales ou les préparations de cellules neuronales, ainsi que l'utilisation de ces cellules neuronales, préparations et compositions dans la recherche ou des applications commerciales, en particulier dans des applications thérapeutiques. Selon un aspect, la préparation de cellules neuronales de cette invention se compose essentiellement de cellules neuronales qui se caractérisent par une morphologie des oligodendrocytes et des cellules Linneg et en ce qu'elles expriment des marqueurs des oligodendrocytes, la 2',3' nucléotide cyclique 3'-phosphohydrolase (CNPase), le marqueur oligodendrocyte O4, la myéline et/ou le galactocérébroside (GalC).
PCT/CA2008/000365 2007-02-26 2008-02-26 Préparations de cellules neuronales et procédés de production et d'utilisation correspondants WO2008104065A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003059276A2 (fr) * 2002-01-14 2003-07-24 The Board Of Trustees Of The University Of Illinois Nouvelles cellules souches multipotentes d'origine mammalienne, methodes de preparation et methodes d'administration desdites cellules
US20050074435A1 (en) * 2001-12-21 2005-04-07 Robert Casper Cellular compositions and methods of making and using them
US7098027B2 (en) * 2000-06-26 2006-08-29 Renomedix Institute, Inc. Method for remyelinating a demyelinized lesion due to injury in the brain or spinal cord
US7166277B1 (en) * 1991-07-08 2007-01-23 Neurospheres Holdings Ltd. Remyelination of neurons using multipotent neural stem cell progeny

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US7166277B1 (en) * 1991-07-08 2007-01-23 Neurospheres Holdings Ltd. Remyelination of neurons using multipotent neural stem cell progeny
US7098027B2 (en) * 2000-06-26 2006-08-29 Renomedix Institute, Inc. Method for remyelinating a demyelinized lesion due to injury in the brain or spinal cord
US20050074435A1 (en) * 2001-12-21 2005-04-07 Robert Casper Cellular compositions and methods of making and using them
WO2003059276A2 (fr) * 2002-01-14 2003-07-24 The Board Of Trustees Of The University Of Illinois Nouvelles cellules souches multipotentes d'origine mammalienne, methodes de preparation et methodes d'administration desdites cellules

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MCKENZIE I.A. ET AL.: "Skin-Derived Precursors Generate Myelinating Schwann Cells for the Injured and Dysmyelinated Nervous System", J. NEUROSCI., vol. 26, no. 24, 14 June 2006 (2006-06-14), pages 6651 - 6660 *
YANDAVA B.D. ET AL.: ""Global" Cell Replacement is Feasible Via Neural Stem Cell Transplantation: Evidence from the Dysmyelinated Shiverer", MOUSE BRAIN, PROC. NATL. ACAD. SCI. U.S.A., vol. 96, June 1999 (1999-06-01), pages 7029 - 7034, XP002614827, DOI: doi:10.1073/pnas.96.12.7029 *

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