WO2004007700A1 - 神経系細胞の製造方法 - Google Patents

神経系細胞の製造方法 Download PDF

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Publication number
WO2004007700A1
WO2004007700A1 PCT/JP2003/007906 JP0307906W WO2004007700A1 WO 2004007700 A1 WO2004007700 A1 WO 2004007700A1 JP 0307906 W JP0307906 W JP 0307906W WO 2004007700 A1 WO2004007700 A1 WO 2004007700A1
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Prior art keywords
cell
cells
scs
stem cells
culture
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PCT/JP2003/007906
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English (en)
French (fr)
Japanese (ja)
Inventor
Takashi Nakayama
Nobuo Inoue
Yasushi Kondo
Yutaka Suzuki
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Tanabe Pharma Corp
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Tanabe Seiyaku Co Ltd
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Priority to IL16577203A priority Critical patent/IL165772A0/xx
Priority to EP03738499A priority patent/EP1516926A4/en
Priority to CA2489206A priority patent/CA2489206C/en
Priority to JP2004521136A priority patent/JP4439396B2/ja
Priority to KR1020047021075A priority patent/KR100846643B1/ko
Priority to HK05111956.4A priority patent/HK1079812B/xx
Priority to AU2003246176A priority patent/AU2003246176B2/en
Priority to US10/518,749 priority patent/US8492145B2/en
Priority to CN038149354A priority patent/CN1662645B/zh
Application filed by Tanabe Seiyaku Co Ltd filed Critical Tanabe Seiyaku Co Ltd
Publication of WO2004007700A1 publication Critical patent/WO2004007700A1/ja
Priority to IL165772A priority patent/IL165772A/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C12N5/0618—Cells of the nervous system
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0623—Stem cells
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/44—Thiols, e.g. mercaptoethanol
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/06—Anti-neoplasic drugs, anti-retroviral drugs, e.g. azacytidine, cyclophosphamide
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells

Definitions

  • the present invention provides a method for producing a nervous system cell capable of obtaining a substantially isolated nervous system cell, a substantially isolated neural stem cell, a substantially isolated nerve cell,
  • the present invention relates to an isolated glial cell, a cell pharmaceutical composition, and a method for treating neurodegenerative diseases or nerve damage.
  • EB embryoid body
  • b FGF basic fibroblast growth factor
  • ES cells are cultured in suspension in the presence of leukemia inhibitory factor (LIF), neura 1 spheres are prepared from neural stem cells present in about 0.2% of ES cells, and then differentiated into neural cells [ Tropepe V. et al., Neuron 30: 65-78 (2001)]
  • LIF leukemia inhibitory factor
  • retinoic acid It is difficult to secure a sufficient amount of cells for use in regenerative medicine from the viewpoint of the teratogenicity of differentiated cells, the time required to produce neural stem cells, the rate of differentiation, the efficiency of yield, etc. .
  • Neural stem cells are multipotent cells capable of differentiating into neural cells and glial cells, and have self-replicating ability, and play an important role in transplantation and regeneration medicine of the nervous system.
  • the N eurosphere method has been established as a method for maintaining and proliferating neural stem cells in an undifferentiated state [Reynolds, BA, et al., J. Neurosci., 12: 4565-4574, (1992), Reynolds , BA) et al.,
  • N 2 additive N2 supplement; insulin, transferrin, selenium and progesterone
  • 20 ngZm 1 epidermal growth factor (EGF) 20 ngZm 1 epidermal growth factor
  • DMEM F-12 serum-free medium containing 20 ng Zm 1 b FGF
  • the proliferated neural stem cells are positive for nestin, an intermediate filament, which is a marker, and form cell aggregates (neuroshere) and can be cultured in a floating state.
  • the above-mentioned neu ro s p h ere is differentiated into nerve cells, astrocytes, oligodendrocytes, etc. when cultured on an adherent substrate plate excluding growth-promoting factors.
  • Neuros ph ere has the disadvantage that it is formed only from some cells, not all.
  • a monolayer culture method in which neural stem cells are cultured and proliferated and differentiated on a plate coated with an adhesive substrate has also been established as a method different from the Neurosphere method.
  • the monolayer culture method a method of performing monolayer culture by concentrating neural stem cells by density gradient centrifugation [Palmer, T. D. et al., J. Neurosci.,
  • the present invention provides at least a neural cell that is substantially isolated, a large amount of the nervous system cell, and a neural cell without being limited to a source of embryonic stem cells.
  • An object of the present invention is to provide a method for producing a nervous system cell that can achieve one.
  • the present invention is substantially isolated and useful as a cell or tissue source in neuroregenerative medicine for neurodegenerative diseases (for example, Parkinson's disease, Alzheimer's disease, etc.), spinal cord injury, cerebral infarction, etc.
  • the purpose is to provide neural stem cells.
  • an object of the present invention is to provide nerve cells useful for regenerative medicine such as nerve cell transplantation treatment for neurodegenerative diseases (for example, Parkinson's disease, Alzheimer's disease, etc.), spinal cord injury, cerebral infarction and the like.
  • the present invention also provides glial cells that are transplanted at the same time as nerve cells and neural stem cells to support the differentiation and growth of nerve cells, and further form a blood-brain barrier to replenish nutrients.
  • neurodegenerative diseases e.g., Parkinson's disease, Alzheimer's disease, etc.
  • spinal cord injury is useful in regenerative medicine nerve cell transplantation therapy or the like for cerebral infarction, during cell therapy
  • An object of the present invention is to provide a cell medicine composition capable of obtaining a stable therapeutic effect, a high therapeutic effect, and the like.
  • the present invention also provides neurodegeneration or nerve damage. It is an object of the present invention to provide a method for treating a neurodegenerative disease or nerve damage capable of treating a condition caused by the above in a stable state. That is, the gist of the present invention is
  • a method for producing a substantially isolated nervous system cell characterized in that embryonic stem cells are cultured in suspension in the presence of a fast mouth conditioned medium or a component substantially equivalent to the conditioned medium,
  • SCS stem cell globulomer
  • step (B) Stem cell spherical aggregate (S) obtained in step (A) in the presence of basic fibroblast growth factor (bFGF) and / or epidermal growth factor (EG F) and in the presence of a cell adhesion molecule.
  • bFGF basic fibroblast growth factor
  • EG F epidermal growth factor
  • the culture in the step (B) is performed by adhering the stem cell spherical aggregate (SCS) obtained in the step (A) and an adhesive culture substrate holding a cell adhesion molecule.
  • SCS stem cell spherical aggregate
  • step (B) Stem cell spherical aggregate (SCS) obtained in step (A) in the presence of basic fibroblast growth factor (bFGF) and / or epidermal growth factor (EG F) and in the presence of a cell adhesion molecule. ), and
  • a method for producing a nerve cell wherein the neural stem cell obtained by the production method according to any one of the above [1] to [7] is cultured while being adhered to an adhesive culture substrate retaining a cell adhesion molecule,
  • a substantially isolated neural stem cell which is induced to differentiate from an embryonic stem cell by the production method of any one of [1] to [7],
  • a cell comprising, as an active ingredient, a substantially isolated neural stem cell that is induced to differentiate from an embryonic stem cell by the production method according to any one of [1] to [7] Pharmaceutical composition,
  • a cell pharmaceutical composition comprising, as an active ingredient, a substantially isolated nerve cell obtained by the production method of [8] or [10]
  • a cell pharmaceutical composition comprising, as an active ingredient, a substantially isolated Darial cell obtained by the production method of [9], and
  • a method for treating a neurodegenerative disease or nerve injury characterized by introducing at least one cell selected from the group consisting of: Brief Description of Drawings
  • Figure 1 shows the expression of nestin, a marker of neural stem cells, in mouse embryonic stem cells (HK cell line) colonies (SCS) cultured in suspension in a mixture of astrocyte conditioned medium and astrocyte basal medium. It is a photograph which shows the result of having investigated the distribution of Br dU which is an index of cell distribution and cell division.
  • a confocal laser scanning fluorescence microscope manufactured by Zeiss was used.
  • panel A is S A phase contrast observation image of CS is shown
  • Panel B shows an immunofluorescence staining image with anti-nestin antibody
  • Panel C shows an immunofluorescence staining image with anti-B r dU antibody
  • Panel D shows Panel B and Panel C The double exposure image of is shown.
  • Fig. 2 is a photograph showing the results of examining the expression of class III / 3 tubulin, a marker of juvenile neurons, in SCS that had been in suspension culture for 7 days. For observation, an upright fluorescent microscope made by Nikon was used. In the figure, panel A shows a phase contrast observation image of SCS, and panel B shows an immunofluorescent staining image with an anti-class IV) 3-tubulin antibody.
  • the scale bar indicates 50 ⁇ m.
  • Fig. 3 is a photograph showing the results of differentiation induction in SCS cultured for 4 days on the surface of the culture dish coated with an adhesive substrate after 4 days of suspension culture (ie, the side containing the medium). It is.
  • Panel A shows a phase contrast image
  • Panel B shows an immunofluorescent staining image with an anti-nestin antibody
  • Panel C shows an immunofluorescent staining image with an anti-neurofilament (NF) antibody
  • Panel D shows a panel B And double exposure image of Panel C.
  • the scale bar indicates 50 m.
  • FIG. 4 is a photograph showing the results of examining the distribution of Br dU incorporation and neurofilament expression in adherent cultured SCS.
  • a confocal laser fluorescence microscope was used for observation.
  • Panel A shows an immunofluorescent staining image with anti-NF antibody
  • Panel B shows an immunofluorescent staining image with anti-B r dU antibody
  • Panel C shows double exposure of Panel A and Panel B. Show the image.
  • the scale bar indicates 50 xm.
  • Fig. 5 is a photograph showing the results of examining the expression of neurotransmitter synthase in the adhered SCS. An upright fluorescent microscope was used for observation.
  • Panel A shows a phase contrast image
  • Panel B shows an immunofluorescent staining image with an anti-NF antibody
  • Panel C shows an immunofluorescent staining image with an anti-tyrosine hydroxylase (TH) antibody
  • Panel D shows a double exposure image of Panel B and Panel C.
  • the scale bar indicates 50 m.
  • Fig. 6 is a photograph showing the results of examining the expression of neurotransmitter synthase in the adhered SCS. An upright fluorescent microscope was used for observation.
  • Panel A shows an immunofluorescent staining image with an anti-glutamate decarboxylase (GAD) antibody
  • Panel B shows an immunofluorescent staining image with an anti-NF antibody
  • Panel C shows double exposure of Panel A and Panel B Show the image.
  • the scale bar indicates 50 m.
  • FIG. 7 is a photograph showing the results of examining the expression of choline acetyltransferase (ChAT). An upright fluorescent microscope was used for observation. Panel A shows an immunofluorescent staining image with anti-ChAT antibody, Panel B shows an immunofluorescent staining image with anti-NF antibody, and Panel C shows a double exposure image of Panel A and Panel B. The scale bar indicates 20 zm.
  • FIG. 8 is a photograph showing the results of examining SCS when adherent culture of SCS and using Neurobasa 1 TM B-27 containing bFGF as the culture medium.
  • Panel A shows a phase contrast image
  • Panel B shows an immunofluorescent staining image with an anti-nestin antibody.
  • the scale bar indicates 5.
  • Fig. 9 is a photograph showing a colony of neural stem cells that have migrated from one SCS.
  • the scale bar indicates 50 j m.
  • FIG. 10 is a photograph showing the results of examining the distribution of Br dU incorporation and nestin expression in SCS that migrates neural stem cells in Neurobasal TM B-27 containing bFGF.
  • Panel A shows an immunofluorescent staining image with anti-B r dU
  • Panel B shows an immunofluorescent staining image with an anti-nestin antibody
  • Panel C shows a double exposure image of Panel A and Panel B.
  • the scale bar indicates 50 / _im.
  • Figure 11 shows the marker of neural stem cell marker nestin and cell fragmentation when b FGF is added to the suspension culture medium from the beginning when preparing SCS from the mouse embryonic stem cell HK cell line. It is a photograph which shows the result of having investigated distribution of dU.
  • Panel A shows an immunofluorescent staining image of anti-nestin antibody.
  • Panel B shows an immunofluorescent staining image of anti-B r dU antibody.
  • Panel C is the double dew of Panel A and Panel B An optical image is shown.
  • the scale bar shows 50 111.
  • FIG. 12 is a photograph showing the results of examining the differentiation of neural stem cells migrated from SCS into neurons.
  • Panel A shows the phase difference between SCS and neural stem cells immediately after replacement of Neuro basa 1 TM B-27 containing bFGF with a mixture of fast-mouth conditioned medium and long-mouth basal medium.
  • Panel B shows the phase contrast image one day after replacement.
  • the scale bar shows 50 111.
  • FIG. 13 is a photograph showing the results of examining the expression of a neurotransmitter synthesizing enzyme in nerve cells derived from migrated neural stem cells.
  • panel A shows a phase contrast image
  • panel B shows an immunofluorescent staining image with anti-NF antibody
  • panel C shows an immunofluorescent staining image with anti-TH antibody
  • panel D shows panel B and A double exposure image with panel C is shown.
  • the scale bar indicates 20 m.
  • FIG. 14 is a photograph showing the results of examining the distribution of TH and GAD expression in neurons derived from migrated neural stem cells.
  • panel A shows a phase contrast image
  • panel B shows an immunofluorescent staining image with an anti-TH antibody
  • panel C shows an immunofluorescent staining image with an anti-GAD antibody
  • panel D shows panel B And double exposure image of panel C.
  • the scale bar is 20
  • FIG. 15 is a photograph showing the results of examining the presence of serotonin, a neurotransmitter, in migrated neural stem cell-derived neurons.
  • panel A shows a phase contrast image
  • panel B shows an immunofluorescent staining image with an anti-serotonin antibody.
  • the scale bar indicates 20 m.
  • FIG. 16 is a photograph showing the results of examining differentiation induction from migrated neural stem cells to astrocytes.
  • panel A shows a phase contrast image centered on SCS
  • panel B shows an immunofluorescent staining image with an anti-GF AP antibody at the same site as panel A
  • panel C at the same site as panel A
  • Panel D shows a phase contrast image at a site away from SCS
  • Panel E shows an immunofluorescent staining image with an anti-GF AP antibody at the same site as Panel D
  • Panel F shows an immunofluorescent staining image with anti-GF AP antibody in the periphery of the colony.
  • Panel G also shows an immunofluorescent staining image (high magnification) with the anti-GF AP antibody in the periphery of the colony.
  • Panel H Shows an immunofluorescent staining image (high magnification) with an anti-GFAP antibody around the colony.
  • the scale bars on panels A to F indicate 50 ⁇ m, and the scale bars on panels G and H indicate 20 m.
  • Fig. 17 is a photograph showing the results of examining the differentiation of dispersed migrating neural stem cells into neurons.
  • Panel A shows an immunofluorescent staining image with anti-nestin antibody
  • Panel B shows an immunofluorescence staining image with anti-TH antibody
  • Panel C shows a double exposure image of Panel A and Panel B .
  • the scale bar indicates 20 m.
  • FIG. 18 is a photograph showing the results of examining the differentiation of cryopreserved neural stem cells into neural cells.
  • Panel A shows a phase contrast image
  • Panel B shows an immunofluorescent staining image with an anti-TH antibody
  • Panel C shows an immunofluorescent staining image with an anti-Ch AT antibody
  • Panel D shows a panel A double exposure image of A and Panel B is shown.
  • the scale bar indicates 20 m.
  • FIG. 19 is a photograph showing the results of suspension culture in a mixture of astrocyte conditioned medium and astrocyte basal medium for a commercially available embryonic stem cell line (129 SV cell line) .
  • the scale bar indicates 50 ⁇ m.
  • FIG. 20 is a photograph showing the distribution of nestin when SCS was prepared in the CMK-6 cell line, an embryonic stem cell line established from Riki cynomolgus monkeys.
  • Panel A in the figure is an immunofluorescent staining image of floating SCS with anti-nestin antibody.
  • Panel B is an immunofluorescent staining image of cells that have migrated with SCS attached using anti-nestin antibodies.
  • the scale bar indicates 50 111.
  • FIG. 21 is a photograph showing the result of suspension culture in a mixture of astrocyte conditioned medium and astrocyte basal medium for CMK-6 cell line derived from Riki quizal.
  • the scale bar indicates 20 z m.
  • Figure 22 shows the SCS obtained from the CMK-6 cell line of the power cynomolgus embryonic stem cell 2 is a photograph showing the result of examining the expression of a neurotransmitter synthase in a neuron migrating from a cell.
  • Panel A shows an immunofluorescent staining image with anti-TH antibody
  • Panel B shows an immunofluorescent staining image with anti-NF antibody
  • Panel C shows a double exposure image of Panel A and Panel B .
  • the scale bar indicates 20 // m.
  • FIG. 23 is a diagram showing the results of examining gene expression associated with differentiation induction from embryonic stem cells to neurons.
  • lane 1 shows the results of undifferentiated embryonic stem cell clones
  • lane 2 shows the SCS formed by suspension culture for 4 days
  • lane 3 shows the results of cell mass obtained by adhesion culture for 5 days.
  • FIG. 24 is a graph showing changes in gene expression over time accompanying induction of differentiation from embryonic stem cells to neurons. Divide the amounts of O ct _ 4, Nestin (N estin in the figure) and TH by the amount of GAPDH to obtain relative expression levels as O ct _4 / GAPDH, nestin ZG APDH, and THZGAPDH. It is shown in the graph with the maximum value of the relative amount expressed as 1.
  • the method for producing a substantially isolated nervous system cell comprises subjecting an embryonic stem cell to suspension culture in the presence of an astrocyte conditioned medium or a component substantially equivalent to the conditioned medium.
  • an astrocyte cell culture supernatant that is, an astrocyte conditioned medium
  • the present invention uses an astrocyte cell culture supernatant, that is, an astrocyte conditioned medium, to perform undifferentiated embryonic stem cells in a short period of time by culturing them in suspension in a short period of time.
  • Inducible and capable of preparing a large amount of neural stem cells, and further differentiation-induced neural stem cells are used to differentiate into neurons, particularly dopaminergic neurons; glial cells, in particular, astrocytic Based on the inventors' surprising discovery that guidance can be performed.
  • a fast mouth conditioned medium or a component substantially equivalent to the conditioned medium is used. From the embryonic stem cells, it exhibits the excellent effect of being able to obtain substantially isolated neural cells efficiently in a short period of time.
  • a neural stem cell can be obtained in a surprisingly short period of time, 2 to 4 days after suspension culture of embryonic stem cells, for example, compared to the SDIA method or the like.
  • embryonic stem cells are cultured in suspension, so that it is possible to obtain neural cells with surprising efficiency and surprisingly short period of time. .
  • the embryonic stem cell can be efficiently produced without inducing ectoderm cells, mesoderm cells, and endoderm cells other than the nervous system cells. Further c is exhibited an excellent effect that it is possible to provide a substantially isolated neuronal cells, according to the production method of the present invention, the embryonic stem cells, through a neural stem cell, neural cell or Daria An excellent effect is that one of the cells can be selectively differentiated.
  • neural stem cells neural stem cells, neural cells (two Yulong), glial cells (e.g., ⁇ stroke site, etc.) c wherein neural stem cells is intended to include like, brain
  • a central nervous system pluripotent undifferentiated cell that has the ability to differentiate into neurons, glial cells, etc. that constitute the spinal cord and the like and has the ability to self-replicate.
  • the neural stem cells can be examined by, for example, detecting the expression of the corresponding gene by a conventional nucleic acid detection method using the expression of a marker such as Nestin, RC 2 or Musashi 1 as an index. Protein expression can be identified by examining immunocytohistochemical methods.
  • the nerve cell that is, a neuron is characterized by the expression of a receptor for a signal transmitter, and is characterized by the expression of a neurofilament, tyrosine hydroxylase, glutamate decarboxylase, choline acetyltransferase, and the like.
  • the morphological features of eurone include cell bodies, dendrites, axons, and axon growth cones.
  • Neurons obtained by the method for producing nervous system cells of the present invention include neurons. More specifically, examples of the neuron include dopaminergic neurones, GABAergic neurons, cholinergic neurons, and the like.
  • the dopaminergic neurons are expected to be applied to, for example, Parkinson's disease.
  • GABAergic neurons are inhibitory neurons and are expected to be applied to suppress overexcitement.
  • the cholinergic neurons are expected to be applied to Alheimer's disease and the like.
  • the glial cell is a cell that fills the gap between neurons and mediates the metabolism of the neurons and also serves as a supporting tissue.
  • the glial cells include astrocytes, oligodendrocytes and microgliocytes in the central nervous system, and mantle cells, Schwann cells and terminal glial cells in the peripheral nervous system.
  • astrocytes can be obtained among the glial cells.
  • the astrocytes are characterized by the expression of Darya fibrillary acidic protein (GFAP).
  • GFAP Darya fibrillary acidic protein
  • N2 additive insulin, transferrin, selenium, progesterone; eg, Pottenstein et al., Proc Natl. Acad. Sci. USA, 76: 514 (1979), etc.
  • DMEM Dulbec
  • DMEM for example, DMEM, F-12, MEM, Neurobasal TM (GIBCO BRL) Product
  • F_12 for example, DMEM, F-12, MEM, Neurobasal TM (GIBCO BRL) Product
  • These media include, for example, Frshney, R. Ian, Culture 'Ob' Animal Cells A Mani Yual Ob 'Basic' Technique, Second Edition (Culture of animal cell A manual of basic technique, 2nd ed.), Alan R. Liss. Inc., 66-84 (1987) and the like.
  • the astrocytes that can be used in the preparation of the astrocyte conditioned medium are not particularly limited.
  • the astrocyte is a known method [Banker, G.
  • substantially equivalent component to the conditioned medium refers to a component that can exert the same action as the conditioned medium. From the astrocyte culture, the basic medium component and astrocyte used. This refers to the components obtained by removing, such as metabolites.
  • the embryonic stem cells used in the method for producing nervous system cells of the present invention are not particularly limited to the type of individual serving as the source of the embryonic stem cells.
  • mammals specifically, for example, mice, monkeys, etc. (For example, cynomolgus monkeys), embryonic stem cells derived from humans, rats and the like.
  • commercially available embryonic stem cells may be used.
  • the obtained nervous system cells are used for cell transplantation treatment or the like, from the viewpoint of biocompatibility, it is desirable that they are embryonic stem cells derived from an individual of the same type as the individual to be applied for cell transplantation treatment or the like.
  • the medium used for culturing embryonic stem cells includes the astrocyte conditioned medium or components substantially equivalent to the conditioned medium, and the astrocyte basal medium, such as DMEM: F-12, DMEM, F -12, and mixtures with any medium such as MEM, Neurobasal TM, etc.
  • the volume ratio is preferably 1: 1 to 1: 3.
  • the suspension culture of embryonic stem cells varies depending on the type of embryonic stem cells used.
  • the size of the culture vessel used for suspension culture of the embryonic stem cells is desirably a 35 mm dish, It is desirable that the concentration of embryonic stem cells in the culture medium is such that the concentration of SCS is 20 or less in a 2 ml culture medium.
  • the condition of the culture gas phase is around 37 in the case of the HK cell line, for example, , 37 ⁇ 0. 2, C_ ⁇ 2 concentration around 5%, e.g., 4.8 to 5.2%, it is desirable that the 100% humidity.
  • a mixture of the astrocyte conditioned medium and the astrocyte basal medium contains 2m 1 In a 35 mm dish, 37: can be performed by culturing about 10 to 20 cells in a humidified atmosphere of 5% CO 2 and 100% in air.
  • the size of mouse-derived embryonic stem cells used here is the size on the feeder cell layer from the viewpoint of ease of operation, stable maintenance of the state of embryonic stem cells, and efficient acquisition of SCS.
  • the diameter is preferably about 400 to 500 m.
  • the size of the power cynomolgus monkey-derived embryonic stem cells used here is 400 to 500 / m in diameter from the viewpoint of ease of manipulation, stable maintenance of the state of embryonic stem cells, and efficient acquisition of SCS. Is desirable.
  • embryonic stem cells those obtained by proliferating in a cell-adhered state on an appropriate medium, further on a feeder cell if necessary, and obtained as a mass of undifferentiated embryonic stem cell colonies can be used.
  • SCS differs depending on the type of embryonic stem cell used, in the case of mouse-derived embryonic stem cells, it is formed under conditions of the suspension culture, for example, 2-7 days, preferably 4-5 days. In particular, in the case of embryonic stem cells derived from force cynomolgus monkeys, they are formed under the conditions of the suspension culture, for example, 4 to 15 days, preferably 10 to 12 days.
  • the formation of SCS can be confirmed by forming the core structure under a stereo microscope or a phase contrast inverted microscope.
  • neural stem cells by the method for producing nervous system cells of the present invention, preferably, from the viewpoint of obtaining neural stem cells more efficiently,
  • the neural stem cell production method 1 by performing the step (B), neural stem cells are obtained as cells migrated from the SCS.
  • the method 2 for producing neural stem cells a large amount of neural stem cells can be obtained in the SCS by performing the step ( ⁇ ′).
  • the medium used for SCS culture includes bFGF and / or EGF, and the astrocyte conditioned medium or a component substantially equivalent to the conditioned medium, For example, Ne urobasal TM
  • the concentration of bFGF in the medium is, for example, 10 to 50 ng Zml, preferably 10 to 20 ng gmm 1 from the viewpoint of sufficiently exerting the ability to suppress cell differentiation and the ability to divide neural stem cells. Is desirable.
  • the concentration of EGF in the culture medium is, for example, 10 to 50 ng Zm 1, preferably 10 to 20 ng Zm 1 from the viewpoint of sufficiently exerting the ability to suppress cell differentiation. desirable.
  • b FGF is mixed at a concentration of 10-20 ng / m 1 and EGF is mixed at a concentration of 10-20 ng Z m 1 May be.
  • a substance exhibiting a differentiation-inhibiting action similar to the bFGF and EGF may be used.
  • the mixing ratio with MEM or the like is preferably 1: 1 to 1: 3 as a volume ratio.
  • cell adhesion molecule examples include polylysine, fibronectin, laminin, vitronectin, Matrigel TM (manufactured by BD Bioscience) and the like.
  • the culture in the step (B) may be performed by adhering the SCS obtained in the step (A) and an adhesive culture substrate holding a cell adhesion molecule, and the cell adhesion on an appropriate support.
  • SCS may be cultured in a culture vessel in which a carrier holding molecules is suspended or arranged in a three-dimensional configuration.
  • the adhesive culture substrate examples include those obtained by coating the surface of the culture side of a conventional cell culture dish with the cell adhesion molecule.
  • the cell adhesion molecule For example, when polylysine is used as a cell adhesion molecule, a final concentration of about 0.1 mg / m1 of polylysine is used. Make sure that the culture side surface of the culture vessel (dish for cell culture) is fully immersed in the pure water, and incubate at room temperature for 1 to 2 hours, and then remove the solution from the culture vessel to remove the adhesive culture substrate. Can be obtained.
  • fibronectin when using fibronectin as a cell adhesion molecule, use phosphate buffered saline containing 5-20 ig / m1 of final concentration of fib mouth nectin and incubate 30-90 minutes at 37.
  • laminin when using laminin as a cell adhesion molecule, use phosphate buffered saline containing laminin at a final concentration of 10 to 100 g / m 1 and incubate at 37 for 2 hours or longer.
  • vitronectin is used as a cell adhesion molecule, phosphate buffered physiological saline containing 1 to 10 gZm 1 of vitronectin may be used and incubated at 37 for 2 hours or more.
  • Matrigel is used as an adhesion molecule, it should be diluted 10 to 20 times with culture medium and incubated at 37 for 1 hour or longer.
  • the adhesion between SCS and the adherent culture substrate can be performed, for example, by adding SCS to the adherent culture substrate containing an appropriate medium.
  • the support may be a support made of a substance having a specific gravity smaller than that of the medium when suspended in a culture vessel.
  • a conventional cell culture dish and Supports made of the same material may be used.
  • the culture conditions in the step (B) can be appropriately set depending on the type of embryonic stem cells that are the source of the SCS used.
  • the size of the culture container is preferably 35 mm dish or 60 mm dish.
  • the number of colonies of embryonic stem cells that is, the number of SCS is, for example, 1 to 20 in a 35 mm dish containing 2 ml of medium, preferably 1 to 5 1 to 2, more preferably 1 to 2, in the case of monkey-derived embryonic stem cells, for example, 1 to 20 cells, preferably 1 to 5 cells in a 35 mm dish containing 2 ml of medium. More preferably, it is 1 to 2 pieces.
  • the conditions of the culture gas phase in the case of mouse, 3 7 before and after, for example, 3 7 ⁇ 0. 2, C0 2 concentration of 5% or so, for example, 4.8 to 5.2%, humidity 1 0 0%
  • the humidity of 1 100% in the case of monkeys, around 3 7, for example, 3 7t: ⁇ 0. 2:, C0 2 concentration of 5% or so, for example, 4.8 to 5.2%, it is desirable that the humidity of 1 100%.
  • the culture time in the step (B) can be appropriately set depending on the type of embryonic stem cell used. In the case of mouse-derived embryonic stem cells, it is preferably 5 to 10 days. If it is 5-20 days, it is desirable.
  • bFGF and / or EGF may be added so that the appropriate concentration is obtained during suspension culture at an appropriate time (for example, 1 day interval, 2 day interval, etc.). .
  • Neural stem cell production method 1 '' depending on the type of embryonic stem cell used, in the case of mouse-derived embryonic stem cells, SCS is used under the conditions of the suspension culture, In 2 to 7 days, neural stem cells are obtained in a surprisingly short period of 2 to 5 days, preferably 2 to 4 days, and in the case of monkeys, especially force-biquizal-derived embryonic stem cells, SCS is Under the conditions of the suspension culture, after the suspension culture is started, for example, it is formed in 4 to 15 days, and the neural stem cells are obtained in a surprisingly short time of 4 to 7 days, preferably 4 to 5 days. Demonstrate the effect.
  • the medium for culturing embryonic stem cells contains bFGF and / or EGF, and the astrocyte conditioned medium or the conditioned medium.
  • Examples include a mixture of components substantially equivalent to the medium and, for example, Neurobasa 1 TM B-2 27, DMEM: F-12, DMEM, F_12, MEM, and the like.
  • the concentration of bFGF in the medium is, for example, 10 to 50 ng / m 1, preferably 10 to 20 from the viewpoint of sufficiently exerting the ability to suppress cell differentiation and the ability to divide neural stem cells. It is desirable to be ng / m 1.
  • the concentration of EGF in the medium is, for example, 10 to 50 ng / ml, preferably 10 to 20 from the viewpoint of sufficiently exerting the ability to inhibit cell differentiation and the ability to divide neural stem cells.
  • n gZm 1 is desirable.
  • mix bFGF with a concentration of 10 to 20 ngZm1 and EGF to a concentration of 10 to 20 ng / m1. May be used.
  • Method for producing neural stem cell 2 suspension culture of embryonic stem cells varies depending on the type of embryonic stem cells used. For example, the size of the culture vessel used for suspension culture of embryonic stem cells is as described above.
  • the dish is a 35 mm dish, and the concentration of embryonic stem cells in the medium is desirably 20 or less in a 2 ml culture solution.
  • the culture time in the above step ( ⁇ ′) can be appropriately set depending on the type of embryonic stem cell used. In the case of mouse-derived embryonic stem cells, it is preferably 4 to 5 days. 7 to 10 days is desirable.
  • bFGF and / or EGF may be added so as to achieve an appropriate concentration during suspension culture at an appropriate time (for example, 1 day interval, 2 day interval, etc.).
  • Neurosenor stem cell production method 2 although depending on the type of embryonic stem cells used, in the case of mouse-derived embryonic stem cells, SCS is, for example, 2-7 days under the conditions of suspension culture.
  • the neural stem cells that are formed are obtained in a surprisingly short period of 2-5 days, preferably 2-4 days. For example, it is formed in 4 to 15 days, and the neural stem cell exhibits an excellent effect that it can be obtained in a surprisingly short period of 4 to 7 days, preferably 4 to 5 days.
  • the obtained cell is a neural stem cell using the expression of a marker such as nestin, RC 2 and Musashi 1 as an index. This can be confirmed by examining the detection method described above, or by examining the expression of the protein by immunological techniques. Furthermore, the obtained cells are neural stem cells, which means that the obtained cells are differentiated by culturing in a medium that does not prevent differentiation into various cells or tissues in the presence of retinoic acid or the like. You may confirm by the form of a differentiation tissue, or expression of a marker specific to this differentiation cell or differentiation tissue. Furthermore, the obtained cells can be transplanted into the central nervous system such as the forebrain, midbrain, retina, and olfactory bulb, and the formation of neurons can be confirmed as an index.
  • a marker such as nestin, RC 2 and Musashi 1 as an index.
  • Examples of the morphology of the differentiated cells or differentiated tissues and the markers specific to the differentiated cells or differentiated tissues include the morphological characteristics and specific markers of the nerve cells and glial cells.
  • the neural stem cell may be promodoxyuridine.
  • a hybrid using a probe comprising a nucleic acid encoding a gene to be detected or a specific fragment thereof, or a probe that specifically binds to the nucleic acid or a specific fragment thereof examples include a detection method based on a determination, and a detection method based on a PC scale using a primer for amplifying the nucleic acid or a specific fragment thereof.
  • Examples of the detection method based on the hybridization include Southern hybridization, DNA array hybridization, Northern hybridization, and the like.
  • detection methods based on PC include RT-PCR.
  • the nucleic acid used as a probe or primer preferably has a sequence of a portion specific to the base sequence of the gene to be detected.
  • the “part specific to the nucleotide sequence of the gene to be detected” is, for example, 20% or less, preferably 15% or less, more preferably 1% of sequence identity with a known sequence that is not the detection target. It can be obtained by selecting a sequence of a portion that is 0% or less, more preferably 5% or less, and particularly preferably 0%.
  • Said sequence identity can be determined by comparing two sequences aligned optimally over the region of the sequence to be compared between the two nucleic acids. Also, the sequence identity number (percentage) determines the number of matching sites by determining the same residues present in both sequences, and then the ratio Divide the number of matching sites by the total number of residues in the sequence region to be compared, and calculate
  • an antibody or an antibody fragment against the protein to be detected or a specific fragment thereof can be used, and an immunological technique such as conventional EL ISA or immunostaining can be performed.
  • Antibodies can be prepared by conventional methods (eg, 1 1992, published by John Wiley & Sons, Inc., John Oh. E. Co 1 igan). ) Edit, Current 'Protocols in Immunology 1 (Current Protocol Immuno no 1 ogy) etc.] and using the marker protein to be detected to detect suitable animals such as rabbits, rats, etc. It can be obtained by immunizing mice, goats, etc.
  • the present invention also includes a substantially isolated neural stem cell derived from embryonic stem cells by such a production method.
  • the neural stem cell of the present invention expresses an excellent property of exhibiting differentiation ability into a neuronal cell, a glial cell, etc. even when cryopreserved.
  • the neural stem cells of the present invention can be differentiated into nerve cells, glial cells, etc., Parkinson's disease, Alzheimer's disease, Down's syndrome, prion disease (for example, Creutzfeldt-Jakob disease, Usi spongiform encephalopathy, etc.), polyglutamine Neurodegenerative diseases such as cerebral spinal muscular atrophy, Huntington's disease, spinocerebellar ataxia, amyotrophic lateral sclerosis; cerebral ischemia, demyelinating disease, head trauma, spinal cord injury, cerebral infarction Application to nerve regeneration medicine is expected.
  • Parkinson's disease Alzheimer's disease, Down's syndrome, prion disease (for example, Creutzfeldt-Jakob disease, Usi spongiform encephalopathy, etc.)
  • polyglutamine Neurodegenerative diseases such as cerebral spinal muscular atrophy, Huntington's disease, spinocerebellar ataxia, amyotrophic lateral sclerosis
  • cerebral ischemia demyelinating disease
  • step (A) In the case of producing a nerve cell by the method for producing a nervous system cell of the present invention, from the viewpoint of obtaining a nerve cell more efficiently, preferably, after the step (A), ( ⁇ ′) b FGF and Z or EGF In the absence, and in the presence of an astrocyte conditioned medium or a component substantially equivalent to the conditioned medium, the SCS obtained in step (A) and an adhesive culture substrate retaining a cell adhesion molecule are used.
  • the step of adhering and culturing can be carried out [also referred to as “Neuron production method I”] By performing this step ( ⁇ ′), a nerve cell can be obtained.
  • the neural stem cells obtained as described above can be used in the absence of bFGF and sputum or EGF, and substantially equivalent to astrocyte conditioned medium or the conditioned medium.
  • a method of culturing by adhering to an adhesive culture substrate holding a cell adhesion molecule in the presence of a natural component [also referred to as “manufacturing method II”] can be mentioned.
  • the astrocyte conditioned medium or a component substantially equivalent to the conditioned medium, and DMEM: F-12, DMEM, F-12 examples thereof include a mixture with any medium such as MEM and Neurobasa 1 TM , and the mixing ratio is preferably 1: 1 to 1: 3 as a volume ratio.
  • the culture conditions can be appropriately set depending on the type of embryonic stem cells that are the source of the SCS used.
  • the size of the culture vessel is desirably a 35 mm dish or a 60 mm dish.
  • the number of SCS is, for example, 1 to 20, preferably 1 to 5, more preferably 1 in a culture medium containing 2 ml of medium in a 35 mm dish.
  • monkey-derived embryonic stem cells for example, 1 to 20, preferably 1 to 5, more preferably 1 to 2 in a 2 ml culture medium with a 35 mm dish It is desirable to be individual.
  • the conditions of the culture gas phase mice cases, 37 back and forth, for example, SY i O. 2, C0 2 concentration of 5% or so, for example, 4.8 to 5.2%, it is 100% humidity preferably, in the case of monkey, back and forth 37, for example, ⁇ 0. 2 "at 37, C0 2 concentration of 5% or so, for example, 4.8 to 5.2%, it is desirable that the 100% humidity.
  • the culture time in the step ( ⁇ ′) is the type of embryonic stem cell used.
  • mouse-derived embryonic stem cells it is preferably 1 to 7 days.
  • monkeys it is preferably 1 to 14 days.
  • the time is preferably 1 to 7 days, and in the case of monkeys, the time is preferably 1 to 14 days.
  • the nerve cells have an excellent effect that they can be obtained in a surprisingly short time of 1 to 7 days, preferably 2 to 5 days. Demonstrate.
  • the obtained cell is a neuronal cell.
  • the expression of the corresponding gene is detected using the expression of neurofilament, tyrosine hydroxylase, glutamate decarboxylase, corinacetyltransferase, etc. It can be confirmed by examining by a method or by examining protein expression by immunological techniques such as conventional ELISA and immunostaining.
  • the morphological characteristics of neurons for example, cell bodies, dendrites, axons, axon growth cones, etc. can be confirmed as indices.
  • the present invention also includes substantially isolated nerve cells obtained by such a production method.
  • the nerve cell of the present invention expresses at least one selected from the group consisting of class III / 3 tubulin, neurofilament, tyrosine hydroxylase, glutamate decarboxylase and choline acetyltransferase. , Parkinson's disease, Alzheimer's disease, Down's syndrome, Prion's disease (eg, Kreuzf Ertoyakob disease, Usi spongiform encephalopathy, etc.) Neurodegenerative diseases such as lateral sclerosis; expected to be applied to nerve regeneration therapy in cerebral ischemia, demyelinating disease, head trauma, spinal cord injury, cerebral infarction, etc.
  • step (C) When producing Daria cells by the method for producing nervous system cells of the present invention, from the viewpoint of obtaining glial cells more efficiently, preferably after the step (B), (C) in the absence of bFGF and / or EGF A step of adhering the SCS obtained in the step (B) and an adhesive culture substrate holding a cell adhesion molecule, and culturing. it can. By performing this step (C), glial cells, particularly astrocytes can be obtained as cells migrated from SCS.
  • Examples of the medium used in the step (C) include Neurobasal TM B-27 [Gibco BRL Co., Ltd.] 1-2% additive, DMEM: F-12N 2 additive, etc. It is done.
  • the culture conditions can be appropriately set depending on the type of embryonic stem cells that serve as the supply source of the SCS used.
  • the size of the culture vessel is desirably a 35 mm dish or a 60 mm dish.
  • the number of SCS is, for example, 1 to 20, preferably 1 to 5, more preferably 1 to 2 in a 2 ml culture medium with a 35 mm dish.
  • monkey-derived embryonic stem cells for example, 1 to 20, preferably 1 to 5, more preferably 1 to 2 in a culture medium of 2 ml in a 35 mm dish desirable.
  • the conditions of the culture gas phase in the case of mouse, at 37 before and after, for example, ⁇ 0. 2 at 37, C_ ⁇ 2 concentration of 5% or so, for example, 4.8 to 5.2%, humidity 1 100% it is desirably, if the monkeys, 37 back and forth, for example, 37t:. ⁇ 0. 2 " C, C0 2 concentration of 5% or so, for example, from 4.8 to 5 2%, it is desirable that the 100% humidity .
  • the culture time in the step (C) can be appropriately set depending on the type of embryonic stem cells used, and in the case of mouse-derived embryonic stem cells, it is preferably 2 to 7 days. In the case of monkeys, 5 to 15 days are desirable.
  • glial cells exhibit an excellent effect that they can be obtained in a surprisingly short time of 2 to 7 days, preferably 5 to 7 days.
  • glial cells in particular, astrocytes
  • GFAP glial fibrillary acidic protein
  • Protein expression can be confirmed by examining immunological techniques such as conventional ELISA and immunostaining. It is also possible to confirm the morphological characteristics of astrocytes, for example, a number of unique star-shaped branched protrusions as indicators.
  • the present invention includes a substantially isolated glial cell obtained by such a production method. Vesicles are also included.
  • the glial cells of the present invention are almost pure astroglial cells, they are transplanted at the same time as nerve cells and neural stem cells to support the differentiation and growth of nerve cells, and further form a blood-brain barrier to supply nutrients. Expected to be applied to such applications.
  • the purity of the nervous system cells obtained by the method for producing nervous system cells of the present invention is determined by, for example, expression of cell-specific markers in the total number of cells by flow cytometry using antibodies or antibody fragments against the respective cell-specific markers. It can be determined by the percentage of cells. According to the method for producing a nervous system cell of the present invention, a substantially isolated nervous system cell can be obtained.
  • the nervous system cell of the present invention can also be provided as a cell pharmaceutical composition.
  • Such cell pharmaceutical compositions are also included in the present invention.
  • the cell pharmaceutical composition of the present invention contains, as an active ingredient, a cell selected from the group consisting of the neural stem cell, neural cell and glial cell of the present invention.
  • the cell pharmaceutical composition of the present invention may appropriately have a pharmacologically acceptable auxiliary agent.
  • the neurotransmitter is coded into a neural stem cell. Obtained by encapsulating a cell into which a nucleic acid, a nucleic acid that codes for a neurotrophic factor, or the like is introduced, or if necessary, a cell obtained by inducing differentiation under appropriate differentiation conditions in a semipermeable membrane capsule Alternatively, a cell pharmaceutical composition may be used.
  • the cells used in the cell pharmaceutical composition in which the active ingredient is encapsulated in the capsule may be the same type of cells or different types of cells for the individual to be applied.
  • a state caused by neurodegeneration or nerve damage for example, neurodegenerative disease (for example, nerve cell transplantation treatment for nerve damage such as Parkinson's disease, Alzheimer's disease, etc.), spinal cord injury, cerebral infarction, etc. can be performed. Accordingly, a method for treating a neurodegenerative disease or nerve injury is also included in the present invention. It is.
  • the method for treating a neurodegenerative disease or nerve injury according to the present invention comprises:
  • At least one cell selected from the group consisting of is introduced into a site of neurodegeneration or nerve damage.
  • At least one cell selected from the group consisting of the neural stem cell, nerve cell and glial cell of the present invention is used.
  • a therapeutic effect can be obtained, and an excellent effect that a state caused by neurodegeneration or nerve injury can be treated in a stable state is exhibited.
  • the therapeutic method of the present invention can be applied to the diseases listed above as applications of the neural stem cell, neural cell and glial cell of the present invention.
  • Introduction of cells into the site of neurodegeneration or nerve damage can be performed by injection or the like.
  • the neural stem cell of the present invention is introduced into the damaged site,
  • Parkinson's disease introducing the dopaminergic neuron, which is a neuron of the present invention, into the striatum, midbrain substantia nigra,
  • Etc. can be performed.
  • the neural stem cell, nerve cell and glial cell of the present invention can also be used for the manufacture of a medicament for treating a neurodegenerative disease or nerve injury.
  • EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to the examples.
  • an astrocyte conditioned medium was used as the neural stem cell and neural cell differentiation induction medium.
  • a basic medium for astrocyte culture astrocyte basic medium
  • DMEM F-12 containing N2 additives (insulin, transferrin, selenium, progesterone) was used.
  • N2 additives insulin, transferrin, selenium, progesterone
  • Astrocytes were prepared using mouse fetal brain and rat fetal brain, and the previously reported method [Banking, G. et al., Cultured Nerve Cells, (1991), ZAM IT Press (The MIT Press) (Cambridge, UK) published].
  • Example 1 Preparation of neural stem cells
  • an HK cell line (less than passage 10) established from a blastocyst of C 5 7 BLZ6 mouse (3.5 days after vaginal plug confirmation) by a conventional method was used.
  • the HK cells have few passage numbers and are unlikely to undergo spontaneous differentiation.
  • fibroblasts prepared from syngeneic mice on the 14th day of pregnancy were treated with 10% (wZ).
  • the plate in which the diameter of the embryonic stem cell colony was expanded to about 400 to 500 m was washed 2 to 3 times with DMEM (without serum and other components).
  • Embryonic stem cell colonies were mechanically detached from the feeder cell layer and picked up using a glass capillary with a fine-tipped tip.
  • the obtained embryonic stem cell colonies were washed 2-3 times with serum-free DMEM in a culture dish.
  • SCS Stem Cell Sphere
  • mouse-derived anti-nestin antibody was incubated at 4 ° C.
  • the obtained mixture and the fluorescently labeled rabbit anti-mouse IgG antibody were incubated for 2 hours at room temperature, and further incubated overnight with 4 with a piotine-labeled mouse anti-Br dU antibody.
  • the obtained mixture and fluorescently labeled streptavidin were incubated at room temperature for 2 hours. Thereafter, the obtained cells were observed with a confocal laser scanning microscope (trade name: LSM5 10) manufactured by Zeiss.
  • Fig. 1 shows an immunofluorescent staining image.
  • a strong signal for nestin a marker of neural stem cells
  • B r dU a weak signal for B r dU
  • the cells between the SCS surface layer and the core are cells in a transitional state in which differentiation is induced from embryonic stem cells to neural stem cells.
  • SCS formed by suspension culture using a mixture of astrocyte conditioned medium and astrocyte basic medium has a layer structure similar to the planetary structure. That is, SCS is a nestin-positive neural stem cell layer corresponding to the crustal layer, a pre-neural stem cell layer that is negative for both nestin and BrdU that corresponds to the mantle layer, and a BrdU-positive embryonic property that corresponds to the nucleus. It can be seen that it has a three-layer structure composed of a stem cell layer. These structures are completely different from the structures of EBs in which one to several embryonic stem cells are formed as aggregates by the droplet method or the like. Is different. In the preparation of the EB, embryonic stem cells are aggregated in the presence of serum, and the endoderm, ectoderm, and mesoderm are caused by various unclear differentiation-inducing factors in the serum. Differentiation is induced.
  • the antibody TUJ 1 that recognizes class III) 3 tubulin which is a marker of young neurons, was used.
  • the observation of immunofluorescence histochemistry was performed with an upright fluorescence microscope (trade name: Eel ipse E800) manufactured by Nikon. It was done.
  • FIG. 2 shows the results of suspension culture of the SCS for 6 to 7 days.
  • neural stem cells were produced and induced to differentiate into young neurons.
  • the surface of the culture side of the culture dish coated with polylysine is further treated with 0.1 mg / m1 laminin or Matrigel TM (BD Bioscience) diluted 10-20 times to adhere.
  • a culture substrate was prepared. 4 days suspension culture was SCS sucked up by Garasukiyabirari primary, pre Asuto port site conditioned medium was transferred to adhesive culture Deishu which had been added, at 37 C_ ⁇ 2 ink Yube Isseki within one, 5% C0 The cells were cultured for several hours in a 2 and 100% humidified atmosphere. As a result, SCS adhered to the culture substrate.
  • the adherent SCS differentiated as a nestin-positive neural stem cell that took in BrdU and actively divides. Furthermore, in the vicinity of SCS, many migrated neurons and neurites that extend from the cell body, which are strongly stained with antibodies against NF, are observed, indicating that nestin-positive neural stem cells are present inside . In addition, as shown in Fig. 4, it can be seen that many NF-positive neurites extend on the adhesion culture surface. In addition, as shown in FIG. 4, it can be seen in FIG. 3 that the SCS site where nestin-positive neural stem cells are present is positive for Br d U and cell division is active.
  • the entire SCS is strongly stained with an antibody against TH, which is a marker of dopaminergic neurons, and many of the extending neurites are TH-positive.
  • the entire SCS is strongly stained with an antibody against GAD, which is a marker of GABAergic neurons, and many neurites are extending. It turns out that it is GAD positive.
  • FIG. 7 it can be seen that SCS is strongly stained by an antibody against ChAT, a marker of cholinergic neurons. That is, FIG.
  • the ratio of SCS suspended in suspension in a mixture of astrocyte conditioned medium and astrocyte basic medium to adherent culture substrate is 90% or more.
  • Adhered SCS is an indicator of neuronal differentiation. The rate of neurite extension was almost 100%. Therefore, even if experimental manipulation errors are taken into consideration, almost all s CS grows in suspension culture using a mixture of tuss-mouth conditioned medium and astrocyte basal medium. It is thought that the differentiation into nerves was determined by the action of cell adhesion molecules in adhesion culture.
  • the astrocyte conditioned medium in adhesion culture has an effect of promoting differentiation into nerve cells, but other medium for culture of nerve cells, for example, DMEM: F-12 / N-2 suppl eme nt, Neurobasa 1 TM B-27 [manufactured by GIBCO BRL] etc., differentiation into nerve cells is also observed. This suggests that when SCS differentiates into neural stem cells by suspension culture, it defaults to neuronal cells, and its properties are promoted by interaction with cell adhesion molecules.
  • DMEM F-12 / N-2 suppl eme nt
  • Neurobasa 1 TM B-27 manufactured by GIBCO BRL
  • neural stem cell layer is formed on the surface layer of SCS was suspension culture, where c continue to differentiate the adhered cultured as suspension culture or ⁇ stroke site conditioned media such as into neurons, from the SC S into neurons the differentiation inhibiting, for growing neural stem cells, the SCS after suspension culture in the presence of b FGF, 3 7t :, C0 2 concentration of 5%. were cultured under 100% humidified atmosphere.
  • the bFGF is a factor that maintains neural stem cells in an undifferentiated state and promotes cell proliferation, and was considered to be effective for neural stem cells formed on the SCS surface layer.
  • SCS was adhered to the adhesive culture substrate as in Example 3.
  • N eurobasal TM with a final concentration of 20 ng / m 1 b FGF as the culture medium
  • B-27 was used. Every 1-2 days, bFGF was freshly added to the culture to 20 ng gml. Using an erecting fluorescence microscope (Nikon), SCS and the surrounding morphology were observed.
  • the cells that migrate are nestin positive and are found to be neural stem cells.
  • the culture medium from a mixture of fast mouth conditioned medium and astrocyte basic medium to Neurobasal TM B-27 containing bFGF, differentiation into neurons is suppressed, and nestin-positive neural stem cells It can be seen that it proliferates and migrates from the SCS.
  • Figure 9 shows the results of observation of neural stem cell colonies that spread from one SCS.
  • a surprisingly large number of neural stem cells can be obtained.
  • Neural stem cells that migrate are closely adhered to each other, migrate in a circular shape with a radius of about 600 m centering on the SCS that adheres to the culture substrate, and a large number of neural stem cells from one SCS It turns out that it is obtained.
  • the neurosphere method and other monolayer culture methods it is difficult to prepare such a large number of neural stem cells in a uniform manner, and it can be seen that the method in this example is excellent.
  • the method of preparing neural stem cells using SCS has an excellent feature that it can be used many times as a seed of neural stem cell colonies on which SCS migrates.
  • neural stem cells can be migrated again from the SCS by picking up the SCS at the center with a glass carrier and transferring it to a new adhesive culture substrate. .
  • bFGF was added to the culture in suspension culture to a final concentration of 20 ng / m 1.
  • bromönidine B r dU was added to the culture to a final concentration of 10.
  • Br dU incorporation is an indicator of cell division. The culture was incubated for 8 hours under the same culture conditions as described above. Subsequently, the obtained cells were cultured for 1 hour in a medium containing no Br dU and washed.
  • the resulting cells were then fixed with 4% (w / v) paraformaldehyde, 0.4M sucrose, 50 mM phosphate buffer pH 7.4 for 30 minutes, and 0.1% (v / v) Triton TM X_1 00 treatment and 10% (wZ v) BSA—Proced with PBS.
  • S C S Cells after blocking (S tem C ell S p h er; hereinafter abbreviated as S C S) and mouse-derived anti-nestin antibody were incubated at 4 overnight.
  • the obtained mixture and the fluorescence-labeled Usagi anti-mouse IgG antibody were incubated at room temperature for 2 hours, and further incubated with piotin-labeled mouse anti-Br dU antibody and 4. Subsequently, the obtained mixture and fluorescently labeled streptavidin were incubated at room temperature for 2 hours. Thereafter, the obtained cells were observed with a confocal laser scanning microscope (trade name: LSM510) manufactured by Zeiss.
  • Figure 11 shows an immunofluorescent staining image of the distribution of nestin, a marker of neural stem cells, and BrdU, which is an indicator of cell division.
  • Example 4 It was investigated whether the neural stem cells obtained in Example 4 differentiated into neural cells in the same manner as neural stem cells formed on the SCS surface layer. Specifically, SCS is cultured in Neurobasa 1 TM B-27 containing b FGF to obtain migrating neural stem cells. For the neural stem cells whose differentiation is suppressed, astrocyte conditioned medium and Mouth site basal medium with the mixture 3 7 back and forth the medium of under about 5% C_ ⁇ 2 concentration of 1 0 0% humidified atmosphere, by replacing, was examined whether induced to differentiate into neural cells. The results are shown in Fig. 12.
  • the neural stem cells that were in close contact with each other were replaced with a mixture of astrocyte conditioned medium and astrocyte basic medium. It can be seen that the neural stem cells that migrated while closely adhering to spread out and differentiate into neurons. In addition, like the neural stem cells formed on the surface of the SCS, it can be seen that the migrated neural stem cells differentiate into neurons using the first mouth site conditioned medium. This result suggests that a large amount of nerve cells can be produced as it is from a large amount of prepared neural stem cells.
  • TH which is a marker of dopaminergic neuron
  • TH is expressed in the majority of NF-positive neurons. That is, it can be seen that not only TH positive neurons differentiate from neural stem cells staying inside the SCS, but also TH positive neurons differentiate from migrated neural stem cells.
  • Fig. 13 in the neural cells derived from the migrated neural stem cells, neurons showing co-expression of TH and GAD were observed, suggesting the presence of immature neurons. .
  • neurons containing the neurotransmitter serotonin were confirmed. From these results, the nerve trunk that was proliferated By using cells,
  • Gene-manipulated neurons can be prepared in large quantities, which was difficult when using primary cultured neurons prepared from the brain.
  • Example 7 Differentiation of proliferated neural stem cells into astrocytes.
  • neural stem cells are multipotent cells, we examined whether they can differentiate into cell types other than neurons by changing the culture conditions.
  • the medium was removed from the 60 mm dish containing neural stem cells obtained in Example 4 and washed with Dulbecco's PBS (without Ca and Mg). Next, in the 60mm dish, 21111? 83 was added and incubated at 37 for 5 minutes. The neural stem cells that had weakly adhered to the culture substrate were peeled off by pipetting, and the neural stem cells were collected by centrifugation at 700 xg for 5 minutes. Next, 2 ml of a mixture of astrocyte conditioned medium and astrocyte basal medium was added to the neural stem cells, suspended, and seeded in a 35 mm dish coated with cell adhesion molecules. As a result, as shown in FIG. 17, the nerve stem cells obtained in Example 4 were once detached from the adhesive culture substrate and cultured in a monolayer, but turned into neurons within a few days. You can see that it differentiates.
  • Example 9 Differentiation of cryopreserved neural stem cells into neural cells
  • Neural stem cells were collected in the same manner as in Example 8. The obtained neural stem cells were suspended in 10% (v / v) DMS 0/90% (v / v) FCS and cryopreserved at 180 in a deep freezer.
  • Fig. 18 shows the results of examining the differentiation of cryopreserved neural stem cells into neural cells.
  • HK cell line which is an embryonic stem cell prepared and established from a blastocyst of C 57 BL / 6 mouse in Examples 1 to 3, a commercially available 129 SV mouse embryonic stem cell (passage number 15; (Supplied by Dainippon Pharmaceutical Co., Ltd.) [Kontgen F. et al., Int. Immunol., 5: 957-964, (1993); Mai i ssen ⁇ . Et al., ⁇ J., 12: 4347- 4355., (1993)], the above example:! Differentiation induction was examined under the same culture conditions as in ⁇ 3.
  • Fig. 19 shows the results of suspension culture in a mixture of astrocyte conditioned medium and astrocyte basal medium and adhesion culture using an adhesive culture substrate.
  • Example 1 Differentiation of force dicyno monkey embryonic stem cells (CMK-6) into neurons
  • CMK-6 cell line [Suemori, H. et al., Dev. Dyn., 222: 273-279, (2001)]
  • Riki cynomolgus monkey is similar to mouse embryonic stem cells.
  • CMK-6 cell line When the colonies of the CMK-6 cell line became 400-500 m in size, they were mechanically peeled from the feeder cell layer by a glass capillary, as in the case of mice. Then, colonies 3 7 before and after the resulting CMK- 6 cell lines, C0 2 concentration of around 5%, in bacterin rear dish 35mm under a 100% humidified atmosphere, ⁇ stroke site conditioned medium and a ⁇ stroke Site basal medium 10-20 colonies were cultured in suspension in 2 ml of this mixture.
  • the colonies of the CMK-6 cell line were flat, and immediately after peeling off, the paper floated in a kinked state. However, after several hours, the CMK-6 cell line formed a spherical morphology due to cell-cell adhesion between embryonic stem cells.
  • CMK-6 cell line Compared with mouse embryonic stem cells, the CMK-6 cell line grew slowly, so it took time for the SCS derived from the CMK-6 cell line to increase even in suspension culture.
  • CMK-6 cell line In order to investigate the state of differentiation into neural stem cells during the formation of SCS of the force cynomolgus monkey embryonic stem cells (CMK-6 cell line), suspended in a mixture of astrocyte conditioned medium and astrocyte basal medium for 10 to 12 days After incubation, the cells were fixed, and the distribution was examined with a rabbit-derived anti-nestin antibody against primates in the same manner as in mice. Furthermore, SCS after suspension culture was allowed to adhere to the adhesion medium, and the cells that migrated after 7 to 10 days were also examined for reactivity to nestin. The results are shown in Fig. 20.
  • the floating SCS is also c stained entirety with anti-nestin antibody, cells that have migrated from the adhesive SCS also stained with anti-nestin antibody, is also efficiently neural stem cells and for mice It was shown that it can be produced by suspension culture with astrocyte conditioned medium and astrocyte basal medium.
  • Figure 21 shows the results of observation of SCS and its surroundings on the first day of adhesion culture with a phase contrast microscope.
  • mice embryonic stem cell-derived SCS As shown in Fig. 21, as with mouse embryonic stem cell-derived SCS, neurons migrated from the SCS derived from cynomolgus monkey embryonic stem cells on the first day after adhesion culture, causing neuronal processes to expand. I can see that In addition, as observed with mouse embryonic stem cell adhesion SCS, it can be seen that SCS differentiates into neurons and neurites are actively extended from SCS.
  • NF-positive neurons are TH-positive, indicating that dopaminergic neurons are induced to differentiate.
  • suspension of astrocytic conditioned medium and astrocyte basal medium in suspension culture and adherent culture using an adhesive culture substrate facilitates the development of the nematode embryonic stem cell CMK — 6 cell line. It turns out that it differentiates into.
  • the method of suspension culture in a mixture of astrocyte conditioned medium and fast mouth basal medium, adhesion culture, and differentiation into nerve cells is a primate power quizal with a different species from mice. It can be seen that it can be applied universally to embryonic stem cells. In addition, this method is considered to be effective across species and universal. In addition, given that the power quiz is a primate, this method may also be applicable to human embryonic stem cells.
  • Undifferentiated embryonic stem cell 37, the air 5% C_ ⁇ 2, 1 100% humidified atmosphere, to form a SCS to 4 days suspension culture, then at 37, 5% in air C ⁇ 2, 100 Adhesion culture was performed for 5 days in a humidified atmosphere to induce differentiation into neurons.
  • MRNA was prepared by a conventional method from undifferentiated embryonic stem cells, SCS formed by suspension culture, and 20 cell clusters obtained by adhesion culture. Using the obtained mRNA as a saddle type, a random hexamer was used as a primer, and reverse transcription was performed for 3760 minutes to synthesize cDNA.
  • Oc t-4 transcription regulator specific to ES cells
  • P a X-6 transcription regulator unique to neural progenitor cells
  • nestin NF— M
  • Nu rrl dominating dopamine neurons
  • nestin expressed in neural stem cells is rapidly expressed in floating SCS, and it can be seen that expression continues in the adhesion SCS stage.
  • This nestin expression profile is in good agreement with the continual division and differentiation of neural stem cells into neurons.
  • NF which is expressed only in neurons
  • TH which is a synthase of dopamine neurons
  • Nurr1 transcription factor
  • differentiated neurons obtained by the method of suspension culture of embryonic stem cells in a mixture of astrocyte conditioned medium and astrocyte basal medium to form SCS, and then adhesion culture Since the expression of the marker gene in the endoderm, mesoderm and ectoderm is hardly changed, it is clear that the method is different from the method of forming and differentiating EB.
  • Undifferentiated embryonic stem cell clones are float-cultured for 4 days in a mixture of astrocyte conditioned medium and astrocyte basal medium to form SCS, and the SCS is adherently cultured for 5 days. Induced differentiation into neuronal cells.
  • Undifferentiated embryonic stem cell clone (suspension culture day 0), SCS on suspension culture days 2 and 4, and 4 or 5 cell clusters on adhesion days 2 and 5 respectively
  • mRNA was prepared by a conventional method. Using the obtained mRNA as a saddle type, reverse transcription was performed to synthesize cDNA.
  • nestin expressed in neural stem cells rapidly increases in floating SCS and continues to be expressed in the adhesion SCS stage.
  • This expression profile of nestin is in good agreement with the fact that neural stem cells divide and differentiate into neural cells.
  • TH which is a synthase of dopamine neurons, increases in gene expression as it differentiates from floating SCS to adhesion SCS.
  • the method for producing a substantially isolated nervous system cell of the present invention it is not limited to the supply source of the embryonic stem cell, and the ectoderm other than the nervous system cell is produced in large quantities from the embryonic stem cell. It is possible to provide efficiently and substantially isolated neural cells without inducing sex cells, mesodermal cells, and endoderm cells, and a large number of stable and stable neural cells suitable for transplantation. Can be supplied to. Therefore, it is expected to be applied to nerve regeneration medicine.
  • the neural cells of the present invention are selectively induced to differentiate from embryonic stem cells via neural stem cells to either one of neural cells or Darya cells, neurodegenerative diseases, spinal cord It can be used as a source of cells or tissues in nerve regeneration medicine for injuries, cerebral infarction, etc.
  • the nerve cell of the present invention it is possible to release neurotransmitters, reconstruct nerve contact, etc. in regenerative medicine such as nerve cell transplantation treatment for neurodegenerative diseases, spinal cord injury, cerebral infarction, etc. To do.
  • the glial cell of the present invention it can be transplanted simultaneously with nerve cells and neural stem cells to support the differentiation and growth of nerve cells, and further, a blood-brain barrier can be formed to replenish nutrients.

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WO2004099395A3 (en) * 2003-05-08 2005-03-17 Cellartis Ab A method for the generation of neural progenitor cells
JP2005287430A (ja) * 2004-03-31 2005-10-20 Japan Science & Technology Agency 脳梗塞疾患モデルマウス
WO2006028049A1 (ja) * 2004-09-06 2006-03-16 Tanabe Seiyaku Co., Ltd. アストロサイト様細胞馴化培地の製造方法
DE102007036334A1 (de) * 2007-08-02 2009-02-05 Electrolux Home Products Corporation N.V. Basiseinheit zum induktiven Aufheizen und Induktionsflüssigkeitserhitzungssystem
RU2646099C2 (ru) * 2013-04-06 2018-03-01 СЕУЛ НЭШНЛ ЮНИВЕРСИТИ Ар энд ДиБи ФАУНДЕЙШН Способ получения индуцированных нервных стволовых клеток, перепрограммированных из клеток, не являющихся нервными, с использованием hmga2
CN104673752A (zh) * 2015-03-14 2015-06-03 李�根 一种人脑干细胞库构建方法
WO2018149127A1 (zh) * 2017-02-16 2018-08-23 上海安集协康生物技术股份有限公司 一种经鼻腔给药用于帕金森病治疗的神经干细胞制剂及其制备方法和应用

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