WO2013187416A1 - METHOD FOR AMPLIFYING iPS CELL SUITABLE FOR NEURAL DIFFERENTIATION, AND METHOD FOR INDUCING NEURAL STEM CELL - Google Patents

METHOD FOR AMPLIFYING iPS CELL SUITABLE FOR NEURAL DIFFERENTIATION, AND METHOD FOR INDUCING NEURAL STEM CELL Download PDF

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WO2013187416A1
WO2013187416A1 PCT/JP2013/066102 JP2013066102W WO2013187416A1 WO 2013187416 A1 WO2013187416 A1 WO 2013187416A1 JP 2013066102 W JP2013066102 W JP 2013066102W WO 2013187416 A1 WO2013187416 A1 WO 2013187416A1
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cells
stem cells
inhibitor
pluripotent stem
cell
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岡野　栄之
和土 赤松
松本　拓也
昌伸 庄司
恒史 中村
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学校法人 慶應義塾
武田薬品工業株式会社
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Definitions

  • the present invention relates to a method for amplifying iPS cells suitable for neural differentiation, a method for inducing neural stem cells, and the like. ⁇ Background of the invention>
  • Induction of neural stem cells from human ES cells and iPS cells has been actively studied in recent years as an indispensable technique for performing neuroregenerative medicine.
  • it is expected to be applied not only for transplantation but also as a pathological model analysis system, and is expected to develop a more efficient and quicker method for inducing differentiation of neural stem cells.
  • an adhesion culture system for example, a method is known in which human ES cells are cultured in the presence of two inhibitors of SMAD signals, Noggin and SB431542, thereby converting more than 80% of human ES cells into the nervous system. (Non-Patent Document 1). In addition, by inhibiting GSK3, TGF- ⁇ and Notch signaling pathways, it is possible to convert monolayer cultured human ES cells into uniform initial neuroepithelium within one week.
  • Non-patent Document 2 Patent Document 1
  • a method of inducing into neural stem cells has been developed (Patent Document 2). Also, it is possible to induce neural progenitor cells by suspension culture of human ES cells under hypoxic (3% O 2 ) conditions, and culturing the neural progenitor cells in the presence of retinoic acid and purmorphamine. It has been reported that spinal motor nerves can be induced by (Non-patent Document 3).
  • the adhesion culture system can induce neural stem cells in a relatively short period (about 1 week), but is not suitable for culturing cells in large quantities.
  • the floating culture system is suitable for obtaining a large amount of neural stem cells, but it takes a long period of two weeks or more to induce neural stem cells.
  • An object of the present invention is to establish a technique for inducing a large amount of undifferentiated cells such as neural stem cells from pluripotent stem cells such as iPS cells in a relatively short period of time.
  • iPS cells were subcultured in the presence of a TGF- ⁇ family inhibitor (SB431542), a GSK3 ⁇ inhibitor (CHIR99021) and a BMP inhibitor (Dorsomorphin), While maintaining pluripotency, we succeeded in improving the neuronal differentiation ability of iPS cells. Furthermore, the iPS cells thus obtained were suspended in a medium containing TGF- ⁇ family inhibitor (SB431542), GSK3 ⁇ inhibitor (CHIR99021), ROCK inhibitor (Y27632), bFGF, LIF and B27 under hypoxic conditions. By culturing, neural stem cells were successfully mass-cultured at high speed (3 days) and efficiently. In particular, we succeeded in efficiently inducing neural stem cells from iPS cells derived from T cells that are difficult to differentiate. As a result of further studies based on these findings, the present invention was completed.
  • the present invention relates to the following.
  • a method for enhancing the neuronal differentiation ability of pluripotent stem cells comprising culturing pluripotent stem cells in the presence of a TGF- ⁇ family inhibitor, a GSK3 ⁇ inhibitor, and a BMP inhibitor.
  • the method according to [1] or [2], wherein the pluripotent stem cell is an induced pluripotent stem cell.
  • the induced pluripotent stem cells are derived from T cells or B cells.
  • the TGF- ⁇ family inhibitor is 4- [4- (1,3-benzodioxol-5-yl) -5- (2-pyridinyl) -1H-imidazol-2-yl] -benzamide or its The method according to any one of [1] to [4], which is a hydrate.
  • the GSK3 ⁇ inhibitor is 6-[[2-[[4- (2,4-dichlorophenyl) -5- (4-methyl-1H-imidazol-2-yl) -2-pyrimidinyl] amino] ethyl] amino The method according to any one of [1] to [5], which is nicotinonitrile.
  • a method for producing neural stem cells comprising culturing pluripotent stem cells in the presence of a TGF- ⁇ family inhibitor and a GSK3 ⁇ inhibitor under an oxygen partial pressure of 2 to 10%.
  • the pluripotent stem cell is an induced pluripotent stem cell.
  • the induced pluripotent stem cells are derived from T cells or B cells.
  • the TGF- ⁇ family inhibitor is 4- [4- (1,3-benzodioxol-5-yl) -5- (2-pyridinyl) -1H-imidazol-2-yl] -benzamide or its The method according to any one of [8] to [10], which is a hydrate.
  • GSK3 ⁇ inhibitor is 6-[[2-[[4- (2,4-dichlorophenyl) -5- (4-methyl-1H-imidazol-2-yl) -2-pyrimidinyl] amino] ethyl] amino The method according to any one of [8] to [11], which is nicotinonitrile.
  • a method for producing neural stem cells comprising the following steps: (1) culturing pluripotent stem cells in the presence of a TGF- ⁇ family inhibitor, a GSK3 ⁇ inhibitor, and a BMP inhibitor to enhance the neuronal differentiation ability of the pluripotent stem cells; and (2) Culturing the pluripotent stem cells with enhanced neuronal differentiation ability obtained in step (1) in the presence of a TGF- ⁇ family inhibitor and a GSK3 ⁇ inhibitor under 2 to 10% oxygen partial pressure.
  • a method for producing motor neural progenitor cells comprising the following steps: (1) obtaining neural stem cells by culturing pluripotent stem cells in the presence of a TGF- ⁇ family inhibitor and a GSK3 ⁇ inhibitor under 2 to 10% oxygen partial pressure, and (2) step ( The neural stem cells obtained in 1) were mixed with retinoic acid, 9-cyclohexyl-N- [4- (4-morpholinyl) phenyl] -2- (1-naphthalenyloxy) -9H-purin-6-amine, TGF -Inducing differentiation of motor progenitor cells by culturing in the presence of ⁇ family inhibitor and GSK3 ⁇ inhibitor under 2-10% oxygen partial pressure.
  • a method for producing dopaminergic neural progenitor cells comprising the following steps: (1) obtaining neural stem cells by culturing pluripotent stem cells in the presence of a TGF- ⁇ family inhibitor and a GSK3 ⁇ inhibitor under 2 to 10% oxygen partial pressure, and (2) step ( The neural stem cells obtained in 1) were transformed into TGF- ⁇ family inhibitors, GSK3 ⁇ inhibitors, Sonic hedgehog, 9-cyclohexyl-N- [4- (4-morpholinyl) phenyl] -2- (1-naphthalenyl) Differentiation induction of dopaminergic neural progenitor cells by culturing in the presence of 2-10% oxygen partial pressure in the presence of (oxy) -9H-purin-6-amine and FGF8.
  • neural undifferentiated cells such as neural stem cells and motor neural progenitor cells can be efficiently induced to differentiate from pluripotent stem cells in a short period of time.
  • neural undifferentiated cells such as neural stem cells and motor neural progenitor cells from T cell-derived iPS cells and B lymphocyte-derived iPS cells that are difficult to differentiate.
  • Photograph of 3D-iPS cell colony Photo of neurospheres (left) derived from human iPS cells and differentiated neurons (right).
  • Method for enhancing neural differentiation ability of pluripotent stem cells (Method 1)
  • the present invention provides a method for enhancing the neuronal differentiation ability of pluripotent stem cells, comprising culturing pluripotent stem cells in the presence of a TGF- ⁇ family inhibitor, a GSK3 ⁇ inhibitor, and a BMP inhibitor. is there.
  • the pluripotent stem cells that can be used in the present invention are stem cells that have pluripotency that can be differentiated into all the cells present in the living body and also have proliferative ability.
  • ES embryonic stem
  • ntES embryonic stem
  • GS cells sperm stem cells
  • EG cells embryonic germ cells
  • iPS cells induced pluripotent stem cells
  • Preferred pluripotent stem cells are ES cells, ntES cells, and iPS cells, most preferably iPS cells.
  • Embryonic stem cells ES cells are stem cells established from the inner cell mass of early embryos (for example, blastocysts) of mammals such as humans and mice, and having the pluripotency and the ability to grow by self-replication.
  • ES cells are embryonic stem cells derived from the inner cell mass of the blastocyst, the embryo after the morula, in the 8-cell stage of a fertilized egg, and have the ability to differentiate into any cell that constitutes an adult, so-called differentiation. And ability to proliferate by self-replication.
  • ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292: 154-156), and then ES cell lines were also established in primates such as humans and monkeys (JA Thomson et al. (1999), Science 282: 1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92: 7844-7848; JA Thomson et al. (1996), Biol. Reprod 55: 254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol, 38: 133-165).
  • ES cells can be established by taking an inner cell mass from a blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder. Cell maintenance by subculture is performed using a medium supplemented with substances such as leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF). be able to.
  • LIF leukemia inhibitory factor
  • bFGF basic fibroblast growth factor
  • a medium for preparing ES cells for example, a DMEM / F-12 medium supplemented with 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acid, 2 mM L-glutamic acid, 20% KSR and 4 ng / ml b-FGF is used.
  • Human ES cells can be maintained in a humid atmosphere at 37 ° C., 2% CO 2 /98% air (O. Fumitaka et al. (2008), Nat. Biotechnol., 26: 215-224).
  • ES cells also need to be passaged every 3-4 days, where passage is eg 0.25% trypsin and 0.1 mg / ml collagenase IV in PBS containing 1 mM CaCl 2 and 20% KSR. Can be used.
  • ES cells can be generally selected by Real-Time PCR method using the expression of gene markers such as alkaline phosphatase, Oct3 / 4, Nanog as an index.
  • gene markers such as alkaline phosphatase, Oct3 / 4, Nanog
  • OCT-3 / 4, NANOG, and ECAD can be used as an index (E. Kroon et al. (2008), Nat. Biotechnol., 26: 443). -452).
  • human ES cell lines such as khES-1, KhES-2, KhES-3, KhES-4, and KhES-5 are available from the Institute of Regenerative Medicine, Kyoto University (Kyoto, Japan).
  • sperm stem cells are testis-derived pluripotent stem cells that are the origin of spermatogenesis. Like ES cells, these cells can be induced to differentiate into various types of cells, and have characteristics such as the ability to create chimeric mice when transplanted into mouse blastocysts (M. Kanatsu-Shinohara et al. ( 2003), Biol. Reprod. 69: 612-616; K. Shinohara et al. (2004), Cell, 119: 1001-1012). It can self-replicate in a medium containing glial cell line-derived neurotrophic factor (GDNF), and spermatogonial stem cells can be generated by repeated passage under the same culture conditions as ES cells. (Takebayashi Masanori et al. (2008), Experimental Medicine, Vol. 26, No. 5 (extra), 41-46, Yodosha (Tokyo, Japan)).
  • GDNF glial cell line-derived neurotrophic factor
  • Embryonic germ cells are cells that are established from embryonic primordial germ cells and have the same pluripotency as ES cells. LIF, bFGF, stem cell factor, etc. Can be established by culturing primordial germ cells in the presence of these substances (Y. Matsui et al. (1992), Cell, 70: 841-847; JL Resnick et al. (1992), Nature, 359: 550 -551).
  • iPS cells Artificial pluripotent stem cells
  • germline cells eg, egg cells, sperm cells, and progenitor cells such as oocyte and spermatogonia
  • progenitor cells such as oocyte and spermatogonia
  • embryonic stem cells embryonic stem cells
  • Differentiated cells may be derived from embryos, fetuses or adults, and may be derived from any animal species such as mice and humans.
  • the nature of the differentiated cell is not particularly limited as long as it is a cell that originally lost a part of the total differentiation ability of a fertilized cell.
  • neural stem cells hematopoietic stem cells, mesenchymal stem cells, dental pulp stem cells, etc.
  • Tissue stem cells (somatic stem cells), (2) tissue precursor cells, (3) fibroblasts (skin cells, etc.), epithelial cells, hepatocytes, lymphocytes (T cells, B cells), endothelial cells, muscle cells
  • Examples include hair cells, gastric mucosa cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), differentiated cells such as brain cells, lung cells, kidney cells, skin cells, and the like.
  • the reprogramming method is not particularly limited, in a preferred method, cells are induced to have pluripotency and self-proliferation ability by introducing a nuclear reprogramming factor.
  • a nuclear reprogramming factor for example, any of the initialization methods described in International Publications WO2005 / 080598, WO2007 / 069666, WO2008 / 118820, and WO2009 / 057831 may be used. The disclosures of these publications are incorporated herein by reference.
  • the nuclear reprogramming factor is not particularly limited, and is selected from the Oct gene group, the Klf gene group, the Sox gene group, the Myc gene group, the Sall ⁇ gene group, the Nanog gene group (mouse NM_028016, human NM_024865), and the Lin gene group. Preferably, it is at least one gene product of a gene in the group.
  • genes belonging to the Oct gene group include Oct3 / 4 (mouse NM_013633, human NM_002701), Oct1A (mouse NM_198934, human NM_002697) and Oct6 (mouse NM_011141, human NM_002699), and genes belonging to the Klf gene group include Examples include Klf1 (mouse NM_010635, human NM_006563), Klf2 (mouse NM_008452, human NM_016270), Klf4 (mouse NM_010637, human NM_004235) and Klf5 (mouse NM_009769, human NM_001730).
  • the genes belonging to the Sox gene group include Sox1 ( Mouse NM_009233, human NM_005986), Sox2 (mouse NM_011443, human NM_003106), Sox3 (mouse NM_009237, human NM_005634), Sox7 (mouse NM_011446, human NM_031439), Sox15 (mouse NM_009235, human NM_006942), Sox17 (mouse NM_02144454, human NM_021444) ) And Sox18 (mouse NM_009236, human NM_018419), and the genes belonging to the Myc gene group include c-Myc (mouse NM_010849, human NM_002).
  • N-Myc (mouse NM_008709, human NM_005378) and L-Myc (mouse NM_008506, human NM_001033081).
  • the genes belonging to the SallS gene group include Sall1 (mouse NM_021390, human NM_002968) and Sall4 (mouse NM_175303).
  • Human NM_020436 and the genes belonging to the Lin gene group include Lin28 (mouse NM_145833, human NM_024674) and Lin28b (mouse NM_001031772, human NM_001004317).
  • the nuclear reprogramming factor other types of gene products may be used, and examples thereof include an immortalization inducing factor.
  • the nuclear reprogramming factor is one or more selected from Oct3 / 4 gene, Klf4 gene, Sox2 gene, c-Myc gene, L-Myc gene, Sall4 gene, Sall1 gene, Nanog gene and Lin28 gene. And at least one product of the gene.
  • genes are expressed in mouse and human sequences with reference to the accession numbers registered in the National Center for Biotechnology Information (NCBI), both of which are highly conserved in vertebrates. Therefore, in this specification, unless the name of an animal is indicated, the gene including a homolog is expressed.
  • genes having mutations including polymorphism are also included as long as they have a function equivalent to that of a wild-type gene product.
  • nuclear reprogramming factor In order to prepare iPS cells using a nuclear reprogramming factor, it is preferable to introduce the nuclear reprogramming factor into somatic cells.
  • the number of nuclear reprogramming factors to be included is 2, 3, preferably 4, or 4 or more.
  • a preferred combination of these factors is either a combination of Oct3 / 4 gene, Sox2 gene and Klf4 gene, or a combination of Oct3 / 4 gene, Sox2 gene, Klf4 gene and c-Myc gene.
  • the nuclear reprogramming factor is a protein that functions in a cell
  • the gene encoding the protein is incorporated into an expression vector, and the expression vector is used in a differentiated cell such as a target somatic cell. Is preferably introduced and the protein is expressed in cells (gene transfer method).
  • the expression vector to be used is not particularly limited, but preferably, a plasmid vector, a viral vector, and an artificial chromosome vector (Suzuki N et al., J Biol Chem. 281 (36): 26615, 2006) are exemplified. Examples include adenovirus vectors, Sendai virus vectors, retrovirus vectors, and lentivirus vectors.
  • a protein called Protein Transduction Domain may be bound to a protein and added to the medium to introduce the protein into the cell (Protein Transduction method).
  • the protein can be introduced into cells using any of various protein introduction reagents (for example, Chariot TM , Bioporter TM, etc.).
  • the nuclear reprogramming factor is a protein secreted extracellularly, the factor may be added to the culture medium of differentiated cells at the stage of preparing iPS cells.
  • any of the nuclear reprogramming factors is expressed in the differentiated cells to be reprogrammed, it is not necessary to introduce the factors from the outside.
  • Cytokines include, for example, SCF (stem cell factor), bFGF, Wnt family, and LIF (leukemia protein factor), and compounds include, for example, Histone deacetylase inhibitor, DNA methylation inhibitor, MEK inhibitor, GSK3 ⁇ And inhibitors, TGF receptor inhibitors and ROCK inhibitors (WO2009 / 117439).
  • a feeder cell is not specifically limited, A mouse embryonic fibroblast (MEF) is illustrated.
  • MEF mouse embryonic fibroblast
  • the medium suitably used for this culture include a medium suitable for culturing cells of animal species from which somatic cells are derived.
  • a medium suitable for culturing cells of animal species from which somatic cells are derived for example, in the case of human cells, 20% alternative serum, 2 mM L-glutamine, 1 ⁇ Preferred is DMEM / F12 medium containing 10 -4 M non-essential amino acids, 1 x 10 -4 M 2-mercaptoethanol, 0.5% penicillin and streptomycin, and 4 ng / ml recombinant human basic fibroblast growth factor (bFGF). .
  • iPS cells are isolated by selecting, for example, cells expressing an undifferentiated cell-specific gene from differentiated cells into which a nuclear reprogramming factor has been introduced, or using cell morphology as an index.
  • a method for selecting a cell expressing an undifferentiated cell-specific gene is not particularly limited. If the undifferentiated cell-specific gene encodes an intracellular protein, the GFP gene, galactosidase gene, neomycin-resistant gene, hygromycin-resistant gene, puromycin-resistant gene, etc. downstream of the promoter of the undifferentiated cell-specific gene
  • One of the marker genes such as the drug resistance gene may be knocked in and expressed as a fusion protein, or cells expressing these marker genes may be selected.
  • an undifferentiated cell-specific gene refers to a gene well known to those skilled in the art that is specifically expressed in embryonic stem cells (ES cells).
  • ES cells embryonic stem cells
  • International Publication WO2005 / 080598, WO2007 / Examples are genes specifically expressed in ES cells disclosed in 069666, WO2008 / 118820, WO2009 / 057831 and Nat Biotechnol. 25, 803, 2007.
  • the undifferentiated cell-specific gene is preferably Oct3 / 4, Sox2, Nanog, Lin28, Rex1, UTF1, Eras, Fgf4, TDGF, Cripto, Dax1, ESG1, GDF3, Sall4, Fbx15, SSEA-1, SSEA-4 Selected from the group consisting of TRA-1-60, TRA-1-81 and alkaline phosphatase (eg, TRA-2-54 and TRA-2-49) in consideration of animal species (eg, SSEA-1 Specific to mice, SSEA-4, TRA-1-60 and TRA-1-81 are specific to humans).
  • a preferred undifferentiated cell-specific gene is the Fbx15 gene or Nanog gene. When cell morphology is used as an index, selection may be performed using colony formation as an index, for example.
  • a cell population or cell line or clone isolated from cells that have been reprogrammed can be used as an iPS cell.
  • cell population In the present specification, the terms cell population, cell line and clone are not distinguished unless otherwise specified.
  • E Cloned embryo-derived ES cells obtained by nuclear transfer ntES cells are cloned embryo-derived ES cells produced by nuclear transfer technology and have almost the same characteristics as ES cells derived from fertilized eggs (T. Wakayama et al. (2001), Science, 292: 740-743; S. Wakayama et al. (2005), Biol. Reprod., 72: 932-936; J. Byrne et al. (2007), Nature, 450: 497-502).
  • an ES cell established from an inner cell mass of a blastocyst derived from a cloned embryo obtained by replacing the nucleus of an unfertilized egg with a nucleus of a somatic cell is a ntES (nuclear transfer ES) cell.
  • ntES nuclear transfer ES
  • a combination of nuclear transfer technology JB Cibelli et al. (1998), Nat. Biotechnol., 16: 642-646) and ES cell production technology (above) is used (Wakayama). Seika et al. (2008), Experimental Medicine, Vol. 26, No. 5 (extra number), 47-52).
  • reprogramming can be performed by injecting a somatic cell nucleus into an enucleated unfertilized egg of a mammal and culturing it for several hours.
  • a TGF- ⁇ family inhibitor is an inhibitor involved in the inhibition of TGF- ⁇ signaling through the binding between TGF- ⁇ and the TGF- ⁇ receptor.
  • TGF- ⁇ inhibitors include proteinaceous inhibitors and small molecule inhibitors. Examples of proteinaceous inhibitors include anti-TGF- ⁇ neutralizing antibodies and anti-TGF- ⁇ receptor neutralizing antibodies.
  • SB431542 (4- [4- (1,3-benzodioxol-5-yl) -5- (2-pyridinyl) -1H-imidazol-2-yl] -benzamide or water thereof SB202190 (above, RK Lindemann et a1., Mo1.
  • TGF- ⁇ family members regulate cellular and developmental processes such as mitosis, cell differentiation, embryonic pattern formation and organogenesis.
  • TGF- ⁇ signaling occurs through the heteromeric receptor complex of type I and type II receptors serine threonine kinase, which activates downstream Smad signaling processes. That is, when TGF- ⁇ binds to the receptor complex, TGFs 11 type receptor phosphorylates TGF ⁇ type I receptor, and this TGF ⁇ type 1 receptor phosphorylates receptor-regulated Smad (R-Smad). To initiate a downstream response. When activated R-Smad forms a multimeric complex with Smad4, activated R-Smad moves to the nucleus, and transcriptional regulation of the target gene is induced.
  • pluripotent stem cells are maintained in a state that is easily induced into neural undifferentiated cells.
  • Glycogen synthase kinase regulates the activity of phosphorylating an enzyme having an action of promoting glycogen synthesis.
  • GSK-3 is a multifunctional serine / threonine kinase found in all eukaryotes, including many cellular responses to Wnt, tyrosine kinases and G protein coupled receptors. It is an important regulator of signal transduction pathways and is involved in a wide range of cellular processes ranging from glycogen metabolism to cell cycle regulation and proliferation.
  • GSK3 ⁇ inhibitors are used among GSK inhibitors.
  • GSK3 ⁇ inhibitors examples include CHIR99021 (6-[[2-[[4- (2,4-dichlorophenyl) -5- (4-methyl-1H-imidazol-2-yl) -2-pyrimidinyl] amino]). Ethyl] amino] nicotinonitrile), SB-415286 (3-[(3-chloro-4-hydroxyphenyl) amino] -4- (2-nitrophenyl) -1H-pyrrole-2,5-dione), SB -2167, indirubin-3'-Monoxime, Kenpaullone, BIO (6-bromoindirubin-3'-oxime) and the like.
  • CHIR99021 (6-[[2-[[4- (2, 4-dichlorophenyl) -5- (4-methyl-1H-imidazol-2-yl) -2-pyrimidinyl] amino] ethyl] amino] nicotinonitrile), Kenpaullone are preferred.
  • a BMP inhibitor is an inhibitor involved in the inhibition of BMP signaling (BMP signaling) through the binding between BMP (bone morphogenetic protein) and a BMP receptor (type I or type II).
  • BMP inhibitors include proteinaceous inhibitors and small molecule inhibitors. Examples of proteinaceous inhibitors include natural inhibitors such as Noggin, chordin, and follistatin.
  • the small molecule inhibitor is a compound that inhibits BMP2, BMP4, BMP6 or BMP7 having the ability to activate the transcription factor SMAD1, SMAD5 or SMAD8.
  • Dorsomorphin (6- [4- (2-piperidine-1- (Ilethoxy) phenyl] -3-pyridin-4-ylpyrazolo [1,5-a] pyrimidine) and derivatives thereof (PB Yu et al. (2007), Circulation, 116: II_60; PB Yu et al. ( 2008), Nat. Chem. Biol., 4: 33-41; J. Hao et al. (2008), PLoS ONE (www.plozone.org), 3 (8): e2940).
  • Dorsomorphin is commercially available, for example from Sigma-Aldrich.
  • Dorsomorphin has the biological activity of inhibiting the above BMP signaling by inhibiting the binding of BMP to the BMP receptor.
  • LDN-193189 (4- (6- (4-piperazin-1-yl) phenyl) pyrazolo [1,5-a] pyrimidin-3-yl) quinoline) and its BMPI receptor kinase inhibitor Derivatives are exemplified (PB Yu et al. (2008), Nat. Med., 14: 1363-1369).
  • LDN-193189 is commercially available, for example, available from Stemgent.
  • Each 6-amine may form a salt if it can be formed.
  • salts include salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, and salts with basic or acidic amino acids.
  • the salt with an inorganic base include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt; aluminum salt and ammonium salt.
  • the salt with an organic base include trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, tromethamine [tris (hydroxymethyl) methylamine], tert-butylamine, cyclohexylamine, benzylamine, And salts with dicyclohexylamine and N, N-dibenzylethylenediamine.
  • the salt with inorganic acid include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid and phosphoric acid.
  • salts with organic acids include formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, and benzenesulfonic acid And salts with p-toluenesulfonic acid.
  • salt with basic amino acid include salts with arginine, lysine and ornithine.
  • salt with acidic amino acid include salts with aspartic acid and glutamic acid.
  • feeder cells are not necessarily required, but feeder cells can also be present.
  • feeder cells include fetal fibroblasts.
  • the fetal fibroblasts include, for example, MEF (mouse fetal fibroblasts), STO cells (mouse fetal fibroblast cell line), SNL cells (STO cell subclone; for example, SNL76 / 7 cells), and the like.
  • MEF mouse fetal fibroblasts
  • STO cells mouse fetal fibroblast cell line
  • SNL cells STO cell subclone; for example, SNL76 / 7 cells
  • the neuronal differentiation ability of pluripotent stem cells can be further improved.
  • a medium a medium used for culturing mammalian cells can be prepared as a basal medium.
  • the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI1640 medium, Fischer's medium, and mixed media thereof. Is included.
  • ES cell culture medium modified for ES cell culture eg, mouse ES cell culture medium (eg TX-WES medium, Thrombo X)
  • primate ES cell culture medium eg primate (human & monkey) ES cell culture medium , Reprocell, Kyoto, Japan, etc.
  • the medium may contain serum or may be serum-free.
  • the serum is not particularly limited as long as it can enhance the neuronal differentiation ability of pluripotent stem cells by the method 1 of the present invention, but is preferably a serum derived from the above mammals (for example, fetal bovine serum, human serum, etc.).
  • serum supplements for example, Knockout Serum Replacement (KSR) (Invitrogen)
  • KSR Knockout Serum Replacement
  • the serum concentration is not particularly limited as long as it can enhance the neuronal differentiation ability of pluripotent stem cells by Method 1 of the present invention, but usually 0.1 to 30 (v / v) % Range.
  • the medium can also be used as necessary, for example, serum proteins (eg, albumin), iron sources (eg, transferrin), fatty acids, growth factors, insulin, collagen precursors, trace elements, minerals (eg, sodium selenate), Reducing agent (for example, 2-mercaptoethanol, 3′-thiolglycerol, etc.), lipid, amino acid (for example, L-glutamine, etc.), non-essential amino acid, vitamins (for example, ascorbic acid, d-biotin, etc.), antibiotic (for example, streptomycin, etc.) , Penicillin, gentamicin, etc.), antioxidants, sugars (eg, glucose, etc.), organic acids (eg, pyruvate, lactic acid, etc.), buffers (eg, HEPES, etc.), steroids (eg, ⁇ -estradiol, progesterone, etc.), polyamines One or more substances (such as putrescine) It may have.
  • serum proteins eg, albumin
  • the medium contains a combination of the above TGF- ⁇ family inhibitor, GSK ⁇ inhibitor and BMP inhibitor.
  • the concentration of the TGF- ⁇ family inhibitor in the medium is appropriately set within a range that enhances the neuronal differentiation ability of pluripotent stem cells, but when using SB431542 as a TGF- ⁇ family inhibitor, usually 50 nM to 100 ⁇ M, Preferably, it is 100 nM to 10 ⁇ M, more preferably 1 to 5 ⁇ M.
  • the concentration of the GSK inhibitor in the medium is appropriately set within a range that enhances the neuronal differentiation ability of pluripotent stem cells.
  • CHIR99021 When CHIR99021 is used as the GSK inhibitor, it is usually 50 nM to 100 ⁇ M, preferably 100 nM to 10 ⁇ M, More preferably, it is 1 to 5 ⁇ M.
  • concentration of the BMP inhibitor in the medium is appropriately set within the range that enhances the neuronal differentiation ability of pluripotent stem cells.
  • Dorsomorphin When Dorsomorphin is used as the BMP inhibitor, it is usually 50 nM to 100 ⁇ M, preferably 100 nM to 10 ⁇ M, More preferably, it is 1 to 5 ⁇ M.
  • the method 1 of the present invention is different from the method of inducing differentiation from a pluripotent stem cell to a neural cell while maintaining the pluripotency of the pluripotent stem cell while enhancing its neuronal differentiation ability. Therefore, in the medium used in the method of the present invention, the LIF used for induction of differentiation of pluripotent stem cells into the initial neuroepithelium (Wenlin Li et al., PNAS (2011), 108: 8299-8304) is It is unnecessary. Accordingly, in one embodiment, the medium used in the method 1 of the present invention does not contain LIF.
  • the medium used in the method of the present invention does not require additives such as N2 and B27 used for neural differentiation of pluripotent stem cells. Therefore, in one aspect, the medium used in Method 1 of the present invention does not contain N2 and / or B27.
  • Enhancement of neuronal differentiation ability can be carried out using the medium in the presence or absence of feeder cells.
  • feeder cells include, for example, MEF (mouse fetal fibroblasts), STO cells (mouse fetal fibroblast cell lines), SNL cells (subclones of STO cells;
  • feeder cells are inactivated by irradiation with radiation (such as gamma rays) or treatment with an anticancer agent (such as mitomycin C) in order to stop cell growth.
  • the method 1 of the present invention enhances the neural differentiation ability while maintaining the pluripotency of the pluripotent stem cell, and is different from the method of inducing differentiation from the pluripotent stem cell to the nervous system cell. . Therefore, in the method 1 of the present invention, as feeder cells, stromal cells (for example, PA6 cells (mouse stromal cell line (RIKEN BRC Cell Cell Bank (Japan))) used for induction of neural differentiation of pluripotent stem cells, MS-5 cells (ExpHematol. 17: 145-53 (1989)), OP9 cells (Science. 265: 1098-1101 (1994)), etc.) are not used.
  • stromal cells for example, PA6 cells (mouse stromal cell line (RIKEN BRC Cell Cell Bank (Japan))
  • MS-5 cells ExpHematol. 17: 145-53 (1989)
  • OP9 cells Science. 265: 1098-1101 (1994)
  • the ROCK (p160-Rho-associated coiled-coil kinase) inhibitor is preferably added to the medium containing cultured pluripotent stem cells immediately before and immediately after the culture for enhancing neuronal differentiation ability.
  • a ROCK inhibitor is a substance that exhibits a very strong cell death inhibitory action upon cell dispersion.
  • Y-27632 ((R)-(+)-trans-N- (4-pyridyl) -4 -(1-aminoethyl) -cyclohexanecarboxamide ⁇ 2HCl ⁇ H 2 O), Fasudil (HA-1077), Thiazovivin, etc. are known (Watanabe et al., Nat. Biotech., 25: 681-686 (2007)).
  • the concentration of the ROCK inhibitor is usually about 50 nM to about 10 ⁇ M.
  • the density of pluripotent stem cells in the medium is preferably within the range of about 5.0 ⁇ 10 4 to about 1.0 ⁇ 10 7 cells, but may be outside this range.
  • the culture may be a three-dimensional culture under non-adhesive conditions, such as suspension culture (for example, dispersion culture, agglutination suspension culture, etc.), or a two-dimensional culture under adhesion conditions, such as plate culture, or two-dimensional culture after two-dimensional culture. Including continuous combination culture in which dimensional culture is performed. Two-dimensional culture can be used when differentiation is induced in the presence of feeder cells, while three-dimensional culture can be used when no feeder cells are present. Usually, adhesion culture is performed on feeder cells.
  • the surface is coated with a cell support material such as collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, Matrigel TM for the purpose of improving adhesion to cells.
  • a cell support material such as collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, Matrigel TM for the purpose of improving adhesion to cells.
  • pluripotent stem cells are cultured in a state suspended in a liquid medium.
  • a cell mass (or embryoid body) of pluripotent stem cells can be formed by aggregation suspension culture.
  • agglutination suspension culture for example, embryoid body culture method (Keller et al., Curr. Opin. Cell Bio 1. 7, 862-869 (1995)), SFEB method (eg, Watanabe et al., Nature Neuroscience 8, 288-296 (2005 ); WO 2005/123902) can be used.
  • Matrige1 method Chambers SM, et a1. Nat Biotechno1. 27: 485, 2009
  • SDIA method Kawasaki H, et a1. Proc Nat1 Acad Sci U S A. 99: 1580-5 (2002) can be used.
  • the above-mentioned media can be used as the culture medium, and the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is performed in an atmosphere of CO 2 -containing air.
  • the CO 2 concentration is preferably about 2-5%.
  • pluripotent stem cells are cultured for 5 days or more under the condition of enhancing neuronal differentiation ability.
  • Enhancement of neuronal differentiation ability in pluripotent stem cells is confirmed by an increase in the expression of nervous system markers (Sox2, nestin, Sox1, etc.) compared to before the start of the culture.
  • nervous system markers Sox2, nestin, Sox1, etc.
  • the expression of nanog is decreased and the expression of brachyury (mesoderm marker) and Sox17 (endoderm marker) is increased compared to before the start of the culture. Therefore, by confirming these expression fluctuations, it is possible to distinguish the enhancement of the neuronal differentiation ability in pluripotent stem cells from the differentiation into the nervous system.
  • the neuronal differentiation ability of pluripotent stem cells can be enhanced.
  • pluripotent stem cells preferably induced pluripotent stem cells
  • it can enhance the neuronal differentiation ability of T cell-derived iPS cells and B lymphocyte-derived iPS cells that are difficult to differentiate, from T cell-derived iPS cells and B lymphocyte-derived iPS cells more efficiently than before, It becomes possible to differentiate undifferentiated cells of the nervous system such as neural stem cells.
  • the pluripotent stem cell with enhanced neuronal differentiation ability can be subcultured while maintaining the enhanced neuronal differentiation ability.
  • the conditions for subculture are the same as the culture for enhancing the neuronal differentiation ability of the pluripotent stem cells described above.
  • Pluripotent stem cells with enhanced neural differentiation ability can be differentiated into neural undifferentiated cells such as neural stem cells by a method for inducing differentiation into neural undifferentiated cells described later.
  • Method for inducing differentiation of undifferentiated cells of nervous system comprises culturing pluripotent stem cells in the presence of a TGF- ⁇ family inhibitor and a GSK3 ⁇ inhibitor under an oxygen partial pressure of 2 to 10% (preferably suspension culture).
  • the present invention provides a method for inducing cell differentiation (that is, a method for producing undifferentiated cells of the nervous system).
  • the pluripotent stem cell is preferably a pluripotent stem cell (preferably an induced pluripotent stem cell) with enhanced neuronal differentiation ability in the method 1 of the present invention.
  • a medium used for culturing mammalian cells can be prepared as a basal medium.
  • the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI1640 medium, Fischer's medium, and mixed media thereof. Is included.
  • ES cell culture medium modified for ES cell culture eg, mouse ES cell culture medium (eg TX-WES medium, Thrombo X)
  • primate ES cell culture medium eg primate (human & monkey) ES cell culture medium , Reprocell, Kyoto, Japan, etc.
  • the medium may contain serum or may be serum-free.
  • the serum is not particularly limited as long as differentiation from pluripotent stem cells to neural undifferentiated cells can be induced by the method 2 of the present invention, but preferably the above-mentioned mammal-derived serum (eg, fetal bovine serum, human serum) Etc.).
  • serum supplements for example, Knockout Serum Replacement (KSR) (Invitrogen)
  • KSR Knockout Serum Replacement
  • the serum concentration is not particularly limited as long as differentiation from pluripotent stem cells to neural undifferentiated cells can be induced by the method 2 of the present invention, but usually 0.1 to 30. It is in the range of (v / v)%.
  • the medium can also be used as necessary, for example, serum proteins (eg, albumin), iron sources (eg, transferrin), fatty acids, growth factors, insulin, collagen precursors, trace elements, minerals (eg, sodium selenate), Reducing agent (for example, 2-mercaptoethanol, 3′-thiolglycerol, etc.), lipid, amino acid (for example, L-glutamine, etc.), non-essential amino acid, vitamins (for example, ascorbic acid, d-biotin, etc.), antibiotic (for example, streptomycin, etc.) , Penicillin, gentamicin, etc.), antioxidants, sugars (eg, glucose, etc.), organic acids (eg, pyruvate, lactic acid, etc.), buffers (eg, HEPES, etc.), steroids (eg, ⁇ -estradiol, progesterone, etc.), polyamines One or more substances (such as putrescine) It may have.
  • serum proteins eg, albumin
  • Suitable media can include MHM media having the following composition: DMEM / F-12 (1: 1), glucose (0.6%), glutamine (2 mM), sodium bicarbonate (3 mM), HEPES (5 mM), insulin (25 Ag / ml), transferrin (100 Ag / ml) , progesterone (20 nM), sodium selenate (30 ng) and putrescine (60 nM).
  • MHM media having the following composition: DMEM / F-12 (1: 1), glucose (0.6%), glutamine (2 mM), sodium bicarbonate (3 mM), HEPES (5 mM), insulin (25 Ag / ml), transferrin (100 Ag / ml) , progesterone (20 nM), sodium selenate (30 ng) and putrescine (60 nM).
  • the medium contains a combination of the above TGF- ⁇ family inhibitor and GSK3 ⁇ inhibitor.
  • the concentration of the TGF- ⁇ family inhibitor in the medium is appropriately set within the range that induces differentiation from pluripotent stem cells to undifferentiated cells of the nervous system, but when using SB431542 as a TGF- ⁇ family inhibitor, Usually, it is 50 nM to 100 ⁇ M, preferably 100 nM to 10 ⁇ M, more preferably 1 to 5 ⁇ M.
  • the concentration of the GSK3 ⁇ inhibitor in the medium is appropriately set within a range that induces differentiation from pluripotent stem cells to undifferentiated cells of the nervous system.
  • CHIR99021 is used as the GSK3 ⁇ inhibitor, it is usually 50 nM to 100 ⁇ M, preferably Is from 100 nM to 10 ⁇ M, more preferably from 1 to 5 ⁇ M.
  • the medium used in Method 2 of the present invention preferably further contains LIF in order to promote differentiation induction from pluripotent stem cells to undifferentiated neural cells.
  • LIF is contained in the medium, its concentration is not particularly limited as long as differentiation from pluripotent stem cells to neural undifferentiated cells can be induced by the method of the present invention, but it is usually from 0.25 ng / ml to 1 ⁇ g / ml, preferably 1 to 50 ⁇ ng / ml, most preferably 3 to 30 ⁇ ng / ml.
  • the medium used in Method 2 of the present invention preferably further contains bFGF in order to promote differentiation induction from pluripotent stem cells to undifferentiated nervous system cells.
  • bFGF is contained in the medium, the concentration is not particularly limited as long as differentiation from pluripotent stem cells to undifferentiated neural cells can be induced by the method of the present invention, but a normal concentration of 0.25 ng / ml ⁇ 1 ⁇ g / ml, preferably 1 to 50 ng / ml, most preferably 3 to 30 ng / ml.
  • the medium used in the method 2 of the present invention preferably further contains a ROCK inhibitor (Y-27632, Fasudil (HA-1077), etc.) in order to suppress cell death.
  • Y-27632 is contained in the medium, the concentration is not particularly limited as long as differentiation from pluripotent stem cells to undifferentiated neural cells can be induced by the method of the present invention, but about 50 nM to about 10 ⁇ M.
  • the medium used in the method 2 of the present invention preferably further contains additives such as N2 and B27 in order to promote differentiation induction from pluripotent stem cells to undifferentiated cells of the nervous system.
  • the medium used in Method 2 of the present invention contains bFGF, LIF, and B27 in addition to the TGF- ⁇ family inhibitor and the GSK3 ⁇ inhibitor.
  • the medium used in Method 2 of the present invention contains bFGF, LIF, B27 and a ROCK inhibitor (preferably Y-27632) in addition to the TGF- ⁇ family inhibitor and the GSK3 ⁇ inhibitor.
  • Differentiation induction into undifferentiated cells of the nervous system can be performed using the medium in the presence or absence of feeder cells.
  • feeder cells examples of the cells include, for example, MEF (mouse fetal fibroblasts), STO cells (mouse fetal fibroblast cell lines), SNL cells (subclones of STO cells; Cells, etc.); PA6 cells (mouse stromal cell line (RIKEN BRC Cell Bank (Japan))), MS-5 cells (Exp. Hematol. 17: 145-53 (1989)), OP9 cells (Science. 265: 1098-1101 (1994)), etc.
  • feeder cells are irradiated with radiation (gamma rays, etc.) or treated with an anticancer agent (mitomycin C, etc.) to stop cell growth. Inactivate.
  • the SDIA method is a method in which ES cells are co-cultured with stromal cells, particularly PA6 cells, to selectively differentiate into neurons, but in the present invention, the above-described method is performed even in the absence of feeder cells.
  • a combination of a TGF- ⁇ family inhibitor and a GSK3 ⁇ inhibitor can be selectively differentiated into undifferentiated cells of the nervous system simply by being present in a differentiation-inducing medium and culturing in a hypoxic (2-5%) environment. In addition to such culture conditions, when feeder cells are used, the efficiency of differentiation into undifferentiated cells of the nervous system can be further improved.
  • ROCK inhibitors are substances that exhibit a very strong cell death inhibitory action when cells are dispersed.
  • Y-27632 and Fasudil are known (Watanabe et a1., Nat). . Biotech., 25: 681-686 (2007)).
  • the concentration of inhibitor is usually about 50 nM to about 10 ⁇ M.
  • the density of pluripotent stem cells in the medium is preferably within the range of about 1.0 ⁇ 10 4 to about 1.0 ⁇ 10 7 cells, but may be outside this range.
  • the culture may be a three-dimensional culture under non-adhesive conditions, such as suspension culture (for example, dispersion culture, agglutination suspension culture, etc.), or a two-dimensional culture under adhesion conditions, such as plate culture, or two-dimensional culture after two-dimensional culture. Including continuous combination culture in which dimensional culture is performed. Two-dimensional culture can be used when differentiation is induced in the presence of feeder cells, while three-dimensional culture can be used when no feeder cells are present.
  • pluripotent stem cells are cultured in suspension.
  • the surface is coated with a cell support material such as collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, Matrigel TM for the purpose of improving adhesion to cells.
  • a cell support material such as collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, Matrigel TM for the purpose of improving adhesion to cells.
  • pluripotent stem cells are cultured in a state suspended in a liquid medium.
  • a cell mass (or embryoid body) of pluripotent stem cells can be formed by aggregation suspension culture.
  • agglutination suspension culture for example, embryoid body culture method (Keller et al., Urr Curr. Opin. Cell Bio 1. 7, 862-869 (1995)), SFEB method (eg, Watanabe et al., Nature Neuroscience 8, 288-296 (2005 ); WO 2005/123902) can be used.
  • pluripotent stem cells that have been separated into a single cell state by enzyme treatment are dispersedly cultured in a state of being suspended in a liquid medium.
  • neurospheres containing undifferentiated neural cells such as neural stem cells can be formed.
  • Matrige1 method (Chambers SM, et a1. Nat Biotechno1. 27: 485, 2009), SDIA method (Kawasaki H, et a1. Neuron. 28: 31-40, 2000, or Kawasaki H, et a1. Proc Nat1 Acad Sci U S A. 99: 1580-5, 2002) can be used.
  • the above-mentioned media can be used as the culture medium, and the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is performed in an atmosphere of CO 2 -containing air.
  • the CO 2 concentration is preferably about 2-5%.
  • pluripotent stem cells are cultured in a hypoxic (2 to 10%, preferably 3%) environment.
  • a hypoxic environment By culturing in a hypoxic environment, differentiation induction from pluripotent stem cells into undifferentiated nervous system cells is promoted.
  • pluripotent stem cells are cultured for 3 days or more under differentiation-inducing conditions.
  • pluripotent stem cells are cultured for a period sufficient for induction of differentiation, neurospheres containing undifferentiated neural cells such as neural stem cells are formed.
  • neural undifferentiated cells such as neural stem cells from pluripotent stem cells in a short period of time.
  • pluripotent stem cells having enhanced neuronal differentiation ability in the above-described method 1 of the present invention are cultured in the method 2 of the present invention.
  • the method 1 of the present invention and the method 2 of the present invention, it is possible to induce differentiation of neural undifferentiated cells such as neural stem cells in a very short period of time.
  • the present invention also provides an undifferentiated nervous system cell produced by the method 2 of the present invention.
  • Neural undifferentiated cells obtainable by the method 2 of the present invention include cells of the central nervous system, neurons such as motor nerves and autonomic nerves, and glial cell precursor cells such as oligodendrocytes and astrocytes.
  • the Neural undifferentiated cells are preferably neural stem cells.
  • Neural undifferentiated cells include neural cell adhesion molecule (NCAM), polysialylated NCAM, A2B5 (expressed in fetal and neonatal neurons), intermediate filament proteins (nestin, vimentin, etc.), transcription factor Pax-6, etc. It can be identified by expression markers of primitive neuroectodermal and neural stem cells, dopamine neuron markers (such as tyrosine hydroxylase (TH)), neuronal markers (such as TuJl) and the like.
  • NCAM neural cell adhesion molecule
  • A2B5 expressed in fetal and neonatal neurons
  • intermediate filament proteins nestin, vimentin, etc.
  • transcription factor Pax-6 etc. It can be identified by expression markers of primitive neuroectodermal and neural stem cells, dopamine neuron markers (such as tyrosine hydroxylase (TH)), neuronal markers (such as TuJl) and the like.
  • TH tyrosine hydroxylase
  • Nervous system undifferentiated cells may be transplanted to a living body after production, or may be completely or partially differentiated into neural cells and glial cells (including astrocytes and oligodendrocytes) and then transplanted into the living body. It may be transplanted.
  • glial cells including astrocytes and oligodendrocytes
  • neural undifferentiated cells such as neural stem cells obtained by the method 2 of the present invention are dispersed with a proteolytic enzyme such as trypsin, and the dispersed neural undifferentiated cells are again treated with the method 2 of the present invention.
  • a proteolytic enzyme such as trypsin
  • Method 3 Method for inducing differentiation of motor neural progenitor cells
  • the present invention relates to neural stem cells obtained by the above-described method 2 of the present invention using TGF- ⁇ family inhibitor, GSK3 ⁇ inhibitor, retinoic acid and purmorphamine (9-cyclohexyl-N- [4- (4-morpholinyl) Differentiation induction of motor neuron progenitors, including culturing in the presence of 2-10% oxygen partial pressure in the presence of phenyl] -2- (1-naphthalenyloxy) -9H-purin-6-amine)
  • the method namely, the manufacturing method of a motor neural progenitor cell
  • the manufacturing method of a motor neural progenitor cell is provided.
  • Method 3 of the present invention are the same as those of Method 2 of the present invention except that retinoic acid and purmorphamine are added to the medium.
  • the concentration of retinoic acid in the medium is not particularly limited as long as it induces differentiation of motor neural progenitor cells, but is usually 10 nM to 100 ⁇ M, preferably 100 nM to 10 ⁇ M, more preferably 0.3 to 3 ⁇ M.
  • the concentration of purmorphamine in the medium is not particularly limited as long as it induces differentiation of motor neural progenitor cells, but is usually 10 nM to 100 ⁇ M, preferably 100 nM to 10 ⁇ M, more preferably 0.3 to 3 ⁇ M.
  • retinoic acid and purmorphamine are added to the medium, and further cultivation is continued.
  • Regenerative motor neural progenitor cells can be induced to differentiate.
  • the further culture period is usually 4 days or longer.
  • pluripotent stem cells preferably iPS cells
  • pluripotent stem cells whose neurodifferentiation ability is enhanced in the above-described method 1 of the present invention are cultured by the method 2 of the present invention, and further in the method 3 of the present invention, a motor neuron precursor Differentiation into cells is induced.
  • motor neural progenitor cells can be induced to differentiate from pluripotent stem cells in a very short period of time.
  • motor neural progenitor cells can be efficiently induced to differentiate from pluripotent stem cells that are difficult to differentiate from nerves, such as iPS cells derived from lymphocytes (T cells, B cells, etc.).
  • the present invention also provides a motor neural progenitor cell produced by the method 3 of the present invention.
  • Motor neuron progenitor cells can be identified by the expression of marker genes such as Oligo2 and Nkx2.2.
  • Motor neuron progenitor cells may be transplanted directly into a living body after production, or may be transplanted into a living body after being completely or partially differentiated into motor nerve cells.
  • motor neuron progenitor cells can be induced to differentiate by culturing them in a medium containing N2, IGF-1, GDNF, BDNF, ascorbic acid, retinoic acid, Shh and (dibutyryl) cAMP.
  • the present invention also provides such a method for inducing differentiation of motor neurons (that is, a method for producing motor neurons), and motor neurons produced by the method.
  • Motor neurons can be identified by the expression of marker genes such as HB9, Isl, SMI-32, and ChAT.
  • Method 4 Method for inducing differentiation of dopaminergic neural progenitor cells
  • the present invention relates to neural stem cells obtained by the above-described method 2 of the present invention in the presence of 2-10% in the presence of TGF- ⁇ family inhibitor, GSK3 ⁇ inhibitor, sonic hedgehog (protein), purmorphamine and FGF8.
  • a method for inducing differentiation of dopaminergic neural progenitor cells ie, a method for producing dopaminergic neural progenitor cells, which comprises culturing under a partial pressure of oxygen.
  • the culture conditions in the method 4 of the present invention are the same as those of the method 2 of the present invention except that sonic hedgehog, purmorphamine and FGF8 are added to the medium.
  • the concentration of sonic hedgehog in the medium is not particularly limited as long as it induces differentiation of dopaminergic neural progenitor cells, but is usually 1 ng / ml to 5 ⁇ g / ml, preferably 10 to 500 ng / ml, most preferably 50 to 300 ng / ml.
  • the concentration of purmorphamine in the medium is not particularly limited as long as it induces differentiation of dopaminergic neural progenitor cells, but it is usually 10 to 100 ⁇ M, preferably 100 to 10 ⁇ M, more preferably 0.3 to 3 ⁇ M.
  • the concentration of FGF8 in the medium is not particularly limited as long as it induces differentiation of dopaminergic neural progenitor cells, but is usually 1 ng / ml to 5 ⁇ g / ml, preferably 10 to 500 ng / ml, most preferably 50 to 300 ng. / ml.
  • sonic hedgehog, purmorphamine and FGF8 are added to the medium, and further culturing is continued.
  • Self-renewable dopaminergic neural progenitor cells can be induced to differentiate.
  • the further culture period is usually 4 days or longer.
  • the pluripotent stem cells (preferably iPS cells) whose neuronal differentiation ability has been enhanced in the above-described method 1 of the present invention are cultured in the method 2 of the present invention, and further in the method 4 of the present invention, Differentiation is induced into progenitor cells.
  • iPS cells derived from lymphocytes
  • the present invention also provides a dopaminergic neural progenitor cell produced by the method 4 of the present invention.
  • Dopaminergic neural progenitor cells can be identified by the dopamine contained in the cells.
  • the dopaminergic neuron progenitor cells may be transplanted to the living body as they are after preparation, or may be transplanted to the living body after being completely or partially differentiated into dopaminergic neuron cells.
  • dopaminergic neuron progenitor cells can be induced to differentiate by culturing dopaminergic neuron progenitor cells in a medium containing B27, BDNF, GDNF, ascorbic acid, TGF- ⁇ , cAMP and DAPT.
  • the present invention also provides a method for inducing differentiation of such a dopaminergic neuron (ie, a method for producing a dopaminergic neuron) and a dopaminergic neuron produced by the method.
  • Dopaminergic neurons can be identified by dopamine contained in the cells.
  • Neural undifferentiated cells are compounds for treating neurological diseases (for example, pharmaceutical compounds, solvents, small molecules, It can also be used for screening of peptides or polynucleotides. For example, adding a candidate pharmaceutical compound alone or in combination with other drugs to neural undifferentiated cells (such as neural stem cells), motor neural progenitor cells, motor neuron cells, dopaminergic neural progenitor cells or dopaminergic neuron cells
  • neural undifferentiated cells such as neural stem cells
  • motor neural progenitor cells motor neuron cells
  • dopaminergic neural progenitor cells or dopaminergic neuron cells
  • the evaluation can be performed based on the morphological or functional change of the cell.
  • neural undifferentiated cells neural stem cells and the like
  • motor neural progenitor cells motor neural progenitor cells
  • motor neuron cells motor neuron cells
  • dopaminergic neuron progenitor cells dopaminergic neuron cells
  • dopaminergic neuron cells are cells that exhibit the same phenotype as the neurological disease to be treated.
  • artificial pluripotent stem cells prepared from somatic cells derived from disease or artificially differentiated cells of the artificial nervous system induced by differentiation of ntES cells transplanted with nuclei of somatic cells derived from diseases, artificial motor neurons A progenitor cell, an artificial motor neuron, an artificial dopamine-producing neural progenitor cell, and an artificial dopamine-producing neuron.
  • SAG N-methyl-N ′-(3-pyridinylbenzyl) -N ′-(3-chlorobenzo [b] thiophene-2- Carbonyl) -1,4-diaminocyclohexane
  • concentration of SAG in the medium when SAG is used is not particularly limited as long as it induces differentiation of motor neuron progenitor cells or dopaminergic neuron progenitor cells, but is usually 10 nM to 100 ⁇ M, preferably 100 nM to 10 ⁇ M, more preferably 100 nM to 2 ⁇ M.
  • the undifferentiated nervous system cells neural stem cells and the like
  • motor neural progenitor cells motor neuron cells
  • dopaminergic neuron progenitor cells dopaminergic neuron cells of the present invention are used in the field of regenerative medicine for normalizing damaged nervous system tissues Can be used effectively. Therefore, this cell can be a therapeutic cell for diseases related to various nervous system cell disorders.
  • diseases include ischemic brain diseases (such as stroke), brain trauma, spinal cord injury, motor neuropathy, neurodegenerative diseases, retinitis pigmentosa, age-related macular degeneration, inner ear deafness, multiple sclerosis, muscle Examples include amyotrophic lateral sclerosis, spinocerebellar degeneration, Huntington's chorea, Alzheimer's disease, Parkinson's disease, epilepsy, and schizophrenia.
  • ischemic brain diseases such as stroke
  • brain trauma such as stroke
  • spinal cord injury motor neuropathy
  • neurodegenerative diseases such as retinitis pigmentosa
  • age-related macular degeneration inner ear deafness
  • multiple sclerosis muscle
  • amyotrophic lateral sclerosis spinocerebellar degeneration
  • Huntington's chorea Alzheimer's disease, Parkinson's disease, epilepsy, and schizophrenia.
  • the method for this purpose include a method for selecting a target cell, for example, a flow cytometry method, treatment with an anticancer agent-containing medium, and the like.
  • a flow cytometry method cell particles are flowed at a high speed in a very thin flow, irradiated with laser light, and light such as fluorescence (when cells are pre-fluorescently labeled) and scattered light are emitted. If a cell sorter is provided for measurement, target cells can be selected and separated.
  • Fluorescent labeling of cells includes antibodies (fluorescent labeling) specific for neural undifferentiated cells, motor neural progenitor cells, motor neuron cells, dopaminergic neuron progenitor cells and dopaminergic neuron cells, respectively ( Anti-Nestin antibody).
  • undifferentiated cells can be removed by treatment with an anticancer agent-containing medium.
  • anticancer agents are mitomycin C, 5-fluorouracil, adriamycin, methotrexate and the like.
  • Example 1 WD39 obtained by introducing Oct3 / 4, Sox2, Klf4, and c-Myc into human fibroblasts by retrovirus was used as human iPS cells inducing 3D (3 Drugs) -iPS .
  • aTKA4 obtained by introducing Oct3 / 4, Sox2, Klf4 and c-Myc into human T cells by Sendai virus was also used.
  • DMEM / F12 containing 20% KSR, 1% NEAA, 0.1% mercaptoethanol, penicillin (50 u / ml) / streptomycin (50 ⁇ g / ml), 2 mM glutamine and 4 ng / ml bFGF. was used.
  • 3D-iPS cells were obtained by culturing human iPS cells in the above passage medium supplemented with 3 ⁇ M SB431542, 3 ⁇ M CHIR99021 and 3 ⁇ M dorsomorphin for 5 days or more. The colony of 3D-iPS cells exhibited a more three-dimensional morphology compared to human iPS cells cultured in a normal passage medium (Fig.
  • Example 2 Formation of neurospheres from human iPS cells As human iPS cells, WD39 derived from fibroblasts and aTKA4 derived from T cells were used. The culture supernatant of human iPS cells that became confluent was aspirated and washed with 6 ml of PBS. After adding 1 ml of the dissociation solution T, it was immediately sucked out and incubated for about 5 minutes.
  • hES-medium 20% KSR, 1% NEAA, 0.1% mercaptoethanol, penicillin (50u / ml) / streptomycin (50 ⁇ g / ml), 2mM glutamine and 4ng / ml bFGF DMEM / F12
  • penicillin 50u / ml
  • streptomycin 50 ⁇ g / ml
  • 2mM glutamine 50 ⁇ g / ml
  • 4ng / ml bFGF DMEM / F12 2mM glutamine
  • 4ng / ml bFGF DMEM / F12 2mM glutamine
  • 10 ml of hES-medium (ROCK-inhibitor Y27632 (+)) was added, pipetted and spread onto gelatin coated dishes and incubated for 1-2 hours.
  • the dish was rotated to collect the floating cells at the center and collected in a 50 ml tube.
  • the cells were centrifuged at 1000 rpm for 5 minutes and the supernatant was aspirated.
  • 1 ml of Tryple Select was added, incubated in a 37 ° C. water bath for 5 minutes, and occasionally stirred by turning the tube. During this incubation, 2 ml of Trypsin Inhibitor was placed in a 15 ml tube and a cell strainer (70 ⁇ m) was prepared.
  • the Tryple Select digested product was pipetted about 10 times with a P1000 pipetteman, then added to Trypsin Inhibitor, volumed up with MHM, and filtered with Cell Strainer. The cells were centrifuged for 5 minutes, the supernatant was aspirated and 3-5 ml of MHM was added. The number of cells was counted and adjusted to a concentration of 10 4 cells / ml by adding MHM medium.
  • the collected human iPS cells were suspended in MHM medium containing B27, 20 ng / ml bFGF, 10 ng / ml hLIF, and 10 ⁇ M Y27632 for 14 days to form neurospheres (here, this method was used as a single cell floating neural network). Differentiation (SND) method). Neurospheres were also formed under conditions (GSKi + TGF- ⁇ i conditions) in which 3 ⁇ M CHIR99021 and 2 ⁇ M SB431542 were further added to the medium in this SND method. The neurosphere differentiation ability of the formed neurosphere was confirmed by culturing for 10 days in an MHM medium containing B27 on a PO / fibronectin-coated plate.
  • GSKi + TGF- ⁇ i As a result, under the GSKi + TGF- ⁇ i condition, neurospheres capable of neuronal differentiation were formed 6 days after the start of culture, faster than the conventional SND method (FIG. 2). From this result, it was suggested that GSKi + TGF- ⁇ i conditions (addition of CHIR99021 and SB431542) promote differentiation induction of undifferentiated neural cells (neural stem cells, etc.) from iPS cells even in suspension culture.
  • Example 3 Examination of addition timing of CHIR99021 and SB431542 As in Example 2, neurospheres containing neural stem cells were formed by culturing human iPS cells by SND method for 6 days under the condition of adding 3 ⁇ M CHIR99021 and 2 ⁇ M SB431542. Then, the neurodifferentiation ability of the formed neurosphere was evaluated in the same manner as in Example 2. Here, after induction of neurospheres by the SND method has already started, CHIR99021 and SB431542 were added, and the relationship between the timing of addition and the induction efficiency of neural stem cells / differentiated cells of the nervous system was examined.
  • Example 4 Effects of hypoxia and Y27632 on neural stem cell differentiation
  • human iPS cells were cultured for 6 days by the SND method.
  • a neurosphere containing was formed.
  • the neurodifferentiation ability of the formed neurosphere was evaluated in the same manner as in Example 2.
  • the influence of hypoxia and ROCK inhibitor (Y27632) on neural stem cell differentiation by this method was evaluated.
  • the addition of the ROCK inhibitor (Y27632) showed no significant change in neural differentiation from neural stem cells.
  • induction of GFAP positive cells was promoted under hypoxic conditions (FIG. 4).
  • Example 5 Formation of neurospheres from 3D-iPS cells Human 3D-iPS cells were prepared by the method described in Example 1. The culture supernatant of human 3D-iPS cells that became confluent was aspirated and washed with 6 ml of PBS. After adding 1 ml of the dissociation solution T, it was immediately sucked out and incubated for about 5 minutes.
  • hES-medium (20% KSR, 1% NEAA, 0.1% mercaptoethanol, penicillin (50u / ml) / streptomycin (50 ⁇ g / ml), 2mM glutamine and 4ng / ml bFGF DMEM / F12) was added and the cells were collected in 50 ml tubes. iPS cells were centrifuged at 1000 rpm for 5 minutes, and the supernatant was aspirated. 10 ml of hES-medium (ROCK-inhibitor Y27632 (+)) was added, pipetted and spread onto gelatin coated dishes and incubated for 1-2 hours.
  • the dish was rotated to collect the floating cells at the center and collected in a 50 ml tube.
  • the cells were centrifuged at 1000 rpm for 5 minutes and the supernatant was aspirated.
  • 1 ml of Tryple Select was added, incubated in a 37 ° C. water bath for 5 minutes, and occasionally stirred by turning the tube. During this incubation, 2 ml of Trypsin Inhibitor was placed in a 15 ml tube and a cell strainer (70 ⁇ m) was prepared.
  • the Tryple Select digested product was pipetted about 10 times with a P1000 pipetteman, then added to Trypsin Inhibitor, volumed up with MHM, and filtered with Cell Strainer. The cells were centrifuged for 5 minutes, the supernatant was aspirated and 3-5 ml of MHM was added. The number of cells was counted and adjusted to a concentration of 10 4 cells / ml by adding MHM medium.
  • the collected human 3D-iPS cells were cultured under low oxygen (3% O 2 ) conditions in MHM medium containing B27, 20 ng / ml bFGF, 10 ng / ml hLIF, 10 ⁇ M Y27632, 3 ⁇ M CHIR99021, and 2 ⁇ M SB431542. (SND method modified).
  • SND method modified As a result, neurospheres capable of neuronal differentiation were successfully induced 3 days after the start of culture.
  • astrocytes could be induced from the neurosphere 4 days after the start of culture (FIG. 5).
  • Example 6 Induction of motor neurons Like Example 5, human 3D-iPS cells prepared by the method described in Example 1 were treated with B27, 20 ng / ml bFGF, 10 ng / ml hLIF, 10 ⁇ M Y27632, 3 ⁇ M CHIR99021, and 2 ⁇ M SB431542. Culturing was carried out under low oxygen (3% O 2 ) conditions in the MHM medium containing. On the third day from the start of culture, 1 ⁇ M retinoic acid and 1 ⁇ M purmorphamine were added to the medium, and further cultured to form self-replicating neurospheres including motor neuron progenitor cells (MPC).
  • MPC motor neuron progenitor cells
  • Example 7 Induction of motor neurons 2 As in Example 5, human 3D iPS cells prepared by the method described in Example 1 were cultured in MHM medium containing B27, 20 ng / ml bFGF, 10 ng / ml hLIF, 10 ⁇ M Y27632, 3 ⁇ M CHIR99021, and 2 ⁇ M SB431542. The cells were cultured under hypoxic (3% O 2 ) conditions. On the 3rd day from the start of the culture, 1 ⁇ M retinoic acid and 1 ⁇ M purmorphamine were added to the medium, and further cultured for 4 days to form neurospheres including motor neuron progenitor cells (MPC) capable of self-replication.
  • MPC motor neuron progenitor cells
  • Neurospheres containing MPC are transferred to DMEM / F12 containing N2, Glutamax, 10ng / ml IGF-1, 10ng / ml GDNF, 10ng / ml BDNF, 200ng / ml ascorbic acid, 50nM retinoic acid, 50ng / ml Shh, 1 ⁇ M cAMP
  • the medium was changed, and the culture was continued on the PO / laminin-coated plate for another 10 days. As a result, motor neurons were induced with high efficiency (FIG. 6).
  • Example 8 Gene expression pattern analysis of human 3D iPS cells By culturing 2 lines of human fibroblast-derived iPS cells (WD39, B7) in the presence of 3 ⁇ M SB431542, 3 ⁇ M CHIR99021 and 3 ⁇ M dorsomorphin according to Example 1 for 5 or 6 days. 3D-iPS cells were prepared and their properties were confirmed by expression of marker genes by qRT-PCR. The results are shown in FIG. Treatment with SB431542, CHIR99021, and dorsomorphin reduced nanog expression, Sox2 (nervous system marker), nestin (neural stem cell marker), Sox1 (neural system marker), brachyury (mesoderm marker) and Sox17 (endoderm marker) Expression was increased.
  • Example 9 Induction of neural stem cells from T cell-derived iPS cells Seki et al. Cell Stem Cell, Volume 7, Issue 1, 11-14, 2 July (2010) and Nishimura K et al. JBC 2011 11; 286 4760-71 IPS cells (TiPS cells) derived from human T cells using Sendai virus according to the methods described above, and human T cells using plasmid vectors according to the methods described in Nature Methods 8,409-412 (2011) TiPS cells derived from the cells were used.
  • 3D-TiPS cells were prepared by culturing 2 human TiPS cell lines in the presence of 3 ⁇ M SB431542, 3 ⁇ M CHIR99021, and 3 ⁇ M dorsomorphin according to Example 1 for 5 days.
  • 3D-TiPS cells were hypoxic (3% O 2 ) in MHM medium containing B27, 20 ng / ml bFGF, 10 ng / ml hLIF, 10 ⁇ M Y27632, 3 ⁇ M CHIR99021, and 2 ⁇ M SB431542.
  • Neurospheres containing neural stem cells were formed by culturing under conditions for 3 days. That is, neural stem cells were induced to differentiate from TiPS cells in 3 days.
  • Example 10 Induction of dopaminergic neural progenitor cells and dopaminergic neuron cells
  • human 3D-iPS cells derived from fibroblasts and T cells prepared by the method described in Example 1 were B27, 20 ng / The cells were cultured under low oxygen (3% O 2 ) conditions in MHM medium containing ml bFGF, 10 ng / ml hLIF, 10 ⁇ M Y27632, 3 ⁇ M CHIR99021, and 2 ⁇ M SB431542.
  • 2 ⁇ M purmorphamine, 100 ng / ml Sonic hedgehog and 100 ng / ml FGF8 were added to the medium, and further cultured for 4 days. Been formed.
  • iPS cells obtained by introducing Oct3 / 4, Sox2, Klf4 and c-Myc by retrovirus into human B lymphoblasts.
  • DMEM / F12 As a passage medium for human LiPS cells, DMEM / F12 containing 20% KSR, 1% NEAA, 0.1% mercaptoethanol, penicillin (50 u / ml) / streptomycin (50 ⁇ g / ml), 2 mM glutamine and 4 ng / ml bFGF. Is used.
  • 3D-LiPS cells By culturing human iPS cells in the above passage medium supplemented with 3 ⁇ M SB431542, 3 ⁇ M CHIR99021 and 3 ⁇ M dorsomorphin for 5 days or longer, 3D-LiPS cells are obtained. 3D-LiPS cells can be passaged while maintaining pluripotency and good neuronal differentiation potential.
  • Example 12 Formation of neurospheres from human LiPS cells
  • the human 3D-LiPS cells obtained in Example 11 are used. Aspirate the culture supernatant of human 3D-LiPS cells that have become Confluent and wash with 6 ml of PBS. After adding 1 ml of dissociation solution T, immediately absorb and incubate for about 5 minutes.
  • the collected human iPS cells are subjected to suspension culture in MHM medium containing B27, 20 ng / ml bFGF, 10 ng / ml hLIF, 10 ⁇ M Y27632, 3 ⁇ M CHIR99021 and 2 ⁇ M SB431542 for 10 days to obtain neurospheres containing neural stem cells.
  • Example 13 Induction of motor neuron progenitor cells and motor neurons from human LiPS cells
  • human 3D-LiPS cells prepared by the method described in Example 11 were treated with B27, 20 ng / ml bFGF, 10 ng / ml hLIF, The cells are cultured under low oxygen (3% O 2 ) conditions in MHM medium containing 10 ⁇ M Y27632, 3 ⁇ M CHIR99021, and 2 ⁇ M SB431542.
  • MHM medium containing 10 ⁇ M Y27632, 3 ⁇ M CHIR99021, and 2 ⁇ M SB431542.
  • 1 ⁇ M retinoic acid and 1 ⁇ M purmorphamine are added to the medium, and further cultured for 4 days to form neurospheres including motor neuron progenitor cells (MPCs) capable of self-replication.
  • MPCs motor neuron progenitor cells
  • Neurospheres containing MPC are transferred to DMEM / F12 containing N2, Glutamax, 10ng / ml IGF-1, 10ng / ml GDNF, 10ng / ml BDNF, 200ng / ml ascorbic acid, 50nM retinoic acid, 50ng / ml Shh, 1 ⁇ M cAMP Motor neurons are induced by changing the medium and continuing the culture on PO / laminin-coated plates for another 10 days.
  • Example 14 Induction of dopaminergic neural progenitor cells and dopaminergic neurons from human LiPS cells
  • human 3D-LiPS cells prepared by the method described in Example 11 (B27, 20 ng / ml bFGF, 10 ng / ml hLIF, 10 ⁇ M Y27632, 3 ⁇ M CHIR99021, and 2 ⁇ M SB431542 in MHM medium under hypoxia (3% O 2 ) condition 2 ⁇ M purmorphamine, 100 ng / ml Sonic
  • a neurosphere containing dopaminergic neuron progenitor cells capable of self-replication is formed.
  • iPS cells (LiPS cells) obtained by introducing Sox2, Klf4, shp53, Lin28 and L-Myc were used as the passage medium for human LiPS cells, 20% KSR, 1% NEAA, DMEM / F12 containing 0.1% mercaptoethanol, penicillin (50 u / ml) / streptomycin (50 ⁇ g / ml), 2 mM glutamine and 4 ng / ml bFGF was used.
  • hES-medium 10 ml of hES-medium (ROCK-inhibitor (+)) was added to LiPS cells, pipetted, placed in a gelatin-coated 10 cm dish, and incubated for 1-2 hours.
  • the STO sticking condition was confirmed with a microscope, and the dish was turned to collect floating cells in the center, and then collected in a 50 ml tube. Centrifugation was performed at 1000 rpm for 5 minutes, and the supernatant was sucked off.
  • 1.5 ml of Tryple Select was added to the LiPS cell pellet, incubated for 5 minutes in a 37 ° C water bath, and the cells were dispersed by occasionally rotating the tube and stirring.
  • the cell suspension was added to a 15 ml tube containing 3 ml of Trypsin Inhibitor, and the volume was increased with MHM, followed by filtration with a Cell Strainer (70 ⁇ m). The cell suspension that passed through was centrifuged for 5 minutes, the supernatant was aspirated, and 3-5 ml of MHM was added. Count the number of cells, add MHM medium to adjust to 10 cells / ⁇ l, add reagents (Table 1 below), and culture in a flask under hypoxic conditions (4% O 2 ) for 2 weeks. A neurosphere containing was formed.
  • Medium change was performed one week after the start of culture. Medium changes were performed as follows.
  • the Sphere was collected in a 50 ml tube and centrifuged at 1000 rpm for 5 minutes. The cells were loosened by sucking the supernatant and gently tapping to leave about 3-5 ml of medium.
  • a medium (MHM + B27 + FGF + LIF + Y27632) was added to the cells, and the cells were cultured in a T75 flask.
  • Cell passage was performed as follows. The Sphere was collected in a 50 ml tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was blotted out, 1.5 ml of Tryple select was added to the cell pellet and incubated in a 37.0 ° C. water bath for 10 minutes. The tube was removed from the water bath and pipetted with a P1000 pipette until there was no white haze. The cell suspension was added to a 15 ml tube containing 3 ml of TI, and the volume was increased to about 10 ml with MHM and passed through a 70 ⁇ m filter. The passed cell suspension was centrifuged at 1000 rpm for 5 minutes.
  • the supernatant was blotted out and loosened by lightly tapping the cell pellet, 3-5 ml of MHM was added, and the number of cells was counted.
  • the cell suspension was diluted with MHM to 50 cells / ⁇ l, dispensed into a 45 ml / T75 flask, and reagents (table 1 above) were added.
  • the cells were cultured for 2 weeks in a hypoxic (4% O 2 ) incubator. One week after the start of culture, medium change was performed in the same manner as described above.
  • the neurospheres formed were suspended at a concentration of 3x10 5 cells / ml, seeded at 500 ⁇ l / well in a 48-well plate coated with PO / fibronectin, and cultured in MHM medium containing B27. Was confirmed (FIG. 9).
  • neural progenitor cells such as neural stem cells and motor neural progenitor cells can be efficiently induced to differentiate from pluripotent stem cells in a short period of time.
  • neural undifferentiated cells such as neural stem cells and motor neural progenitor cells from T cell-derived iPS cells and B lymphocyte-derived iPS cells that are difficult to differentiate.

Abstract

The present invention relates to a method for increasing the neural differentiation ability of a pluripotent stem cell, which comprises culturing the pluripotent stem cell in the presence of a TGF-β family inhibitor, a GSK3β inhibitor and a BMP inhibitor. The present invention also relates to a method for inducing the differentiation of a pluripotent stem cell into a undifferentiated neural cell, which comprises culturing the pluripotent stem cell in the presence of a TGF-β family inhibitor and a GSK3β inhibitor under an oxygen partial pressure of 2 to 10%.

Description

神経系分化に適したiPS細胞の増幅方法、及び神経幹細胞の誘導方法Method for amplifying iPS cells suitable for neural differentiation and method for inducing neural stem cells
　本発明は、神経系分化に適したiPS細胞の増幅方法、及び神経幹細胞の誘導方法等に関する。
＜発明の背景＞ The present invention relates to a method for amplifying iPS cells suitable for neural differentiation, a method for inducing neural stem cells, and the like.
<Background of the invention>
　ヒトES細胞やiPS細胞からの神経幹細胞の誘導は神経の再生医療を行う上で欠くことのできない技術として近年盛んに研究されている。また移植用の用途としてだけでなく病態モデルの解析系としても応用が期待されており、より効率的でより速やかな神経幹細胞の分化誘導法の開発が期待されている。 Induction of neural stem cells from human ES cells and iPS cells has been actively studied in recent years as an indispensable technique for performing neuroregenerative medicine. In addition, it is expected to be applied not only for transplantation but also as a pathological model analysis system, and is expected to develop a more efficient and quicker method for inducing differentiation of neural stem cells.
　ヒトES細胞やiPS細胞から神経幹細胞を誘導する方法としては、接着培養系と浮遊培養系の2種類が存在する。接着培養系としては、例えば、ヒトES細胞を、SMADシグナルの２つの阻害剤、Noggin及びSB431542の存在下で接着培養することにより、80％を超えるヒトES細胞を神経系に転換する方法が知られている（非特許文献１）。また、GSK3、TGF-β及びNotchシグナル経路を阻害することにより、１週間以内に、単層の培養したヒトES細胞を、均一な初期神経上皮に転換し得ること、この初期神経上皮がLIF、GSK3β阻害剤（CHIR99021）およびTGF-βファミリー阻害剤（SB431542）の存在下で、高い神経分化能を維持しながら自己複製可能であることが報告されている（非特許文献２、特許文献１）。 There are two methods for inducing neural stem cells from human ES cells and iPS cells: an adhesion culture system and a suspension culture system. As an adhesion culture system, for example, a method is known in which human ES cells are cultured in the presence of two inhibitors of SMAD signals, Noggin and SB431542, thereby converting more than 80% of human ES cells into the nervous system. (Non-Patent Document 1). In addition, by inhibiting GSK3, TGF-β and Notch signaling pathways, it is possible to convert monolayer cultured human ES cells into uniform initial neuroepithelium within one week. In the presence of a GSK3β inhibitor (CHIR99021) and a TGF-β family inhibitor (SB431542), it has been reported that self-replication is possible while maintaining high neuronal differentiation potential (Non-patent Document 2, Patent Document 1). .
　本発明者らは、胚性幹細胞をノギン蛋白質の存在下又は非存在下で浮遊培養して胚様体を形成させ、これを繊維芽細胞増殖因子及びソニックヘッジホッグ蛋白質の存在下で浮遊培養して神経幹細胞に誘導する方法を開発している（特許文献２）。また、低酸素（3% O2）条件下でヒトES細胞を浮遊培養することにより、神経前駆細胞を誘導し得ること、当該神経前駆細胞をレチノイン酸及びプルモルファミンの存在下で培養することにより脊髄運動神経を誘導し得ることが報告されている（非特許文献３）。 The present inventors suspension-cultured embryonic stem cells in the presence or absence of noggin protein to form embryoid bodies, and suspended-cultured them in the presence of fibroblast growth factor and sonic hedgehog protein. A method of inducing into neural stem cells has been developed (Patent Document 2). Also, it is possible to induce neural progenitor cells by suspension culture of human ES cells under hypoxic (3% O 2 ) conditions, and culturing the neural progenitor cells in the presence of retinoic acid and purmorphamine. It has been reported that spinal motor nerves can be induced by (Non-patent Document 3).
　しかしながら、接着培養系は、比較的短期間（１週間程度）で神経幹細胞を誘導することができるが、細胞を大量に培養することには向いていない。一方、浮遊培養系によれば、大量の神経幹細胞を得るには適しているが、神経幹細胞の誘導に２週間以上もの長期間を要する。 However, the adhesion culture system can induce neural stem cells in a relatively short period (about 1 week), but is not suitable for culturing cells in large quantities. On the other hand, the floating culture system is suitable for obtaining a large amount of neural stem cells, but it takes a long period of two weeks or more to induce neural stem cells.
WO2012/034101WO2012 / 034101 特開2002-291469号公報JP 2002-291469 A
　本発明の目的は、iPS細胞等の多能性幹細胞から神経幹細胞等の神経系未分化細胞を、比較的短期間で且つ大量に、誘導する技術を確立することである。 An object of the present invention is to establish a technique for inducing a large amount of undifferentiated cells such as neural stem cells from pluripotent stem cells such as iPS cells in a relatively short period of time.
　上記課題を解決するべく、鋭意検討したところ、iPS細胞を、TGF-βファミリー阻害剤（SB431542）、GSK3β阻害剤（CHIR99021）及びBMP阻害剤（Dorsomorphin）の存在下で継代培養することにより、多能性を維持しつつ、iPS細胞の神経分化能を向上させることに成功した。さらに、こうして得られたiPS細胞を、TGF-βファミリー阻害剤（SB431542）、GSK3β阻害剤（CHIR99021）、ROCK阻害剤（Y27632）、bFGF、LIF及びB27を含む培地中で低酸素条件下、浮遊培養することにより、神経幹細胞を高速（３日）かつ効率的に大量培養することに成功した。特に、神経分化しにくいT細胞由来iPS細胞から、効率的に神経幹細胞を誘導することに成功した。これらの知見に基づき、更に検討を重ねた結果、本発明を完成した。 As a result of intensive studies to solve the above problems, iPS cells were subcultured in the presence of a TGF-β family inhibitor (SB431542), a GSK3β inhibitor (CHIR99021) and a BMP inhibitor (Dorsomorphin), While maintaining pluripotency, we succeeded in improving the neuronal differentiation ability of iPS cells. Furthermore, the iPS cells thus obtained were suspended in a medium containing TGF-β family inhibitor (SB431542), GSK3β inhibitor (CHIR99021), ROCK inhibitor (Y27632), bFGF, LIF and B27 under hypoxic conditions. By culturing, neural stem cells were successfully mass-cultured at high speed (3 days) and efficiently. In particular, we succeeded in efficiently inducing neural stem cells from iPS cells derived from T cells that are difficult to differentiate. As a result of further studies based on these findings, the present invention was completed.
　即ち、本発明は以下に関する。
［１］TGF-βファミリー阻害剤、GSK3β阻害剤及びBMP阻害剤の存在下で、多能性幹細胞を培養することを含む、多能性幹細胞の神経分化能の亢進方法。
［２］LIFの不在下で、多能性幹細胞を培養する、［１］記載の方法。
［３］多能性幹細胞が人工多能性幹細胞である、［１］又は［２］記載の方法。
［４］人工多能性幹細胞がT細胞又はB細胞由来である、［３］記載の方法。
［５］TGF-βファミリー阻害剤が４－［４－（１，３－ベンゾジオキソール－５－イル）－５－（２－ピリジニル）－１Ｈ－イミダゾール－２－イル］－ベンズアミド又はその水和物である、［１］～［４］のいずれか記載の方法。
［６］GSK3β阻害剤が６－［［２－［［４－（２，４－ジクロロフェニル）－５－（４－メチル－１Ｈ－イミダゾール－２－イル）－２－ピリミジニル］アミノ］エチル］アミノ］ニコチノニトリルである、［１］～［５］のいずれか記載の方法。
［７］BMP阻害剤が６－［４－（２－ピペリジン－１－イルエトキシ）フェニル］－３－ピリジン－４－イルピラゾロ［１，５－ａ］ピリミジンである、［１］～［６］のいずれか記載の方法。
［８］多能性幹細胞を、TGF-βファミリー阻害剤及びGSK3β阻害剤の存在下で、2～10％の酸素分圧下、培養することを含む、神経幹細胞の製造方法。
［９］多能性幹細胞が人工多能性幹細胞である、［８］記載の方法。
［１０］人工多能性幹細胞がT細胞又はB細胞由来である、［９］記載の方法。
［１１］TGF-βファミリー阻害剤が４－［４－（１，３－ベンゾジオキソール－５－イル）－５－（２－ピリジニル）－１Ｈ－イミダゾール－２－イル］－ベンズアミド又はその水和物である、［８］～［１０］のいずれか記載の方法。
［１２］GSK3β阻害剤が６－［［２－［［４－（２，４－ジクロロフェニル）－５－（４－メチル－１Ｈ－イミダゾール－２－イル）－２－ピリミジニル］アミノ］エチル］アミノ］ニコチノニトリルである、［８］～［１１］のいずれか記載の方法。
［１３］以下の工程を含む、神経幹細胞の製造方法：
（１）TGF-βファミリー阻害剤、GSK3β阻害剤及びBMP阻害剤の存在下で、多能性幹細胞を培養することにより、当該多能性幹細胞の神経分化能を亢進すること、及び
（２）工程（１）で得られた、神経分化能が亢進した多能性幹細胞を、TGF-βファミリー阻害剤及びGSK3β阻害剤の存在下で、2～10％の酸素分圧下、培養すること。
［１４］以下の工程を含む、運動神経前駆細胞の製造方法：
（１）多能性幹細胞を、TGF-βファミリー阻害剤及びGSK3β阻害剤の存在下で、2～10％の酸素分圧下、培養することにより、神経幹細胞を得ること、及び
（２）工程（１）で得られた神経幹細胞を、レチノイン酸、９－シクロヘキシル－Ｎ－［４－（４－モルホリニル）フェニル］－２－（１－ナフタレニルオキシ）－９Ｈ－プリン－６－アミン、TGF-βファミリー阻害剤及びGSK3β阻害剤の存在下で、2～10％の酸素分圧下、培養することにより、運動神経前駆細胞を分化誘導すること。
［１５］以下の工程を含む、ドパミン産生神経前駆細胞の製造方法：
（１）多能性幹細胞を、TGF-βファミリー阻害剤及びGSK3β阻害剤の存在下で、2～10％の酸素分圧下、培養することにより、神経幹細胞を得ること、及び
（２）工程（１）で得られた神経幹細胞を、TGF-βファミリー阻害剤、GSK3β阻害剤、ソニックヘッジホッグ、９－シクロヘキシル－Ｎ－［４－（４－モルホリニル）フェニル］－２－（１－ナフタレニルオキシ）－９Ｈ－プリン－６－アミン及びFGF8の存在下で、2～10％の酸素分圧下、培養することにより、ドパミン産生神経前駆細胞を分化誘導すること。 That is, the present invention relates to the following.
[1] A method for enhancing the neuronal differentiation ability of pluripotent stem cells, comprising culturing pluripotent stem cells in the presence of a TGF-β family inhibitor, a GSK3β inhibitor, and a BMP inhibitor.
[2] The method according to [1], wherein pluripotent stem cells are cultured in the absence of LIF.
[3] The method according to [1] or [2], wherein the pluripotent stem cell is an induced pluripotent stem cell.
[4] The method according to [3], wherein the induced pluripotent stem cells are derived from T cells or B cells.
[5] The TGF-β family inhibitor is 4- [4- (1,3-benzodioxol-5-yl) -5- (2-pyridinyl) -1H-imidazol-2-yl] -benzamide or its The method according to any one of [1] to [4], which is a hydrate.
[6] The GSK3β inhibitor is 6-[[2-[[4- (2,4-dichlorophenyl) -5- (4-methyl-1H-imidazol-2-yl) -2-pyrimidinyl] amino] ethyl] amino The method according to any one of [1] to [5], which is nicotinonitrile.
[7] The [1] to [6], wherein the BMP inhibitor is 6- [4- (2-piperidin-1-ylethoxy) phenyl] -3-pyridin-4-ylpyrazolo [1,5-a] pyrimidine Any one of the methods.
[8] A method for producing neural stem cells, comprising culturing pluripotent stem cells in the presence of a TGF-β family inhibitor and a GSK3β inhibitor under an oxygen partial pressure of 2 to 10%.
[9] The method according to [8], wherein the pluripotent stem cell is an induced pluripotent stem cell.
[10] The method according to [9], wherein the induced pluripotent stem cells are derived from T cells or B cells.
[11] The TGF-β family inhibitor is 4- [4- (1,3-benzodioxol-5-yl) -5- (2-pyridinyl) -1H-imidazol-2-yl] -benzamide or its The method according to any one of [8] to [10], which is a hydrate.
[12] GSK3β inhibitor is 6-[[2-[[4- (2,4-dichlorophenyl) -5- (4-methyl-1H-imidazol-2-yl) -2-pyrimidinyl] amino] ethyl] amino The method according to any one of [8] to [11], which is nicotinonitrile.
[13] A method for producing neural stem cells, comprising the following steps:
(1) culturing pluripotent stem cells in the presence of a TGF-β family inhibitor, a GSK3β inhibitor, and a BMP inhibitor to enhance the neuronal differentiation ability of the pluripotent stem cells; and (2) Culturing the pluripotent stem cells with enhanced neuronal differentiation ability obtained in step (1) in the presence of a TGF-β family inhibitor and a GSK3β inhibitor under 2 to 10% oxygen partial pressure.
[14] A method for producing motor neural progenitor cells, comprising the following steps:
(1) obtaining neural stem cells by culturing pluripotent stem cells in the presence of a TGF-β family inhibitor and a GSK3β inhibitor under 2 to 10% oxygen partial pressure, and (2) step ( The neural stem cells obtained in 1) were mixed with retinoic acid, 9-cyclohexyl-N- [4- (4-morpholinyl) phenyl] -2- (1-naphthalenyloxy) -9H-purin-6-amine, TGF -Inducing differentiation of motor progenitor cells by culturing in the presence of β family inhibitor and GSK3β inhibitor under 2-10% oxygen partial pressure.
[15] A method for producing dopaminergic neural progenitor cells, comprising the following steps:
(1) obtaining neural stem cells by culturing pluripotent stem cells in the presence of a TGF-β family inhibitor and a GSK3β inhibitor under 2 to 10% oxygen partial pressure, and (2) step ( The neural stem cells obtained in 1) were transformed into TGF-β family inhibitors, GSK3β inhibitors, Sonic hedgehog, 9-cyclohexyl-N- [4- (4-morpholinyl) phenyl] -2- (1-naphthalenyl) Differentiation induction of dopaminergic neural progenitor cells by culturing in the presence of 2-10% oxygen partial pressure in the presence of (oxy) -9H-purin-6-amine and FGF8.
　本発明によれば、多能性幹細胞から、神経幹細胞や運動神経前駆細胞等の神経系未分化細胞を短期間で、効率的に分化誘導することができる。特に、神経分化しにくいT細胞由来iPS細胞やBリンパ球由来iPS細胞から、効率的に神経幹細胞や運動神経前駆細胞等の神経系未分化細胞を分化誘導することができる。 According to the present invention, neural undifferentiated cells such as neural stem cells and motor neural progenitor cells can be efficiently induced to differentiate from pluripotent stem cells in a short period of time. In particular, it is possible to efficiently induce differentiation of neural undifferentiated cells such as neural stem cells and motor neural progenitor cells from T cell-derived iPS cells and B lymphocyte-derived iPS cells that are difficult to differentiate.
3D-iPS細胞コロニーの写真。Photograph of 3D-iPS cell colony. ヒトiPS細胞から誘導されたニューロスフェア（左）及びそれから分化した神経細胞（右）の写真。緑：Tuj1(神経細胞)、赤：GFAP、青：Hoechist。Photo of neurospheres (left) derived from human iPS cells and differentiated neurons (right). Green: Tuj1 (nerve cell), Red: GFAP, Blue: Hoechist. CHIR99021及びSB431542を神経幹細胞誘導２日目添加した場合と４日目に添加した場合の、神経幹細胞からの分化誘導の比較。緑：Tuj1(神経細胞)、赤：GFAP、青：Hoechist。Comparison of differentiation induction from neural stem cells when CHIR99021 and SB431542 are added on the second day of neural stem cell induction and on the fourth day. Green: Tuj1 (nerve cell), Red: GFAP, Blue: Hoechist. 低酸素神経幹細胞培養による神経分化への影響。緑：Tuj1(神経細胞)、赤：GFAP、青：Hoechist。Effect of hypoxic neural stem cell culture on neuronal differentiation. Green: Tuj1 (nerve cell), Red: GFAP, Blue: Hoechist. iPS細胞及び3D-iPS細胞から誘導された、培養開始３日後及び４日後のニューロスフェアから誘導された神経細胞。緑：Tuj1(神経細胞)、赤：GFAP、青：Hoechist。Neuronal cells derived from neurospheres derived from iPS cells and 3D-iPS cells 3 days and 4 days after the start of culture. Green: Tuj1 (nerve cell), Red: GFAP, Blue: Hoechist. 運動神経細胞の誘導。HB9、Isl１及びTuJ-1三重陽性細胞が運動神経細胞である。Induction of motor neurons. HB9, Isl1 and TuJ-1 triple positive cells are motor neurons. iPS細胞及び3D-iPS細胞におけるマーカー遺伝子の発現。Expression of marker genes in iPS cells and 3D-iPS cells. ヒトT細胞由来iPS細胞から誘導されたドパミン産生ニューロンの写真。A photograph of dopaminergic neurons derived from human T cell-derived iPS cells. ヒトB細胞由来iPS細胞から誘導されたニューロンの写真。A photograph of neurons derived from human B cell-derived iPS cells.
１．多能性幹細胞の神経分化能の亢進方法（方法１）
　本発明は、TGF-βファミリー阻害剤、GSK3β阻害剤及びBMP阻害剤の存在下で、多能性幹細胞を培養することを含む、多能性幹細胞の神経分化能の亢進方法を提供するものである。 1. Method for enhancing neural differentiation ability of pluripotent stem cells (Method 1)
The present invention provides a method for enhancing the neuronal differentiation ability of pluripotent stem cells, comprising culturing pluripotent stem cells in the presence of a TGF-β family inhibitor, a GSK3β inhibitor, and a BMP inhibitor. is there.
＜多能性幹細胞＞
　本発明で使用可能な多能性幹細胞は、生体に存在するすべての細胞に分化可能である多能性を有し、かつ、増殖能を併せもつ幹細胞であり、それらには、以下のものに限定されないが、例えばヒトやマウス等の哺乳動物の胚性幹(ES)細胞、核移植により得られるクローン胚由来の胚性幹（ntES）細胞、***幹細胞（GS細胞）、胚性生殖細胞（EG細胞）、人工多能性幹細胞（iPS細胞）などが含まれる。好ましい多能性幹細胞は、ES細胞、ntES細胞、及びiPS細胞であり、最も好ましくはiPS細胞である。 <Pluripotent stem cells>
The pluripotent stem cells that can be used in the present invention are stem cells that have pluripotency that can be differentiated into all the cells present in the living body and also have proliferative ability. Although not limited, for example, embryonic stem (ES) cells of mammals such as humans and mice, embryonic stem (ntES) cells derived from nuclear embryos, sperm stem cells (GS cells), embryonic germ cells ( EG cells), induced pluripotent stem cells (iPS cells) and the like. Preferred pluripotent stem cells are ES cells, ntES cells, and iPS cells, most preferably iPS cells.
（A）胚性幹細胞
　ES細胞は、ヒトやマウス等の哺乳動物の初期胚（例えば胚盤胞）の内部細胞塊から樹立された、多能性と自己複製による増殖能を有する幹細胞である。 (A) Embryonic stem cells ES cells are stem cells established from the inner cell mass of early embryos (for example, blastocysts) of mammals such as humans and mice, and having the pluripotency and the ability to grow by self-replication.
　ES細胞は、受精卵の8細胞期、桑実胚後の胚である胚盤胞の内部細胞塊に由来する胚由来の幹細胞であり、成体を構成するあらゆる細胞に分化する能力、いわゆる分化多能性と、自己複製による増殖能とを有している。ES細胞は、マウスで1981年に発見され（M.J. Evans and M.H. Kaufman (1981), Nature 292:154-156）、その後、ヒト、サルなどの霊長類でもES細胞株が樹立された（J.A. Thomson et al. (1999), Science 282:1145-1147；J.A. Thomson et al. (1995), Proc. Natl. Acad. Sci. USA,92:7844-7848；J.A. Thomson et al. (1996), Biol. Reprod., 55:254-259；J.A. Thomson and V.S. Marshall (1998), Curr. Top. Dev. Biol., 38:133-165）。 ES cells are embryonic stem cells derived from the inner cell mass of the blastocyst, the embryo after the morula, in the 8-cell stage of a fertilized egg, and have the ability to differentiate into any cell that constitutes an adult, so-called differentiation. And ability to proliferate by self-replication. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292: 154-156), and then ES cell lines were also established in primates such as humans and monkeys (JA Thomson et al. (1999), Science 282: 1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92: 7844-7848; JA Thomson et al. (1996), Biol. Reprod 55: 254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol, 38: 133-165).
　ES細胞は、対象動物の受精卵の胚盤胞から内部細胞塊を取出し、内部細胞塊を線維芽細胞のフィーダー上で培養することによって樹立することができる。また、継代培養による細胞の維持は、白血病抑制因子（leukemia inhibitory factor (LIF)）、塩基性線維芽細胞成長因子（basic fibroblast growth factor (bFGF)）などの物質を添加した培地を用いて行うことができる。ヒト及びサルのES細胞の樹立と維持の方法については、例えば、H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345:926-932；M. Ueno et al. (2006), Proc. Natl. Acad. Sci. USA, 103:9554-9559；H. Suemori et al.（2001）, Dev. Dyn., 222:273-279；H.Kawasaki et al. (2002), Proc. Natl. Acad. Sci. USA, 99:1580-1585などに記載されている。 ES cells can be established by taking an inner cell mass from a blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder. Cell maintenance by subculture is performed using a medium supplemented with substances such as leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF). be able to. Methods for establishing and maintaining human and monkey ES cells are described in, for example, H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345: 926-932; M. Ueno et al. (2006 ), Proc. Natl. Acad. Sci. USA, 103: 9554-9559; H. Suemori et al. (2001), Dev. Dyn., 222: 273-279; H. Kawasaki et al. (2002), Proc Natl. Acad. Sci. USA, 99: 1580-1585.
　ES細胞作成のための培地として、例えば0.1mM 2-メルカプトエタノール、0.1mM 非必須アミノ酸、2mM L-グルタミン酸、20% KSR及び4ng/ml b-FGFを補充したDMEM/F-12培地を使用し、37℃、2% CO2/98%空気の湿潤雰囲気下でヒトES細胞を維持することができる（O. Fumitaka et al. (2008), Nat. Biotechnol., 26:215-224）。また、ES細胞は、3～4日おきに継代する必要があり、このとき、継代は、例えば1mM CaCl2及び20% KSRを含有するPBS中の0.25% トリプシン及び0.1mg/ml コラゲナーゼIVを用いて行うことができる。 As a medium for preparing ES cells, for example, a DMEM / F-12 medium supplemented with 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acid, 2 mM L-glutamic acid, 20% KSR and 4 ng / ml b-FGF is used. Human ES cells can be maintained in a humid atmosphere at 37 ° C., 2% CO 2 /98% air (O. Fumitaka et al. (2008), Nat. Biotechnol., 26: 215-224). ES cells also need to be passaged every 3-4 days, where passage is eg 0.25% trypsin and 0.1 mg / ml collagenase IV in PBS containing 1 mM CaCl 2 and 20% KSR. Can be used.
　ES細胞の選択は、一般に、アルカリホスファターゼ、Oct3/4、Nanogなどの遺伝子マーカーの発現を指標にしてReal-Time PCR法で行うことができる。特に、ヒトES細胞の選択では、OCT-3/4、NANOG、ECADなどの遺伝子マーカーの発現を指標とすることができる（E.Kroon et al. (2008), Nat. Biotechnol., 26:443-452）。 ES cells can be generally selected by Real-Time PCR method using the expression of gene markers such as alkaline phosphatase, Oct3 / 4, Nanog as an index. In particular, in the selection of human ES cells, the expression of gene markers such as OCT-3 / 4, NANOG, and ECAD can be used as an index (E. Kroon et al. (2008), Nat. Biotechnol., 26: 443). -452).
　例えばkhES-1、KhES-2、KhES-3、KhES-4、KhES-5などのヒトES細胞株は、京都大学再生医科学研究所（京都、日本）から入手可能である。 For example, human ES cell lines such as khES-1, KhES-2, KhES-3, KhES-4, and KhES-5 are available from the Institute of Regenerative Medicine, Kyoto University (Kyoto, Japan).
（B）***幹細胞
　***幹細胞は、精巣由来の多能性幹細胞であり、***形成のための起源となる細胞である。この細胞は、ES細胞と同様に、種々の系列の細胞に分化誘導可能であり、例えばマウス胚盤胞に移植するとキメラマウスを作出できるなどの性質をもつ（M. Kanatsu-Shinohara et al. (2003), Biol. Reprod. 69:612-616；K. Shinohara et al. (2004), Cell, 119:1001-1012）。神経膠細胞系由来神経栄養因子（glial cell line-derived neurotrophic factor (GDNF)）を含む培地で自己複製可能であるし、またES細胞と同様の培養条件下で継代を繰り返すことによって***幹細胞を得ることができる（竹林正則ら(2008),実験医学,26巻,5号（増刊）,41～46頁,羊土社（東京、日本））。 (B) Sperm stem cells Sperm stem cells are testis-derived pluripotent stem cells that are the origin of spermatogenesis. Like ES cells, these cells can be induced to differentiate into various types of cells, and have characteristics such as the ability to create chimeric mice when transplanted into mouse blastocysts (M. Kanatsu-Shinohara et al. ( 2003), Biol. Reprod. 69: 612-616; K. Shinohara et al. (2004), Cell, 119: 1001-1012). It can self-replicate in a medium containing glial cell line-derived neurotrophic factor (GDNF), and spermatogonial stem cells can be generated by repeated passage under the same culture conditions as ES cells. (Takebayashi Masanori et al. (2008), Experimental Medicine, Vol. 26, No. 5 (extra), 41-46, Yodosha (Tokyo, Japan)).
（C）胚性生殖細胞
　胚性生殖細胞は、胎生期の始原生殖細胞から樹立される、ES細胞と同様な多能性をもつ細胞であり、LIF、bFGF、幹細胞因子（stem cell factor）などの物質の存在下で始原生殖細胞を培養することによって樹立しうる（Y. Matsui et al. (1992), Cell, 70:841-847；J.L. Resnick et al. (1992), Nature, 359:550-551）。 (C) Embryonic germ cells Embryonic germ cells are cells that are established from embryonic primordial germ cells and have the same pluripotency as ES cells. LIF, bFGF, stem cell factor, etc. Can be established by culturing primordial germ cells in the presence of these substances (Y. Matsui et al. (1992), Cell, 70: 841-847; JL Resnick et al. (1992), Nature, 359: 550 -551).
（D）人工多能性幹細胞
　人工多能性幹細胞（iPS細胞）とは、生殖系列にある細胞（例えば、卵細胞、***細胞、並びに卵原細胞および精原細胞等それらの前駆細胞）または発生初期胚由来の未分化細胞（例えば、胚性幹細胞）以外の分化細胞を初期化することにより、人工的に誘導された多分化能および自己増殖能を有する細胞のことである。分化細胞は、胚由来であっても胎児由来であっても成体由来であってもよく、また、マウス、ヒト等どのような動物種に由来しても構わない。分化細胞の性状としては、本来、受精細胞が有する全分化能を一部でも失った細胞であれば特に限定されず、例えば、(1) 神経幹細胞、造血幹細胞、間葉系幹細胞、歯髄幹細胞等の組織幹細胞（体性幹細胞）、(2) 組織前駆細胞、(3) 線維芽細胞（皮膚細胞等）、上皮細胞、肝細胞、リンパ球（T細胞、B細胞）、内皮細胞、筋肉細胞、毛細胞、胃粘膜細胞、腸細胞、脾細胞、膵細胞（膵外分泌細胞等）、脳細胞、肺細胞、腎細胞、皮膚細胞等の分化した細胞などが例示できる。 (D) Artificial pluripotent stem cells Artificial pluripotent stem cells (iPS cells) are germline cells (eg, egg cells, sperm cells, and progenitor cells such as oocyte and spermatogonia) or early development. It is a cell that has artificially induced multipotency and self-proliferation ability by reprogramming differentiated cells other than embryo-derived undifferentiated cells (eg, embryonic stem cells). Differentiated cells may be derived from embryos, fetuses or adults, and may be derived from any animal species such as mice and humans. The nature of the differentiated cell is not particularly limited as long as it is a cell that originally lost a part of the total differentiation ability of a fertilized cell.For example, (1) neural stem cells, hematopoietic stem cells, mesenchymal stem cells, dental pulp stem cells, etc. Tissue stem cells (somatic stem cells), (2) tissue precursor cells, (3) fibroblasts (skin cells, etc.), epithelial cells, hepatocytes, lymphocytes (T cells, B cells), endothelial cells, muscle cells, Examples include hair cells, gastric mucosa cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), differentiated cells such as brain cells, lung cells, kidney cells, skin cells, and the like.
　初期化方法は特に限定されないが、好ましい方法においては、細胞を、核初期化因子を導入することにより、多分化能および自己増殖能を有するように誘導する。例えば国際公開WO2005/080598、WO2007/069666、WO2008/118820およびWO2009/057831に記載された初期化方法のいずれかを用いてもよい。これらの刊行物の開示内容は、参照により、本明細書に組み込まれる。 Although the reprogramming method is not particularly limited, in a preferred method, cells are induced to have pluripotency and self-proliferation ability by introducing a nuclear reprogramming factor. For example, any of the initialization methods described in International Publications WO2005 / 080598, WO2007 / 069666, WO2008 / 118820, and WO2009 / 057831 may be used. The disclosures of these publications are incorporated herein by reference.
　核初期化因子は、特に限定されないが、Oct遺伝子群、Klf遺伝子群、Sox遺伝子群、Myc遺伝子群、Sall 遺伝子群、Nanog遺伝子群（マウスNM_028016、ヒトNM_024865）およびLin遺伝子群のそれぞれから選択された群の中の遺伝子の少なくとも１つの遺伝子産物であることが好ましい。Oct遺伝子群に属する遺伝子としては、Oct3/4（マウスNM_013633、ヒトNM_002701）、Oct1A（マウスNM_198934、ヒトNM_002697）およびOct6（マウスNM_011141、ヒトNM_002699）が挙げられ、Klf遺伝子群に属する遺伝子としては、Klf1（マウスNM_010635、ヒトNM_006563）、Klf2（マウスNM_008452、ヒトNM_016270）、Klf4（マウスNM_010637、ヒトNM_004235）およびKlf5（マウスNM_009769、ヒトNM_001730）が挙げられ、Sox遺伝子群に属する遺伝子としては、Sox1（マウスNM_009233、ヒトNM_005986）、Sox2（マウスNM_011443、ヒトNM_003106）、Sox3（マウスNM_009237、ヒトNM_005634）、Sox7（マウスNM_011446、ヒトNM_031439）、Sox15（マウスNM_009235、ヒトNM_006942）、Sox17（マウスNM_011441、ヒトNM_022454）およびSox18（マウスNM_009236、ヒトNM_018419）が挙げられ、Myc遺伝子群に属する遺伝子としては、c-Myc（マウスNM_010849、ヒトNM_002467）、N-Myc（マウスNM_008709、ヒトNM_005378）およびL-Myc（マウスNM_008506、ヒトNM_001033081）が挙げられ、Sall 遺伝子群に属する遺伝子としては、Sall1（マウスNM_021390、ヒトNM_002968）およびSall4（マウスNM_175303、ヒトNM_020436）が挙げられ、Lin遺伝子群に属する遺伝子としては、Lin28（マウスNM_145833、ヒトNM_024674）およびLin28b（マウスNM_001031772、ヒトNM_001004317）が挙げられる。核初期化因子としては、他の種類の遺伝子産物を使用してもよく、例えば、不死化誘導因子などが挙げられる。 The nuclear reprogramming factor is not particularly limited, and is selected from the Oct gene group, the Klf gene group, the Sox gene group, the Myc gene group, the Sall 群 gene group, the Nanog gene group (mouse NM_028016, human NM_024865), and the Lin gene group. Preferably, it is at least one gene product of a gene in the group. Examples of genes belonging to the Oct gene group include Oct3 / 4 (mouse NM_013633, human NM_002701), Oct1A (mouse NM_198934, human NM_002697) and Oct6 (mouse NM_011141, human NM_002699), and genes belonging to the Klf gene group include Examples include Klf1 (mouse NM_010635, human NM_006563), Klf2 (mouse NM_008452, human NM_016270), Klf4 (mouse NM_010637, human NM_004235) and Klf5 (mouse NM_009769, human NM_001730). The genes belonging to the Sox gene group include Sox1 ( Mouse NM_009233, human NM_005986), Sox2 (mouse NM_011443, human NM_003106), Sox3 (mouse NM_009237, human NM_005634), Sox7 (mouse NM_011446, human NM_031439), Sox15 (mouse NM_009235, human NM_006942), Sox17 (mouse NM_02144454, human NM_021444) ) And Sox18 (mouse NM_009236, human NM_018419), and the genes belonging to the Myc gene group include c-Myc (mouse NM_010849, human NM_002). 467), N-Myc (mouse NM_008709, human NM_005378) and L-Myc (mouse NM_008506, human NM_001033081). The genes belonging to the SallS gene group include Sall1 (mouse NM_021390, human NM_002968) and Sall4 (mouse NM_175303). , Human NM_020436), and the genes belonging to the Lin gene group include Lin28 (mouse NM_145833, human NM_024674) and Lin28b (mouse NM_001031772, human NM_001004317). As the nuclear reprogramming factor, other types of gene products may be used, and examples thereof include an immortalization inducing factor.
　より好ましくは、核初期化因子としては、Oct3/4遺伝子、Klf4遺伝子、Sox2遺伝子、c-Myc遺伝子、L-Myc遺伝子、Sall4遺伝子、Sall1遺伝子、Nanog遺伝子およびLin28遺伝子から選択される１つ以上の遺伝子の少なくとも１つの産物が挙げられ得る。 More preferably, the nuclear reprogramming factor is one or more selected from Oct3 / 4 gene, Klf4 gene, Sox2 gene, c-Myc gene, L-Myc gene, Sall4 gene, Sall1 gene, Nanog gene and Lin28 gene. And at least one product of the gene.
　これらの遺伝子は、本明細書中、National Center for Biotechnology Information（NCBI）に登録されたアクセション番号を参照してマウスおよびヒトの配列で表したが、いずれも、脊椎動物で高度に保存されている遺伝子であり、従って本明細書では、特に動物名を示さない限り、ホモログを含めた遺伝子を表すものとする。また、polymorphismを含め、変異を有する遺伝子も、野生型の遺伝子産物と同等の機能を有する限り包含される。 These genes are expressed in mouse and human sequences with reference to the accession numbers registered in the National Center for Biotechnology Information (NCBI), both of which are highly conserved in vertebrates. Therefore, in this specification, unless the name of an animal is indicated, the gene including a homolog is expressed. In addition, genes having mutations including polymorphism are also included as long as they have a function equivalent to that of a wild-type gene product.
　核初期化因子を用いてiPS細胞を調製するには、上記核初期化因子を体細胞へ導入することが好ましい。含有する核初期化因子の数は、２個、３個、好ましくは４個、もしくは４個以上である。それらの因子の好ましい組み合わせは、Oct3/4遺伝子、Sox2遺伝子およびKlf4遺伝子の組み合わせ、もしくは、Oct3/4遺伝子、Sox2遺伝子、Klf4遺伝子およびc-Myc遺伝子の組み合わせのいずれかである。 In order to prepare iPS cells using a nuclear reprogramming factor, it is preferable to introduce the nuclear reprogramming factor into somatic cells. The number of nuclear reprogramming factors to be included is 2, 3, preferably 4, or 4 or more. A preferred combination of these factors is either a combination of Oct3 / 4 gene, Sox2 gene and Klf4 gene, or a combination of Oct3 / 4 gene, Sox2 gene, Klf4 gene and c-Myc gene.
　核初期化因子が細胞内で機能するタンパク質である場合は、核初期化因子を導入するために、そのタンパク質をコードする遺伝子を発現ベクターに組み込み、対象とする体細胞などの分化細胞に発現ベクターを導入し、そのタンパク質を細胞内で発現させることが好ましい（遺伝子導入法）。使用する発現ベクターは特に限定されないが、好ましくはプラスミドベクター、ウイルスベクターおよび人工染色体ベクター（Suzuki N et al., J Biol Chem. 281(36):26615, 2006）が例示され、ウイルスベクターとして、好ましくはアデノウイルスベクター、センダイウィルスベクター、レトロウイルスベクターおよびレンチウイルスベクターが例示される。あるいは、Protein Transduction Domain（PTD）と呼ばれるペプチドをタンパク質に結合させ、培地に添加することにより、そのタンパク質を細胞内に導入してもよい（Protein Transduction 法）。また、例えば、精製したタンパク質を導入するために、各種のタンパク質導入試薬（例えば、ＣｈａｒｉｏｔＴＭ、ＢｉｏｐｏｒｔｅｒＴＭ等）のいずれかを用いてそのタンパク質を細胞内に導入することができる。核初期化因子が細胞外に分泌されるタンパク質である場合は、iPS細胞の調製段階で、分化細胞の培地にその因子を添加すればよい。なお、初期化すべき分化細胞で、核初期化因子のいずれかが発現している場合は、その因子に関しては外部から導入する必要が無い。 If the nuclear reprogramming factor is a protein that functions in a cell, in order to introduce the nuclear reprogramming factor, the gene encoding the protein is incorporated into an expression vector, and the expression vector is used in a differentiated cell such as a target somatic cell. Is preferably introduced and the protein is expressed in cells (gene transfer method). The expression vector to be used is not particularly limited, but preferably, a plasmid vector, a viral vector, and an artificial chromosome vector (Suzuki N et al., J Biol Chem. 281 (36): 26615, 2006) are exemplified. Examples include adenovirus vectors, Sendai virus vectors, retrovirus vectors, and lentivirus vectors. Alternatively, a protein called Protein Transduction Domain (PTD) may be bound to a protein and added to the medium to introduce the protein into the cell (Protein Transduction method). Further, for example, in order to introduce a purified protein, the protein can be introduced into cells using any of various protein introduction reagents (for example, Chariot ™ , Bioporter ™, etc.). When the nuclear reprogramming factor is a protein secreted extracellularly, the factor may be added to the culture medium of differentiated cells at the stage of preparing iPS cells. When any of the nuclear reprogramming factors is expressed in the differentiated cells to be reprogrammed, it is not necessary to introduce the factors from the outside.
　場合によっては、核初期化因子を置換する目的もしくは誘導効率を上げる目的で、サイトカインまたは化合物を添加してもよい。サイトカインとしては、例えば、SCF（stem cell factor）、bFGF、WntファミリーおよびLIF（leukemia inhibitory factor）が挙げられ、化合物としては、例えば、Histone Deacetylase阻害剤、DNAメチル化阻害剤、MEK阻害剤、GSK3β阻害剤、TGF受容体阻害剤およびROCK阻害剤が挙げられる（WO2009/117439）。 In some cases, cytokines or compounds may be added for the purpose of replacing the nuclear reprogramming factor or increasing the induction efficiency. Cytokines include, for example, SCF (stem cell factor), bFGF, Wnt family, and LIF (leukemia protein factor), and compounds include, for example, Histone deacetylase inhibitor, DNA methylation inhibitor, MEK inhibitor, GSK3β And inhibitors, TGF receptor inhibitors and ROCK inhibitors (WO2009 / 117439).
　体細胞へ初期化因子を導入した後、フィーダー細胞上へ移して培養してもよい。フィーダー細胞は、特に限定されないが、マウス胚性線維芽細胞（MEF）が例示される。この培養に好適に用いられる培地としては、体細胞が由来する動物種の細胞を培養するのに適した培地が挙げられ、例えばヒト細胞の場合、20％代替血清、2mM L-グルタミン、1 x 10-4 M非必須アミノ酸、1 x 10-4 M 2-メルカプトエタノール、0.5% ペニシリンおよびストレプトマイシン、並びに4ng/ml組換えヒトbasic fibroblast growth factor（bFGF）を含むDMEM/F12培地であることが好ましい。 After introducing the reprogramming factor into somatic cells, it may be transferred onto feeder cells and cultured. Although a feeder cell is not specifically limited, A mouse embryonic fibroblast (MEF) is illustrated. Examples of the medium suitably used for this culture include a medium suitable for culturing cells of animal species from which somatic cells are derived. For example, in the case of human cells, 20% alternative serum, 2 mM L-glutamine, 1 × Preferred is DMEM / F12 medium containing 10 -4 M non-essential amino acids, 1 x 10 -4 M 2-mercaptoethanol, 0.5% penicillin and streptomycin, and 4 ng / ml recombinant human basic fibroblast growth factor (bFGF). .
　その後、核初期化因子を導入した分化細胞から、例えば未分化細胞特異的遺伝子を発現している細胞を選択すること、もしくは細胞の形態を指標にすることにより、iPS細胞を単離する。未分化細胞特異的遺伝子を発現している細胞を選択する方法は特に限定されない。未分化細胞特異的遺伝子が細胞内タンパク質をコードしている場合は、未分化細胞特異的遺伝子のプロモーターの下流にGFP遺伝子、ガラクトシダーゼ遺伝子、並びにネオマイシン耐性遺伝子、ハイグロマイシン耐性遺伝子およびピューロマイシン耐性遺伝子等の薬剤耐性遺伝子などのマーカー遺伝子の１つをノックインして融合タンパク質として発現させてもよく、それらマーカー遺伝子を発現している細胞を選択してもよい。マーカーが薬剤耐性遺伝子の場合は、薬剤で選択することにより容易に目的の細胞を単離することができる。未分化細胞特異的遺伝子が細胞膜タンパク質をコードしている場合は、特異的抗体を用いるか、またはそのタンパク質の酵素活性を用いて、そのタンパク質を発現している細胞を選択することができる。本明細書中で使用される未分化細胞特異的遺伝子とは、胚性幹細胞（ES細胞）に特異的に発現している当業者に周知の遺伝子を指し、例えば国際公開WO2005/080598、WO2007/069666、WO2008/118820、 WO2009/057831およびNat Biotechnol. 25, 803, 2007に開示されたES細胞に特異的に発現している遺伝子が例示される。未分化細胞特異的遺伝子は、好ましくは、Oct3/4、Sox2、Nanog、Lin28、Rex1、UTF1、Eras、Fgf4、TDGF、Cripto、Dax1、ESG1、GDF3、Sall4、Fbx15、SSEA-1、SSEA-4、TRA-1-60、TRA-1-81およびアルカリホスファターゼ（例えば、TRA-2-54およびTRA-2-49）からなる群より動物種を考慮して選択される（例えば、SSEA-1はマウスに特異的であり、SSEA-4、TRA-1-60およびTRA-1-81はヒトに特異的である）。好ましい未分化細胞特異的遺伝子は、Fbx15遺伝子またはNanog遺伝子である。細胞の形態を指標にする場合、選択は、例えば、コロニーの形成を指標として行なってもよい。 Thereafter, iPS cells are isolated by selecting, for example, cells expressing an undifferentiated cell-specific gene from differentiated cells into which a nuclear reprogramming factor has been introduced, or using cell morphology as an index. A method for selecting a cell expressing an undifferentiated cell-specific gene is not particularly limited. If the undifferentiated cell-specific gene encodes an intracellular protein, the GFP gene, galactosidase gene, neomycin-resistant gene, hygromycin-resistant gene, puromycin-resistant gene, etc. downstream of the promoter of the undifferentiated cell-specific gene One of the marker genes such as the drug resistance gene may be knocked in and expressed as a fusion protein, or cells expressing these marker genes may be selected. When the marker is a drug resistance gene, the target cell can be easily isolated by selecting with a drug. When the undifferentiated cell-specific gene encodes a cell membrane protein, a cell expressing the protein can be selected using a specific antibody or using the enzyme activity of the protein. As used herein, an undifferentiated cell-specific gene refers to a gene well known to those skilled in the art that is specifically expressed in embryonic stem cells (ES cells). For example, International Publication WO2005 / 080598, WO2007 / Examples are genes specifically expressed in ES cells disclosed in 069666, WO2008 / 118820, WO2009 / 057831 and Nat Biotechnol. 25, 803, 2007. The undifferentiated cell-specific gene is preferably Oct3 / 4, Sox2, Nanog, Lin28, Rex1, UTF1, Eras, Fgf4, TDGF, Cripto, Dax1, ESG1, GDF3, Sall4, Fbx15, SSEA-1, SSEA-4 Selected from the group consisting of TRA-1-60, TRA-1-81 and alkaline phosphatase (eg, TRA-2-54 and TRA-2-49) in consideration of animal species (eg, SSEA-1 Specific to mice, SSEA-4, TRA-1-60 and TRA-1-81 are specific to humans). A preferred undifferentiated cell-specific gene is the Fbx15 gene or Nanog gene. When cell morphology is used as an index, selection may be performed using colony formation as an index, for example.
　このようにして、初期化された細胞から単離された細胞集団または細胞株もしくはクローンをiPS細胞として用いることができる。 In this way, a cell population or cell line or clone isolated from cells that have been reprogrammed can be used as an iPS cell.
　本明細書において、細胞集団、細胞株およびクローンという語は、特に定めのない限り区別されない。 In the present specification, the terms cell population, cell line and clone are not distinguished unless otherwise specified.
（E）核移植により得られたクローン胚由来のES細胞
　ntES細胞は、核移植技術によって作製されたクローン胚由来のES細胞であり、受精卵由来のES細胞とほぼ同じ特性を有している（T. Wakayama et al. (2001), Science, 292:740-743；S. Wakayama et al. (2005), Biol. Reprod., 72:932-936；J. Byrne et al. (2007), Nature, 450:497-502）。即ち、未受精卵の核を体細胞の核と置換することによって得られたクローン胚由来の胚盤胞の内部細胞塊から樹立されたES細胞がntES（nuclear transfer ES）細胞である。ntES細胞の作製のためには、核移植技術（J.B. Cibelli et al. (1998), Nat. Biotechnol., 16:642-646）とES細胞作製技術（上記）との組み合わせが利用される（若山清香ら(2008), 実験医学, 26巻,5号（増刊）,47-52頁）。核移植においては、哺乳動物の除核した未受精卵に、体細胞の核を注入し、数時間培養することで再プログラム化することができる。 (E) Cloned embryo-derived ES cells obtained by nuclear transfer ntES cells are cloned embryo-derived ES cells produced by nuclear transfer technology and have almost the same characteristics as ES cells derived from fertilized eggs (T. Wakayama et al. (2001), Science, 292: 740-743; S. Wakayama et al. (2005), Biol. Reprod., 72: 932-936; J. Byrne et al. (2007), Nature, 450: 497-502). That is, an ES cell established from an inner cell mass of a blastocyst derived from a cloned embryo obtained by replacing the nucleus of an unfertilized egg with a nucleus of a somatic cell is a ntES (nuclear transfer ES) cell. For the production of ntES cells, a combination of nuclear transfer technology (JB Cibelli et al. (1998), Nat. Biotechnol., 16: 642-646) and ES cell production technology (above) is used (Wakayama). Seika et al. (2008), Experimental Medicine, Vol. 26, No. 5 (extra number), 47-52). In nuclear transfer, reprogramming can be performed by injecting a somatic cell nucleus into an enucleated unfertilized egg of a mammal and culturing it for several hours.
＜TGF-βファミリー阻害剤＞
　本明細書において、TGF-βファミリー阻害剤とは、TGF-βとTGF-β受容体との結合を介するTGF-βシグナル伝達の阻害に関与する阻害剤である。TGF-β阻害剤には、タンパク質性阻害剤及び低分子阻害剤が包含される。タンパク質性阻害剤としては、抗TGF-β中和抗体、抗TGF-β受容体中和抗体等を挙げることができる。低分子阻害剤としては、SB431542（４－［４－（１，３－ベンゾジオキソール－５－イル）－５－（２－ピリジニル）－１Ｈ－イミダゾール－２－イル］－ベンズアミド又はその水和物）、SB202190(以上、R.K. Lindemann et a1.， Mo1. Cancer 2:20 (2003))、SB505124(GlaxoSmithKline)、NPC30345、SD093、SD908、SD208(Scios)、LY2109761、LY364947、LY580276(Lilly Research Laboratories)などが包含され、SB431542（４－［４－（１，３－ベンゾジオキソール－５－イル）－５－（２－ピリジニル）－１Ｈ－イミダゾール－２－イル］－ベンズアミド又はその水和物）が好ましい。 <TGF-β family inhibitor>
In the present specification, a TGF-β family inhibitor is an inhibitor involved in the inhibition of TGF-β signaling through the binding between TGF-β and the TGF-β receptor. TGF-β inhibitors include proteinaceous inhibitors and small molecule inhibitors. Examples of proteinaceous inhibitors include anti-TGF-β neutralizing antibodies and anti-TGF-β receptor neutralizing antibodies. As a small molecule inhibitor, SB431542 (4- [4- (1,3-benzodioxol-5-yl) -5- (2-pyridinyl) -1H-imidazol-2-yl] -benzamide or water thereof SB202190 (above, RK Lindemann et a1., Mo1. Cancer 2:20 (2003)), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories) SB431542 (4- [4- (1,3-benzodioxol-5-yl) -5- (2-pyridinyl) -1H-imidazol-2-yl] -benzamide or its hydration Are preferred.
　TGF-βファミリーのメンバーは、有糸***、細胞分化、胚パターン形成および器官形成などの細胞過程および発生過程を調節する。例えば、TGF-β シグナル伝達は、I型及びII型受容体のセリンートレオニンキナーゼのヘテロメリックな受容体複合体を介して行われ、この複合体は下流のSmadシグナル伝達過程を活性化する。すなわち、受容体複合体にTGF-βが結合すると、TGFs 11型受容体がTGFβI型受容体をリン酸化し、このTGFβ1型受容体が、受容体調節されるSmad(R-Smad)をリン酸化することによって、下流応答が開始される。活性化R-SmadはSmad4と多量体複合体を形成することによって、活性化R-Smadは核に移動し、標的遺伝子の転写調節が誘導される。 TGF-β family members regulate cellular and developmental processes such as mitosis, cell differentiation, embryonic pattern formation and organogenesis. For example, TGF-β signaling occurs through the heteromeric receptor complex of type I and type II receptors serine threonine kinase, which activates downstream Smad signaling processes. That is, when TGF-β binds to the receptor complex, TGFs 11 type receptor phosphorylates TGFβ type I receptor, and this TGFβ type 1 receptor phosphorylates receptor-regulated Smad (R-Smad). To initiate a downstream response. When activated R-Smad forms a multimeric complex with Smad4, activated R-Smad moves to the nucleus, and transcriptional regulation of the target gene is induced.
　このような、TGF-βファミリーシグナル伝達が、BMPシグナル伝達及びGSK-3とともに阻害されると、多能性幹細胞は神経系未分化細胞へ誘導されやすい状態に保たれる。 When such TGF-β family signaling is inhibited together with BMP signaling and GSK-3, pluripotent stem cells are maintained in a state that is easily induced into neural undifferentiated cells.
＜GSK3β阻害剤＞
　グリコーゲン合成酵素キナーゼ（glycogen synthase kinase: GSK）は、グリコーゲンの合成を促す作用を有する酵素をリン酸化して、その活性を調節するものである。該グリコーゲン合成酵素キナーゼの中でも、GSK-3はすべての真核生物に見られる多機能性セリン／スレオニンキナーゼであって、Wnt、チロシンキナーゼ及びＧタンパク質共役受容体に対する細胞性の応答を含め多くのシグナル伝達経路の重要なレギュレーターであり、グリコーゲン代謝から細胞サイクルの調節及び増殖に至る広い範囲の細胞プロセスに関与している。本発明では、GSK阻害剤の中でも、GSK3β阻害剤が用いられる。GSK3β阻害剤としては、例えば、CHIR99021（６－［［２－［［４－（２，４－ジクロロフェニル）－５－（４－メチル－１Ｈ－イミダゾール－２－イル）－２－ピリミジニル］アミノ］エチル］アミノ］ニコチノニトリル）、SB-415286（３－［（３－クロロ－４－ヒドロキシフェニル）アミノ］－４－（２－ニトロフェニル）－１Ｈ－ピロール－２，５－ジオン）、SB-2167、indirubin-3’-Monoxime、Kenpaullone、BIO（６－ブロモインジルビン－３'－オキシム）等が挙げられるが、これらの中でも特にCHIR99021（６－［［２－［［４－（２，４－ジクロロフェニル）－５－（４－メチル－１Ｈ－イミダゾール－２－イル）－２－ピリミジニル］アミノ］エチル］アミノ］ニコチノニトリル）、Kenpaulloneが好ましい。 <GSK3β inhibitor>
Glycogen synthase kinase (GSK) regulates the activity of phosphorylating an enzyme having an action of promoting glycogen synthesis. Among the glycogen synthase kinases, GSK-3 is a multifunctional serine / threonine kinase found in all eukaryotes, including many cellular responses to Wnt, tyrosine kinases and G protein coupled receptors. It is an important regulator of signal transduction pathways and is involved in a wide range of cellular processes ranging from glycogen metabolism to cell cycle regulation and proliferation. In the present invention, among GSK inhibitors, GSK3β inhibitors are used. Examples of GSK3β inhibitors include CHIR99021 (6-[[2-[[4- (2,4-dichlorophenyl) -5- (4-methyl-1H-imidazol-2-yl) -2-pyrimidinyl] amino]). Ethyl] amino] nicotinonitrile), SB-415286 (3-[(3-chloro-4-hydroxyphenyl) amino] -4- (2-nitrophenyl) -1H-pyrrole-2,5-dione), SB -2167, indirubin-3'-Monoxime, Kenpaullone, BIO (6-bromoindirubin-3'-oxime) and the like. Among these, CHIR99021 (6-[[2-[[4- (2, 4-dichlorophenyl) -5- (4-methyl-1H-imidazol-2-yl) -2-pyrimidinyl] amino] ethyl] amino] nicotinonitrile), Kenpaullone are preferred.
＜BMP阻害剤＞
　本明細書において、BMP阻害剤とは、BMP (bone morphogenetic protein)とBMP受容体（I型又はII型）との結合を介するBMPシグナル伝達（BMP signaling）の阻害に関与する阻害剤である。BMP阻害剤には、タンパク質性阻害剤及び低分子阻害剤が包含される。タンパク質性阻害剤としては、天然の阻害剤であるNoggin、chordin、follistatin等を挙げることが出来る。低分子阻害剤としては、転写因子SMAD1、SMAD5又はSMAD8を活性化する能力をもつBMP2、BMP4、BMP6又はBMP7を阻害する化合物である、例えばDorsomorphin（６－［４－（２－ピペリジン－１－イルエトキシ）フェニル］－３－ピリジン－４－イルピラゾロ［１，５－ａ］ピリミジン）及びその誘導体が包含される（P.B. Yu et al. (2007), Circulation, 116:II_60；P.B. Yu et al. (2008), Nat. Chem. Biol., 4:33-41；J. Hao et al. (2008), PLoS ONE (www.plozone.org), 3(8):e2940）。Dorsomorphinは市販されており、例えばSigma-Aldrichなどから入手可能である。Dorsomorphinは、BMP受容体へのBMPの結合を阻害することによって上記のBMPシグナル伝達を阻害する生物活性を有する。この他にも、BMPI受容体キナーゼ阻害剤としてLDN-193189（4-(6-(4-piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl）quinoline）及びその誘導体が例示される（P.B. Yu et al. (2008), Nat. Med., 14:1363-1369）。LDN-193189は市販されており、例えばStemgent社などから入手可能である。 <BMP inhibitor>
In this specification, a BMP inhibitor is an inhibitor involved in the inhibition of BMP signaling (BMP signaling) through the binding between BMP (bone morphogenetic protein) and a BMP receptor (type I or type II). BMP inhibitors include proteinaceous inhibitors and small molecule inhibitors. Examples of proteinaceous inhibitors include natural inhibitors such as Noggin, chordin, and follistatin. The small molecule inhibitor is a compound that inhibits BMP2, BMP4, BMP6 or BMP7 having the ability to activate the transcription factor SMAD1, SMAD5 or SMAD8. For example, Dorsomorphin (6- [4- (2-piperidine-1- (Ilethoxy) phenyl] -3-pyridin-4-ylpyrazolo [1,5-a] pyrimidine) and derivatives thereof (PB Yu et al. (2007), Circulation, 116: II_60; PB Yu et al. ( 2008), Nat. Chem. Biol., 4: 33-41; J. Hao et al. (2008), PLoS ONE (www.plozone.org), 3 (8): e2940). Dorsomorphin is commercially available, for example from Sigma-Aldrich. Dorsomorphin has the biological activity of inhibiting the above BMP signaling by inhibiting the binding of BMP to the BMP receptor. In addition, LDN-193189 (4- (6- (4-piperazin-1-yl) phenyl) pyrazolo [1,5-a] pyrimidin-3-yl) quinoline) and its BMPI receptor kinase inhibitor Derivatives are exemplified (PB Yu et al. (2008), Nat. Med., 14: 1363-1369). LDN-193189 is commercially available, for example, available from Stemgent.
　上記GSK3β阻害剤およびBMP阻害剤として例示された各種化合物、および後述する９－シクロヘキシル－Ｎ－［４－（４－モルホリニル）フェニル］－２－（１－ナフタレニルオキシ）－９Ｈ－プリン－６－アミンは、形成可能である場合にはそれぞれ塩を形成していてもよい。
　そのような塩としては、例えば、無機塩基との塩、有機塩基との塩、無機酸との塩、有機酸との塩、塩基性または酸性アミノ酸との塩が挙げられる。
　無機塩基との塩の好適な例としては、ナトリウム塩、カリウム塩等のアルカリ金属塩；カルシウム塩、マグネシウム塩等のアルカリ土類金属塩；アルミニウム塩、アンモニウム塩が挙げられる。
　有機塩基との塩の好適な例としては、トリメチルアミン、トリエチルアミン、ピリジン、ピコリン、エタノールアミン、ジエタノールアミン、トリエタノールアミン、トロメタミン［トリス（ヒドロキシメチル）メチルアミン］、ｔｅｒｔ－ブチルアミン、シクロヘキシルアミン、ベンジルアミン、ジシクロヘキシルアミン、Ｎ，Ｎ－ジベンジルエチレンジアミンとの塩が挙げられる。
　無機酸との塩の好適な例としては、塩酸、臭化水素酸、硝酸、硫酸、リン酸との塩が挙げられる。
　有機酸との塩の好適な例としては、ギ酸、酢酸、トリフルオロ酢酸、フタル酸、フマル酸、シュウ酸、酒石酸、マレイン酸、クエン酸、コハク酸、リンゴ酸、メタンスルホン酸、ベンゼンスルホン酸、ｐ－トルエンスルホン酸との塩が挙げられる。
　塩基性アミノ酸との塩の好適な例としては、アルギニン、リジン、オルニチンとの塩が挙げられる。
　酸性アミノ酸との塩の好適な例としては、アスパラギン酸、グルタミン酸との塩が挙げられる。 Various compounds exemplified as the above GSK3β inhibitor and BMP inhibitor, and 9-cyclohexyl-N- [4- (4-morpholinyl) phenyl] -2- (1-naphthalenyloxy) -9H-purine- described later Each 6-amine may form a salt if it can be formed.
Examples of such salts include salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, and salts with basic or acidic amino acids.
Preferable examples of the salt with an inorganic base include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt; aluminum salt and ammonium salt.
Preferable examples of the salt with an organic base include trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, tromethamine [tris (hydroxymethyl) methylamine], tert-butylamine, cyclohexylamine, benzylamine, And salts with dicyclohexylamine and N, N-dibenzylethylenediamine.
Preferable examples of the salt with inorganic acid include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid and phosphoric acid.
Preferable examples of salts with organic acids include formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, and benzenesulfonic acid And salts with p-toluenesulfonic acid.
Preferable examples of the salt with basic amino acid include salts with arginine, lysine and ornithine.
Preferable examples of the salt with acidic amino acid include salts with aspartic acid and glutamic acid.
＜フィーダー細胞＞
　本発明では、フィーダー細胞は必ずしも必要ないが、フィーダー細胞を存在させることも可能である。フィーダー細胞としては、例えば胎仔線維芽細胞などが挙げられる。胎仔線維芽細胞には、例えばMEF（マウス胎仔線維芽細胞）、STO細胞（マウス胎仔線維芽細胞株）、SNL細胞（STO細胞のサブクローン;例えばSNL76/7細胞）などが含まれる。本発明では、フィーダー細胞が不在であっても、上記のTGF-βファミリー阻害剤、GSK阻害剤及びBMP阻害剤の組み合わせを培地に存在させるだけで、多能性幹細胞の神経分化能を亢進させることができる。このような培養条件に加えて、フィーダー細胞を使用すると、多能性幹細胞の神経分化能をさらに向上させることができる。 <Feeder cells>
In the present invention, feeder cells are not necessarily required, but feeder cells can also be present. Examples of feeder cells include fetal fibroblasts. The fetal fibroblasts include, for example, MEF (mouse fetal fibroblasts), STO cells (mouse fetal fibroblast cell line), SNL cells (STO cell subclone; for example, SNL76 / 7 cells), and the like. In the present invention, even in the absence of feeder cells, only the combination of the above TGF-β family inhibitor, GSK inhibitor and BMP inhibitor is present in the medium to enhance the neuronal differentiation ability of pluripotent stem cells. be able to. In addition to such culture conditions, when feeder cells are used, the neuronal differentiation ability of pluripotent stem cells can be further improved.
　しかし、たとえそうであっても、ヒトなどの哺乳動物への移植を考えた場合、ドナーに対して異種の細胞の使用はできる限り避けることが望ましいことは言うまでもない。 However, even if so, it goes without saying that it is desirable to avoid the use of heterogeneous cells as much as possible when considering transplantation into mammals such as humans.
＜多能性幹細胞の神経分化能の亢進＞
(A)培地
　培地としては、哺乳動物細胞の培養に用いられる培地を基礎培地として調製することができる。基礎培地としては、例えばIMDM培地、Medium 199培地、Eagle's Minimum Essential Medium (EMEM)培地、αMEM培地、Dulbecco's modified Eagle's Medium (DMEM)培地、Ham's F12培地、RPMI1640培地、Fischer's培地、およびこれらの混合培地などが包含される。 <Enhancement of neuronal differentiation ability of pluripotent stem cells>
(A) Medium As a medium, a medium used for culturing mammalian cells can be prepared as a basal medium. Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI1640 medium, Fischer's medium, and mixed media thereof. Is included.
　またES細胞培養用に改変された培地（例えばマウスES細胞培養用培地(例えばTX-WES培地、トロンボX社)、霊長類ES細胞培養用培地(例えば霊長類(ヒト&サル) ES細胞用培地、リプロセル、京都、日本)等）を用いてもよい。 Medium modified for ES cell culture (eg, mouse ES cell culture medium (eg TX-WES medium, Thrombo X)), primate ES cell culture medium (eg primate (human & monkey) ES cell culture medium , Reprocell, Kyoto, Japan, etc.).
　培地には、血清が含有されていてもよいし、あるいは無血清でもよい。血清としては、本発明の方法１により多能性幹細胞の神経分化能を亢進し得る範囲において特に限定されないが、好ましくは上記哺乳動物由来の血清（例えばウシ胎仔血清、ヒト血清等）である。また血清の代替添加物（例えばKnockout Serum Replacement (KSR)（Invitrogen社製）等）を用いてもよい。培地に血清が含まれる場合、血清の濃度は、本発明の方法１により多能性幹細胞の神経分化能を亢進し得る範囲において特に限定されないが、通常、０．１～３０（ｖ／ｖ）％の範囲である。 The medium may contain serum or may be serum-free. The serum is not particularly limited as long as it can enhance the neuronal differentiation ability of pluripotent stem cells by the method 1 of the present invention, but is preferably a serum derived from the above mammals (for example, fetal bovine serum, human serum, etc.). Alternatively, serum supplements (for example, Knockout Serum Replacement (KSR) (Invitrogen)) may be used. When serum is contained in the medium, the serum concentration is not particularly limited as long as it can enhance the neuronal differentiation ability of pluripotent stem cells by Method 1 of the present invention, but usually 0.1 to 30 (v / v) % Range.
　培地はまた、必要に応じて、例えば、血清蛋白質（例えばアルブミン等）、鉄源（例えばトランスフェリン等）、脂肪酸、成長因子、インスリン、コラーゲン前駆体、微量元素、ミネラル（例えばセレン酸ナトリウム等）、還元剤（例えば２－メルカプトエタノール、3'－チオールグリセロール等）、脂質、アミノ酸（例えばＬ－グルタミン等）、非必須アミノ酸、ビタミン類（例えばアスコルビン酸、ｄ－ビオチン等）、抗生物質（例えばストレプトマイシン、ペニシリン、ゲンタマイシン等）、抗酸化剤、糖類（例えばグルコース等）、有機酸（例えばピルビン酸、乳酸等）、緩衝剤（例えばＨＥＰＥＳ等）、ステロイド（例えばβ－エストラジオール、プロゲステロン等）、ポリアミン類（例えばプトレシン等）等の1つ以上の物質も含有しうる。 The medium can also be used as necessary, for example, serum proteins (eg, albumin), iron sources (eg, transferrin), fatty acids, growth factors, insulin, collagen precursors, trace elements, minerals (eg, sodium selenate), Reducing agent (for example, 2-mercaptoethanol, 3′-thiolglycerol, etc.), lipid, amino acid (for example, L-glutamine, etc.), non-essential amino acid, vitamins (for example, ascorbic acid, d-biotin, etc.), antibiotic (for example, streptomycin, etc.) , Penicillin, gentamicin, etc.), antioxidants, sugars (eg, glucose, etc.), organic acids (eg, pyruvate, lactic acid, etc.), buffers (eg, HEPES, etc.), steroids (eg, β-estradiol, progesterone, etc.), polyamines One or more substances (such as putrescine) It may have.
　培地には、上記のTGF-βファミリー阻害剤、GSKβ阻害剤及びBMP阻害剤の組み合わせが含まれる。培地中のTGF-βファミリー阻害剤の濃度は、多能性幹細胞の神経分化能を亢進する範囲で適宜設定されるが、TGF-βファミリー阻害剤として、SB431542を用いる場合、通常50nM～100μM、好ましくは100nM～10μM、より好ましくは1～5μMである。培地中のGSK阻害剤の濃度は、多能性幹細胞の神経分化能を亢進する範囲で適宜設定されるが、GSK阻害剤として、CHIR99021を用いる場合、通常50nM～100μM、好ましくは100nM～10μM、より好ましくは1～5μMである。培地中のBMP阻害剤の濃度は、多能性幹細胞の神経分化能を亢進する範囲で適宜設定されるが、BMP阻害剤として、Dorsomorphinを用いる場合、通常50nM～100μM、好ましくは100nM～10μM、より好ましくは1～5μMである。 The medium contains a combination of the above TGF-β family inhibitor, GSKβ inhibitor and BMP inhibitor. The concentration of the TGF-β family inhibitor in the medium is appropriately set within a range that enhances the neuronal differentiation ability of pluripotent stem cells, but when using SB431542 as a TGF-β family inhibitor, usually 50 nM to 100 μM, Preferably, it is 100 nM to 10 μM, more preferably 1 to 5 μM. The concentration of the GSK inhibitor in the medium is appropriately set within a range that enhances the neuronal differentiation ability of pluripotent stem cells. When CHIR99021 is used as the GSK inhibitor, it is usually 50 nM to 100 μM, preferably 100 nM to 10 μM, More preferably, it is 1 to 5 μM. The concentration of the BMP inhibitor in the medium is appropriately set within the range that enhances the neuronal differentiation ability of pluripotent stem cells. When Dorsomorphin is used as the BMP inhibitor, it is usually 50 nM to 100 μM, preferably 100 nM to 10 μM, More preferably, it is 1 to 5 μM.
　本発明の方法１では、多能性幹細胞の多能性を維持しつつ、その神経分化能を亢進するのであって、多能性幹細胞から神経系細胞へ分化を誘導する方法とは異なる。従って、本発明の方法に用いられる培地には、多能性幹細胞の初期神経上皮への分化誘導（Wenlin Li et al., PNAS (2011), 108:8299-8304）の際に使用するLIFは不要である。従って、一態様において、本発明の方法１に用いられる培地にはLIFが含まれない。 The method 1 of the present invention is different from the method of inducing differentiation from a pluripotent stem cell to a neural cell while maintaining the pluripotency of the pluripotent stem cell while enhancing its neuronal differentiation ability. Therefore, in the medium used in the method of the present invention, the LIF used for induction of differentiation of pluripotent stem cells into the initial neuroepithelium (Wenlin Li et al., PNAS (2011), 108: 8299-8304) is It is unnecessary. Accordingly, in one embodiment, the medium used in the method 1 of the present invention does not contain LIF.
　また、本発明の方法に用いられる培地には、多能性幹細胞の神経分化の際に使用するN2やB27等の添加物は不要である。従って、一態様において、本発明の方法１に用いられる培地にはN2及び／又はB27が含まれない。 In addition, the medium used in the method of the present invention does not require additives such as N2 and B27 used for neural differentiation of pluripotent stem cells. Therefore, in one aspect, the medium used in Method 1 of the present invention does not contain N2 and / or B27.
 (B) 神経分化能の亢進
　ES細胞、iPS細胞などの多能性幹細胞の神経分化能の亢進に際して、これらの細胞を、上記文献に記載の方法を用いて作製し、培養する。ヒトES細胞やヒトiPS細胞の培養時には、霊長類ES細胞用培地(リプロセル(京都、日本))を好ましく使用できる。 (B) Enhancement of neural differentiation ability When enhancing the neuronal differentiation ability of pluripotent stem cells such as ES cells and iPS cells, these cells are prepared and cultured using the method described in the above-mentioned document. When culturing human ES cells or human iPS cells, a medium for primate ES cells (Reprocell (Kyoto, Japan)) can be preferably used.
　神経分化能の亢進は、前記培地を用いて、フィーダー細胞の存在下又は非存在下のいずれかで行うことができる。フィーダー細胞が存在する場合、該細胞として、上に例示の、例えばMEF(マウス胎仔線維芽細胞〉、STO細胞(マウス胎仔線維芽細胞株)、SNL細胞(STO細胞のサブクローン; 例えばSNL76/7細胞など)等を使用することができる。フィーダー細胞については、一般に、細胞増殖を停止させるために放射線（ガンマ線等）照射や抗癌剤（マイトマイシンＣ等）処理等で不活化する。 Enhancement of neuronal differentiation ability can be carried out using the medium in the presence or absence of feeder cells. When feeder cells are present, examples of the cells include, for example, MEF (mouse fetal fibroblasts), STO cells (mouse fetal fibroblast cell lines), SNL cells (subclones of STO cells; In general, feeder cells are inactivated by irradiation with radiation (such as gamma rays) or treatment with an anticancer agent (such as mitomycin C) in order to stop cell growth.
　尚、本発明の方法１では、多能性幹細胞の多能性を維持しつつ、その神経分化能を亢進するのであって、多能性幹細胞から神経系細胞へ分化を誘導する方法とは異なる。従って、本発明の方法１においては、フィーダー細胞として、多能性幹細胞の神経系分化誘導の際に使用するストローマ細胞（例えばPA6細胞(マウスストローマ細胞株(理研BRC Cell Bank (日本)))、MS-5細胞(ExpHematol. 17:145-53 (1989))、OP9細胞(Science. 265:1098-1101 (1994)) など）は使用されない。 The method 1 of the present invention enhances the neural differentiation ability while maintaining the pluripotency of the pluripotent stem cell, and is different from the method of inducing differentiation from the pluripotent stem cell to the nervous system cell. . Therefore, in the method 1 of the present invention, as feeder cells, stromal cells (for example, PA6 cells (mouse stromal cell line (RIKEN BRC Cell Cell Bank (Japan))) used for induction of neural differentiation of pluripotent stem cells, MS-5 cells (ExpHematol. 17: 145-53 (1989)), OP9 cells (Science. 265: 1098-1101 (1994)), etc.) are not used.
　神経分化能の亢進のための培養の直前および直後に、好ましくは、培養多能性幹細胞を含む培地にROCK(p160-Rho-associated coiled-coil kinase)阻害剤を添加して処理する。ROCK阻害剤は、細胞の分散の際に非常に強力な細胞死抑制作用を示す物質であり、例えば、Y-27632（(R)-(＋)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide・2HCl・H2O）、Fasudil(HA-1077)、Thiazovivinなどが知られている（Watanabe et a1. ， Nat. Biotech.， 25:681-686 (2007)）。ROCK阻害剤の濃度は、通常、約50nM～約10μMである。 The ROCK (p160-Rho-associated coiled-coil kinase) inhibitor is preferably added to the medium containing cultured pluripotent stem cells immediately before and immediately after the culture for enhancing neuronal differentiation ability. A ROCK inhibitor is a substance that exhibits a very strong cell death inhibitory action upon cell dispersion. For example, Y-27632 ((R)-(+)-trans-N- (4-pyridyl) -4 -(1-aminoethyl) -cyclohexanecarboxamide · 2HCl · H 2 O), Fasudil (HA-1077), Thiazovivin, etc. are known (Watanabe et al., Nat. Biotech., 25: 681-686 (2007)). . The concentration of the ROCK inhibitor is usually about 50 nM to about 10 μM.
　多能性幹細胞の培地中の密度は、約5.0ｘ104～約1.0ｘ107細胞の範囲内であることが好ましいが、この範囲の外であってもよい。 The density of pluripotent stem cells in the medium is preferably within the range of about 5.0 × 10 4 to about 1.0 × 10 7 cells, but may be outside this range.
　培養は、非接着性条件下での三次元培養、例えば浮遊培養(例えば、分散培養、凝集浮遊培養など)、または接着条件下での二次元培養、例えば平板培養、あるいは、三次元培養後に二次元培養を行うという連続的な組み合わせ培養、などを包含する。フィーダー細胞の存在下で分化誘導する場合には、二次元培養を使用することができるが、一方、フィーダー細胞が不在の場合には、三次元培養を使用することができる。通常は、フィーダー細胞上で接着培養する。 The culture may be a three-dimensional culture under non-adhesive conditions, such as suspension culture (for example, dispersion culture, agglutination suspension culture, etc.), or a two-dimensional culture under adhesion conditions, such as plate culture, or two-dimensional culture after two-dimensional culture. Including continuous combination culture in which dimensional culture is performed. Two-dimensional culture can be used when differentiation is induced in the presence of feeder cells, while three-dimensional culture can be used when no feeder cells are present. Usually, adhesion culture is performed on feeder cells.
　細胞接着性の培養器では、細胞との接着性を向上させる目的で、その表面を、細胞支持物質、例えばコラーゲン、ゼラチン、ポリ－L－リジン、ポリ－D－リジン、ラミニン、フィブロネクチン、マトリゲルTM (ベクトン・ディキンソン)などの物質でコーティングすることができる。 In the cell-adhesive incubator, the surface is coated with a cell support material such as collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, Matrigel ™ for the purpose of improving adhesion to cells. Can be coated with materials such as (Becton Dickinson).
　分散培養では、多能性幹細胞は液体培地に懸濁した状態で培養される。また、凝集浮遊培養により、多能性幹細胞の細胞塊(または、胚様体)を形成することができる。凝集浮遊培養については、例えば胚様体培養法(Kellerら， Curr. Opin. Cell Bio1. 7， 862-869(1995))、SFEB法(例、Watanabeら， Nature Neuroscience 8，288-296 (2005)；WO 2005/123902) などを利用することができる。 In dispersion culture, pluripotent stem cells are cultured in a state suspended in a liquid medium. In addition, a cell mass (or embryoid body) of pluripotent stem cells can be formed by aggregation suspension culture. Regarding the agglutination suspension culture, for example, embryoid body culture method (Keller et al., Curr. Opin. Cell Bio 1. 7, 862-869 (1995)), SFEB method (eg, Watanabe et al., Nature Neuroscience 8, 288-296 (2005 ); WO 2005/123902) can be used.
　接着培養では、例えば Matrige1法(Chambers SM，et a1. Nat Biotechno1. 27: 485，2009)、SDIA法(Kawasaki H， et a1. Neuron. 28: 31-40， 2000、またはKawasaki H，et a1. Proc Nat1 Acad Sci U S A. 99:1580-5， 2002) などを利用することができる。 In the adhesion culture, for example, Matrige1 method (Chambers SM, et a1. Nat Biotechno1. 27: 485, 2009), SDIA method (Kawasaki H, et a1. Neuron. 28: 31-40, 2000, or Kawasaki H, et a1. Proc Nat1 Acad Sci U S A. 99: 1580-5 (2002) can be used.
　培養条件について、培地は上記の培地が使用されうるし、培養温度は、以下に限定されないが、約30～40℃、好ましくは約37℃であり、CO2含有空気の雰囲気下で培養が行われ、CO2濃度は、好ましくは約2～5%である。通常は、多能性幹細胞が、神経分化能の亢進条件下において5日以上培養される。 Regarding the culture conditions, the above-mentioned media can be used as the culture medium, and the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is performed in an atmosphere of CO 2 -containing air. The CO 2 concentration is preferably about 2-5%. Usually, pluripotent stem cells are cultured for 5 days or more under the condition of enhancing neuronal differentiation ability.
　多能性幹細胞における神経分化能の亢進は、上記培養開始前と比較して、神経系マーカー（Sox2、nestin、Sox1等）の発現が上昇することにより確認される。また、神経分化能が亢進した多能性幹細胞においては、上記培養開始前と比較して、nanogの発現が減少し、brachyury（中胚葉マーカー）及びSox17（内胚葉マーカー）の発現が上昇していることから、これらの発現変動を確認することによって、多能性幹細胞における神経分化能の亢進を、神経系への分化と区別することができる。 Enhancement of neuronal differentiation ability in pluripotent stem cells is confirmed by an increase in the expression of nervous system markers (Sox2, nestin, Sox1, etc.) compared to before the start of the culture. In addition, in pluripotent stem cells with enhanced neuronal differentiation ability, the expression of nanog is decreased and the expression of brachyury (mesoderm marker) and Sox17 (endoderm marker) is increased compared to before the start of the culture. Therefore, by confirming these expression fluctuations, it is possible to distinguish the enhancement of the neuronal differentiation ability in pluripotent stem cells from the differentiation into the nervous system.
　本発明の方法１によれば、多能性幹細胞、好ましくは人工多能性幹細胞の神経分化能を亢進することができる。特に、神経分化しにくいT細胞由来iPS細胞およびBリンパ球由来iPS細胞の神経分化能を亢進することができるので、従前よりも効率的にT細胞由来iPS細胞およびBリンパ球由来iPS細胞から、神経幹細胞等の神経系未分化細胞を分化させることが可能となる。 According to the method 1 of the present invention, the neuronal differentiation ability of pluripotent stem cells, preferably induced pluripotent stem cells, can be enhanced. In particular, since it can enhance the neuronal differentiation ability of T cell-derived iPS cells and B lymphocyte-derived iPS cells that are difficult to differentiate, from T cell-derived iPS cells and B lymphocyte-derived iPS cells more efficiently than before, It becomes possible to differentiate undifferentiated cells of the nervous system such as neural stem cells.
　尚、神経分化能が亢進した多能性幹細胞は、その亢進した神経分化能を維持した状態で、継代培養可能である。継代培養の条件は、上述の多能性幹細胞の神経分化能の亢進のための培養と同じである。 In addition, the pluripotent stem cell with enhanced neuronal differentiation ability can be subcultured while maintaining the enhanced neuronal differentiation ability. The conditions for subculture are the same as the culture for enhancing the neuronal differentiation ability of the pluripotent stem cells described above.
　神経分化能が亢進した多能性幹細胞は、後述する神経系未分化細胞への分化誘導方法等により、神経幹細胞等の神経系未分化細胞へ分化させることができる。 Pluripotent stem cells with enhanced neural differentiation ability can be differentiated into neural undifferentiated cells such as neural stem cells by a method for inducing differentiation into neural undifferentiated cells described later.
２．神経系未分化細胞の分化誘導方法（方法２）
　本発明は、多能性幹細胞を、TGF-βファミリー阻害剤及びGSK3β阻害剤の存在下で、2～10％の酸素分圧下、培養（好ましくは浮遊培養）することを含む、神経系未分化細胞を分化誘導する方法（即ち、神経系未分化細胞の製造方法）を提供するものである。 2. Method for inducing differentiation of undifferentiated cells of nervous system (Method 2)
The present invention comprises culturing pluripotent stem cells in the presence of a TGF-β family inhibitor and a GSK3β inhibitor under an oxygen partial pressure of 2 to 10% (preferably suspension culture). The present invention provides a method for inducing cell differentiation (that is, a method for producing undifferentiated cells of the nervous system).
　各用語の定義は、上記１．にて記載の通りである。 The definition of each term is as described in 1. above. As described in.
　本発明の方法２においては、多能性幹細胞としては、好ましくは、上記本発明の方法１において神経分化能が亢進された多能性幹細胞（好ましくは人工多能性幹細胞）が用いられる。 In the method 2 of the present invention, the pluripotent stem cell is preferably a pluripotent stem cell (preferably an induced pluripotent stem cell) with enhanced neuronal differentiation ability in the method 1 of the present invention.
 (A)培地
　培地としては、哺乳動物細胞の培養に用いられる培地を基礎培地として調製することができる。基礎培地としては、例えばIMDM培地、Medium 199培地、Eagle's Minimum Essential Medium (EMEM)培地、αMEM培地、Dulbecco's modified Eagle's Medium (DMEM)培地、Ham's F12培地、RPMI1640培地、Fischer's培地、およびこれらの混合培地などが包含される。 (A) Medium As a medium, a medium used for culturing mammalian cells can be prepared as a basal medium. Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI1640 medium, Fischer's medium, and mixed media thereof. Is included.
　またES細胞培養用に改変された培地（例えばマウスES細胞培養用培地(例えばTX-WES培地、トロンボX社)、霊長類ES細胞培養用培地(例えば霊長類(ヒト&サル) ES細胞用培地、リプロセル、京都、日本)等）を用いてもよい。 Medium modified for ES cell culture (eg, mouse ES cell culture medium (eg TX-WES medium, Thrombo X)), primate ES cell culture medium (eg primate (human & monkey) ES cell culture medium , Reprocell, Kyoto, Japan, etc.).
　培地には、血清が含有されていてもよいし、あるいは無血清でもよい。血清としては、本発明の方法２により多能性幹細胞から神経系未分化細胞への分化を誘導し得る範囲において特に限定されないが、好ましくは上記哺乳動物由来の血清（例えばウシ胎仔血清、ヒト血清等）である。また血清の代替添加物（例えばKnockout Serum Replacement (KSR)（Invitrogen社製）等）を用いてもよい。培地に血清が含まれる場合、血清の濃度は、本発明の方法２により多能性幹細胞から神経系未分化細胞への分化を誘導し得る範囲において特に限定されないが、通常、０．１～３０（ｖ／ｖ）％の範囲である。 The medium may contain serum or may be serum-free. The serum is not particularly limited as long as differentiation from pluripotent stem cells to neural undifferentiated cells can be induced by the method 2 of the present invention, but preferably the above-mentioned mammal-derived serum (eg, fetal bovine serum, human serum) Etc.). Alternatively, serum supplements (for example, Knockout Serum Replacement (KSR) (Invitrogen)) may be used. When serum is contained in the medium, the serum concentration is not particularly limited as long as differentiation from pluripotent stem cells to neural undifferentiated cells can be induced by the method 2 of the present invention, but usually 0.1 to 30. It is in the range of (v / v)%.
　培地はまた、必要に応じて、例えば、血清蛋白質（例えばアルブミン等）、鉄源（例えばトランスフェリン等）、脂肪酸、成長因子、インスリン、コラーゲン前駆体、微量元素、ミネラル（例えばセレン酸ナトリウム等）、還元剤（例えば２－メルカプトエタノール、3'－チオールグリセロール等）、脂質、アミノ酸（例えばＬ－グルタミン等）、非必須アミノ酸、ビタミン類（例えばアスコルビン酸、ｄ－ビオチン等）、抗生物質（例えばストレプトマイシン、ペニシリン、ゲンタマイシン等）、抗酸化剤、糖類（例えばグルコース等）、有機酸（例えばピルビン酸、乳酸等）、緩衝剤（例えばＨＥＰＥＳ等）、ステロイド（例えばβ－エストラジオール、プロゲステロン等）、ポリアミン類（例えばプトレシン等）等の1つ以上の物質も含有しうる。 The medium can also be used as necessary, for example, serum proteins (eg, albumin), iron sources (eg, transferrin), fatty acids, growth factors, insulin, collagen precursors, trace elements, minerals (eg, sodium selenate), Reducing agent (for example, 2-mercaptoethanol, 3′-thiolglycerol, etc.), lipid, amino acid (for example, L-glutamine, etc.), non-essential amino acid, vitamins (for example, ascorbic acid, d-biotin, etc.), antibiotic (for example, streptomycin, etc.) , Penicillin, gentamicin, etc.), antioxidants, sugars (eg, glucose, etc.), organic acids (eg, pyruvate, lactic acid, etc.), buffers (eg, HEPES, etc.), steroids (eg, β-estradiol, progesterone, etc.), polyamines One or more substances (such as putrescine) It may have.
　好適な培地として、以下の組成を有するMHM培地を挙げることができる：
DMEM/F-12 (1:1), glucose (0.6%), glutamine (2 mM), sodium bicarbonate (3 mM), HEPES (5mM), insulin (25 Ag/ml), transferrin (100 Ag/ml), progesterone (20 nM), sodium selenate (30 ng)及びputrescine (60nM)。 Suitable media can include MHM media having the following composition:
DMEM / F-12 (1: 1), glucose (0.6%), glutamine (2 mM), sodium bicarbonate (3 mM), HEPES (5 mM), insulin (25 Ag / ml), transferrin (100 Ag / ml) , progesterone (20 nM), sodium selenate (30 ng) and putrescine (60 nM).
　培地には、上記のTGF-βファミリー阻害剤及びGSK3β阻害剤の組み合わせが含まれる。培地中のTGF-βファミリー阻害剤の濃度は、多能性幹細胞から神経系未分化細胞への分化を誘導する範囲で適宜設定されるが、TGF-βファミリー阻害剤として、SB431542を用いる場合、通常50nM～100μM、好ましくは100nM～10μM、より好ましくは1～5μMである。培地中のGSK3β阻害剤の濃度は、多能性幹細胞から神経系未分化細胞への分化を誘導する範囲で適宜設定されるが、GSK3β阻害剤として、CHIR99021を用いる場合、通常50nM～100μM、好ましくは100nM～10μM、より好ましくは1～5μMである。 The medium contains a combination of the above TGF-β family inhibitor and GSK3β inhibitor. The concentration of the TGF-β family inhibitor in the medium is appropriately set within the range that induces differentiation from pluripotent stem cells to undifferentiated cells of the nervous system, but when using SB431542 as a TGF-β family inhibitor, Usually, it is 50 nM to 100 μM, preferably 100 nM to 10 μM, more preferably 1 to 5 μM. The concentration of the GSK3β inhibitor in the medium is appropriately set within a range that induces differentiation from pluripotent stem cells to undifferentiated cells of the nervous system. When CHIR99021 is used as the GSK3β inhibitor, it is usually 50 nM to 100 μM, preferably Is from 100 nM to 10 μM, more preferably from 1 to 5 μM.
　本発明の方法２において用いられる培地は、多能性幹細胞から神経系未分化細胞への分化誘導を促進するために、好ましくは更にLIFを含む。LIFが培地中に含まれる場合には、その濃度は、本発明の方法により多能性幹細胞から神経系未分化細胞への分化を誘導し得る範囲において特に限定されないが、通常0.25 ng/ml～1 μg/ml、好ましくは1～50 ng/ml、最も好ましくは3～30 ng/mlである。 The medium used in Method 2 of the present invention preferably further contains LIF in order to promote differentiation induction from pluripotent stem cells to undifferentiated neural cells. When LIF is contained in the medium, its concentration is not particularly limited as long as differentiation from pluripotent stem cells to neural undifferentiated cells can be induced by the method of the present invention, but it is usually from 0.25 ng / ml to 1 μg / ml, preferably 1 to 50 μng / ml, most preferably 3 to 30 μng / ml.
　本発明の方法２において用いられる培地は、多能性幹細胞から神経系未分化細胞への分化誘導を促進するために、好ましくは更にbFGFを含む。bFGFが培地中に含まれる場合には、その濃度は、本発明の方法により多能性幹細胞から神経系未分化細胞への分化を誘導し得る範囲において特に限定されないが、通常濃度0.25 ng/ml～1 μg/ml、好ましくは1～50 ng/ml、最も好ましくは3～30 ng/mlである。 The medium used in Method 2 of the present invention preferably further contains bFGF in order to promote differentiation induction from pluripotent stem cells to undifferentiated nervous system cells. When bFGF is contained in the medium, the concentration is not particularly limited as long as differentiation from pluripotent stem cells to undifferentiated neural cells can be induced by the method of the present invention, but a normal concentration of 0.25 ng / ml ˜1 μg / ml, preferably 1 to 50 ng / ml, most preferably 3 to 30 ng / ml.
　本発明の方法２において用いられる培地は、細胞死抑制のために、好ましくは更にROCK阻害剤（Y-27632、Fasudil(HA-1077)など）を含む。Y-27632が培地中に含まれる場合には、その濃度は、本発明の方法により多能性幹細胞から神経系未分化細胞への分化を誘導し得る範囲において特に限定されないが、約50nM～約10μMである。 The medium used in the method 2 of the present invention preferably further contains a ROCK inhibitor (Y-27632, Fasudil (HA-1077), etc.) in order to suppress cell death. When Y-27632 is contained in the medium, the concentration is not particularly limited as long as differentiation from pluripotent stem cells to undifferentiated neural cells can be induced by the method of the present invention, but about 50 nM to about 10 μM.
　本発明の方法２において用いられる培地は、多能性幹細胞から神経系未分化細胞への分化誘導を促進するために、好ましくは更にN2やB27等の添加物を含む。 The medium used in the method 2 of the present invention preferably further contains additives such as N2 and B27 in order to promote differentiation induction from pluripotent stem cells to undifferentiated cells of the nervous system.
　一態様において、本発明の方法２において用いられる培地は、TGF-βファミリー阻害剤及びGSK3β阻害剤に加えて、bFGF、LIF、及びB27を含む。 In one embodiment, the medium used in Method 2 of the present invention contains bFGF, LIF, and B27 in addition to the TGF-β family inhibitor and the GSK3β inhibitor.
　一態様において、本発明の方法２において用いられる培地は、TGF-βファミリー阻害剤及びGSK3β阻害剤に加えて、bFGF、LIF、B27及びROCK阻害剤（好ましくはY-27632）を含む。 In one embodiment, the medium used in Method 2 of the present invention contains bFGF, LIF, B27 and a ROCK inhibitor (preferably Y-27632) in addition to the TGF-β family inhibitor and the GSK3β inhibitor.
(B) 分化誘導法
　ES細胞、iPS細胞などの多能性細胞から神経系未分化細胞への分化誘導に際して、これらの細胞を、上記文献に記載の方法を用いて作製し、培養する。ヒトES細胞やヒトiPS細胞の培養時には、霊長類ES細胞用培地(リプロセル(京都、日本))を好ましく使用できる。 (B) Differentiation induction method Upon differentiation induction from pluripotent cells such as ES cells and iPS cells into undifferentiated cells of the nervous system, these cells are prepared and cultured using the method described in the above-mentioned document. When culturing human ES cells or human iPS cells, a medium for primate ES cells (Reprocell (Kyoto, Japan)) can be preferably used.
　神経系未分化細胞への分化誘導は、前記培地を用いて、フィーダー細胞の存在下又は非存在下のいずれかで行うことができる。フィーダー細胞が存在する場合、該細胞として、上に例示の、例えばMEF(マウス胎仔線維芽細胞〉、STO細胞(マウス胎仔線維芽細胞株)、SNL細胞(STO細胞のサブクローン; 例えばSNL76/7細胞など)等の線維芽細胞；PA6細胞(マウスストローマ細胞株(理研BRC Cell Bank (日本)))、MS-5細胞(Exp Hematol. 17:145-53 (1989))、OP9細胞(Science. 265:1098-1101 (1994))等のストローマ細胞を使用することができる。フィーダー細胞については、一般に、細胞増殖を停止させるために放射線（ガンマ線等）照射や抗癌剤（マイトマイシンＣ等）処理等で不活化する。 Differentiation induction into undifferentiated cells of the nervous system can be performed using the medium in the presence or absence of feeder cells. When feeder cells are present, examples of the cells include, for example, MEF (mouse fetal fibroblasts), STO cells (mouse fetal fibroblast cell lines), SNL cells (subclones of STO cells; Cells, etc.); PA6 cells (mouse stromal cell line (RIKEN BRC Cell Bank (Japan))), MS-5 cells (Exp. Hematol. 17: 145-53 (1989)), OP9 cells (Science. 265: 1098-1101 (1994)), etc. In general, feeder cells are irradiated with radiation (gamma rays, etc.) or treated with an anticancer agent (mitomycin C, etc.) to stop cell growth. Inactivate.
　SDIA法は、ES細胞をストローマ細胞、特にPA6細胞と一緒に共培養することによってほぼ選択的に神経細胞へ分化させる方法であるが、本発明では、フィーダー細胞が不在であっても、上記のTGF-βファミリー阻害剤及びGSK3β阻害剤の組み合わせを分化誘導培地に存在させ、低酸素（２～５％）環境下で培養するだけで選択的に神経系未分化細胞に分化させることができる。このような培養条件に加えて、フィーダー細胞を使用すると、神経系未分化細胞への分化効率をさらに向上させることができる。 The SDIA method is a method in which ES cells are co-cultured with stromal cells, particularly PA6 cells, to selectively differentiate into neurons, but in the present invention, the above-described method is performed even in the absence of feeder cells. A combination of a TGF-β family inhibitor and a GSK3β inhibitor can be selectively differentiated into undifferentiated cells of the nervous system simply by being present in a differentiation-inducing medium and culturing in a hypoxic (2-5%) environment. In addition to such culture conditions, when feeder cells are used, the efficiency of differentiation into undifferentiated cells of the nervous system can be further improved.
　分化誘導の直前および直後に、好ましくは、培養多能性幹細胞を含む培地にROCK(p160-Rho-associated coiled-coil kinase)阻害剤を添加して処理する。ROCK阻害剤は、細胞の分散の際に非常に強力な細胞死抑制作用を示す物質であり、例えば、Y-27632、Fasudil(HA-1077)などが知られている（Watanabe et a1.， Nat. Biotech.， 25:681-686 (2007))。阻害剤の濃度は、通常、約50nM～約10μMである。 Immediately before and after induction of differentiation, preferably, a ROCK (p160-Rho-associated-coiled-coil-kinase) inhibitor is added to the medium containing cultured pluripotent stem cells for treatment. ROCK inhibitors are substances that exhibit a very strong cell death inhibitory action when cells are dispersed. For example, Y-27632 and Fasudil (HA-1077) are known (Watanabe et a1., Nat). . Biotech., 25: 681-686 (2007)). The concentration of inhibitor is usually about 50 nM to about 10 μM.
　多能性幹細胞の培地中の密度は、約1.0ｘ104～約1.0ｘ107細胞の範囲内であることが好ましいが、この範囲の外であってもよい。 The density of pluripotent stem cells in the medium is preferably within the range of about 1.0 × 10 4 to about 1.0 × 10 7 cells, but may be outside this range.
　培養は、非接着性条件下での三次元培養、例えば浮遊培養(例えば、分散培養、凝集浮遊培養など)、または接着条件下での二次元培養、例えば平板培養、あるいは、三次元培養後に二次元培養を行うという連続的な組み合わせ培養、などを包含する。フィーダー細胞の存在下で分化誘導する場合には、二次元培養を使用することができるが、一方、フィーダー細胞が不在の場合には、三次元培養を使用することができる。好ましくは、多能性幹細胞は浮遊培養される。 The culture may be a three-dimensional culture under non-adhesive conditions, such as suspension culture (for example, dispersion culture, agglutination suspension culture, etc.), or a two-dimensional culture under adhesion conditions, such as plate culture, or two-dimensional culture after two-dimensional culture. Including continuous combination culture in which dimensional culture is performed. Two-dimensional culture can be used when differentiation is induced in the presence of feeder cells, while three-dimensional culture can be used when no feeder cells are present. Preferably, pluripotent stem cells are cultured in suspension.
　細胞接着性の培養器では、細胞との接着性を向上させる目的で、その表面を、細胞支持物質、例えばコラーゲン、ゼラチン、ポリ－L－リジン、ポリ－D－リジン、ラミニン、フィブロネクチン、マトリゲルTM (ベクトン・ディキンソン)などの物質でコーティングすることができる。 In the cell-adhesive incubator, the surface is coated with a cell support material such as collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, Matrigel ™ for the purpose of improving adhesion to cells. Can be coated with materials such as (Becton Dickinson).
　分散培養では、多能性幹細胞は液体培地に懸濁した状態で培養される。また、凝集浮遊培養により、多能性幹細胞の細胞塊(または、胚様体)を形成することができる。凝集浮遊培養については、例えば胚様体培養法(Kellerら， Curr. Opin. Cell Bio1. 7， 862-869 (1995))、SFEB法(例、Watanabeら， Nature Neuroscience 8，288-296 (2005)；WO 2005/123902) などを利用することができる。好ましくは、酵素処理によりシングルセルの状態にまでバラバラになった多能性幹細胞が、液体培地に懸濁した状態で分散培養される。多能性幹細胞を、本発明の方法２により分散培養することにより、神経幹細胞等の神経系未分化細胞を含むニューロスフェアを形成することができる。 In dispersion culture, pluripotent stem cells are cultured in a state suspended in a liquid medium. In addition, a cell mass (or embryoid body) of pluripotent stem cells can be formed by aggregation suspension culture. Regarding the agglutination suspension culture, for example, embryoid body culture method (Keller et al., Urr Curr. Opin. Cell Bio 1. 7, 862-869 (1995)), SFEB method (eg, Watanabe et al., Nature Neuroscience 8, 288-296 (2005 ); WO 2005/123902) can be used. Preferably, pluripotent stem cells that have been separated into a single cell state by enzyme treatment are dispersedly cultured in a state of being suspended in a liquid medium. By culturing the pluripotent stem cells by the method 2 of the present invention, neurospheres containing undifferentiated neural cells such as neural stem cells can be formed.
　接着培養では、例えば Matrige1法(Chambers SM，et a1. Nat Biotechno1. 27: 485，2009)、SDIA法(Kawasaki H， et a1. Neuron. 28: 31-40, 2000、またはKawasaki H，et a1. Proc Nat1 Acad Sci U S A. 99:1580-5, 2002) などを利用することができる。 In adhesion culture, for example, Matrige1 method (Chambers SM, et a1. Nat Biotechno1. 27: 485, 2009), SDIA method (Kawasaki H, et a1. Neuron. 28: 31-40, 2000, or Kawasaki H, et a1. Proc Nat1 Acad Sci U S A. 99: 1580-5, 2002) can be used.
　培養条件について、培地は上記の培地が使用されうるし、培養温度は、以下に限定されないが、約30～40℃、好ましくは約37℃であり、CO2含有空気の雰囲気下で培養が行われ、CO2濃度は、好ましくは約2～5%である。 Regarding the culture conditions, the above-mentioned media can be used as the culture medium, and the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is performed in an atmosphere of CO 2 -containing air. The CO 2 concentration is preferably about 2-5%.
　本発明の方法２においては、低酸素（2～10%、好ましくは3%）環境下で、多能性幹細胞が培養される。低酸素環境下で培養することにより、多能性幹細胞からの神経系未分化細胞への分化誘導が促進される。 In the method 2 of the present invention, pluripotent stem cells are cultured in a hypoxic (2 to 10%, preferably 3%) environment. By culturing in a hypoxic environment, differentiation induction from pluripotent stem cells into undifferentiated nervous system cells is promoted.
　通常は、多能性幹細胞が、分化誘導条件下において3日以上培養される。分化誘導に十分な期間多能性幹細胞を培養することにより、神経幹細胞等の神経系未分化細胞を含むニューロスフェアが形成される。 Usually, pluripotent stem cells are cultured for 3 days or more under differentiation-inducing conditions. By culturing pluripotent stem cells for a period sufficient for induction of differentiation, neurospheres containing undifferentiated neural cells such as neural stem cells are formed.
　本発明の方法２により、短期間で多能性幹細胞から神経幹細胞等の神経系未分化細胞を分化誘導することができる。 According to the method 2 of the present invention, it is possible to induce differentiation of neural undifferentiated cells such as neural stem cells from pluripotent stem cells in a short period of time.
　一態様において、上述の本発明の方法１において神経分化能が亢進した多能性幹細胞（好ましくは、iPS細胞）が、本発明の方法２において培養される。このように、本発明の方法１と本発明の方法２とを組み合わせることにより、極めて短期間で神経幹細胞等の神経系未分化細胞を分化誘導することができる。また、リンパ球（T細胞、B細胞等）由来のiPS細胞等の神経分化しにくい多能性幹細胞からも、効率的に神経幹細胞等の神経系未分化細胞を分化誘導することができる。 In one embodiment, pluripotent stem cells (preferably iPS cells) having enhanced neuronal differentiation ability in the above-described method 1 of the present invention are cultured in the method 2 of the present invention. Thus, by combining the method 1 of the present invention and the method 2 of the present invention, it is possible to induce differentiation of neural undifferentiated cells such as neural stem cells in a very short period of time. In addition, it is possible to efficiently induce differentiation of neural undifferentiated cells such as neural stem cells from pluripotent stem cells that are difficult to differentiate into nerves, such as iPS cells derived from lymphocytes (T cells, B cells, etc.).
　本発明は、本発明の方法２によって作製された神経系未分化細胞も提供する。 The present invention also provides an undifferentiated nervous system cell produced by the method 2 of the present invention.
　本発明の方法２によって得ることができる神経系未分化細胞には、中枢神経系の細胞、運動神経、自律神経などの神経細胞およびオリゴデンドロサイト、アストロサイトなどのグリア細胞前駆体細胞が包含される。神経系未分化細胞は好ましくは神経幹細胞である。 Neural undifferentiated cells obtainable by the method 2 of the present invention include cells of the central nervous system, neurons such as motor nerves and autonomic nerves, and glial cell precursor cells such as oligodendrocytes and astrocytes. The Neural undifferentiated cells are preferably neural stem cells.
　神経系未分化細胞は、神経細胞接着分子(NCAM)、ポリシアリル化NCAM、A2B5(胎児や新生児の神経細胞に発現する)、中間体フィラメントタンパク質(ネスチン、ビメンチンなど)、転写因子Pax-6などの原始的神経外胚葉および神経幹細胞の発現マーカー、ドパミンニューロンマーカー(チロシンハイドロキシラーゼ(TH)など)、神経マーカー(TuJlなど)などによって同定されうる。 Neural undifferentiated cells include neural cell adhesion molecule (NCAM), polysialylated NCAM, A2B5 (expressed in fetal and neonatal neurons), intermediate filament proteins (nestin, vimentin, etc.), transcription factor Pax-6, etc. It can be identified by expression markers of primitive neuroectodermal and neural stem cells, dopamine neuron markers (such as tyrosine hydroxylase (TH)), neuronal markers (such as TuJl) and the like.
　神経系未分化細胞は、作製後、そのまま生体へ移植されてもよいし、あるいは神経細胞やグリア細胞(アストロサイトおよびオリゴデンドロサイトを含む)に完全にまたは部分的に分化させた後で生体に移植されてもよい。 Nervous system undifferentiated cells may be transplanted to a living body after production, or may be completely or partially differentiated into neural cells and glial cells (including astrocytes and oligodendrocytes) and then transplanted into the living body. It may be transplanted.
　本発明の方法２により得られた神経幹細胞等の神経系未分化細胞を含むニューロスフェアをトリプシン等のタンパク質分解酵素により分散し、分散された神経系未分化細胞を再び、本発明の方法２と同一条件で培養することにより、神経系未分化細胞を継代培養することができる。 The neurospheres containing neural undifferentiated cells such as neural stem cells obtained by the method 2 of the present invention are dispersed with a proteolytic enzyme such as trypsin, and the dispersed neural undifferentiated cells are again treated with the method 2 of the present invention. By culturing under the same conditions, neural undifferentiated cells can be subcultured.
３．運動神経前駆細胞の分化誘導方法（方法３）
　本発明は、上記本発明の方法２により得られた神経幹細胞を、TGF-βファミリー阻害剤、GSK3β阻害剤、レチノイン酸及びプルモルファミン（９－シクロヘキシル－Ｎ－［４－（４－モルホリニル）フェニル］－２－（１－ナフタレニルオキシ）－９Ｈ－プリン－６－アミン）の存在下で、2～10％の酸素分圧下、培養することを含む、運動神経前駆細胞を分化誘導する方法（即ち、運動神経前駆細胞の製造方法）を提供するものである。 3. Method for inducing differentiation of motor neural progenitor cells (Method 3)
The present invention relates to neural stem cells obtained by the above-described method 2 of the present invention using TGF-β family inhibitor, GSK3β inhibitor, retinoic acid and purmorphamine (9-cyclohexyl-N- [4- (4-morpholinyl) Differentiation induction of motor neuron progenitors, including culturing in the presence of 2-10% oxygen partial pressure in the presence of phenyl] -2- (1-naphthalenyloxy) -9H-purin-6-amine) The method (namely, the manufacturing method of a motor neural progenitor cell) is provided.
　本発明の方法３における培養条件は、培地にレチノイン酸及びプルモルファミンが添加されることを除き、本発明の方法２と同一である。 The culture conditions in Method 3 of the present invention are the same as those of Method 2 of the present invention except that retinoic acid and purmorphamine are added to the medium.
　培地中のレチノイン酸濃度は、運動神経前駆細胞を分化誘導する範囲で特に限定されないが、通常10nM～100μM、好ましくは100nM～10μM、より好ましくは0.3～3μMである。培地中のプルモルファミン濃度は、運動神経前駆細胞を分化誘導する範囲で特に限定されないが、通常10nM～100μM、好ましくは100nM～10μM、より好ましくは0.3～3μMである。 The concentration of retinoic acid in the medium is not particularly limited as long as it induces differentiation of motor neural progenitor cells, but is usually 10 nM to 100 μM, preferably 100 nM to 10 μM, more preferably 0.3 to 3 μM. The concentration of purmorphamine in the medium is not particularly limited as long as it induces differentiation of motor neural progenitor cells, but is usually 10 nM to 100 μM, preferably 100 nM to 10 μM, more preferably 0.3 to 3 μM.
　本発明の方法２において、神経幹細胞を含むニューロスフェアが得られた段階（例えば、培養開始から３日以降）で、培地にレチノイン酸及びプルモルファミンを添加して更に培養を続けることにより、自己再生可能な運動神経前駆細胞を分化誘導することができる。更なる培養期間は、通常、4日以上である。 In the method 2 of the present invention, at the stage where a neurosphere containing neural stem cells is obtained (for example, after 3 days from the start of culture), retinoic acid and purmorphamine are added to the medium, and further cultivation is continued. Regenerative motor neural progenitor cells can be induced to differentiate. The further culture period is usually 4 days or longer.
　一態様において、上述の本発明の方法１において神経分化能が亢進した多能性幹細胞（好ましくは、iPS細胞）が、本発明の方法２により培養され、更に本発明の方法３において運動神経前駆細胞へ分化誘導される。このように、本発明の方法１～３を組み合わせることにより、極めて短期間で多能性幹細胞から運動神経前駆細胞を分化誘導することができる。また、リンパ球（T細胞、B細胞等）由来のiPS細胞等の神経分化しにくい多能性幹細胞からも、効率的に運動神経前駆細胞を分化誘導することができる。 In one embodiment, pluripotent stem cells (preferably iPS cells) whose neurodifferentiation ability is enhanced in the above-described method 1 of the present invention are cultured by the method 2 of the present invention, and further in the method 3 of the present invention, a motor neuron precursor Differentiation into cells is induced. Thus, by combining the methods 1 to 3 of the present invention, motor neural progenitor cells can be induced to differentiate from pluripotent stem cells in a very short period of time. In addition, motor neural progenitor cells can be efficiently induced to differentiate from pluripotent stem cells that are difficult to differentiate from nerves, such as iPS cells derived from lymphocytes (T cells, B cells, etc.).
　本発明は、本発明の方法３によって作製された運動神経前駆細胞も提供する。 The present invention also provides a motor neural progenitor cell produced by the method 3 of the present invention.
　運動神経前駆細胞は、Oligo2、Nkx2.2等のマーカー遺伝子の発現によって同定されうる。 Motor neuron progenitor cells can be identified by the expression of marker genes such as Oligo2 and Nkx2.2.
　運動神経前駆細胞は、作製後、そのまま生体へ移植されてもよいし、あるいは運動神経細胞に完全にまたは部分的に分化させた後で生体に移植されてもよい。 Motor neuron progenitor cells may be transplanted directly into a living body after production, or may be transplanted into a living body after being completely or partially differentiated into motor nerve cells.
　例えば、運動神経前駆細胞を、N2、IGF-1、GDNF、BDNF、アスコルビン酸、レチノイン酸、Shh及び（ジブチリル）cAMPを含む培地中で培養することにより、運動神経細胞を分化誘導することができる。本発明は、このような運動神経細胞の分化誘導方法（即ち、運動神経細胞の製造方法）、及び当該方法により作製された運動神経細胞も提供する。 For example, motor neuron progenitor cells can be induced to differentiate by culturing them in a medium containing N2, IGF-1, GDNF, BDNF, ascorbic acid, retinoic acid, Shh and (dibutyryl) cAMP. . The present invention also provides such a method for inducing differentiation of motor neurons (that is, a method for producing motor neurons), and motor neurons produced by the method.
　運動神経細胞は、HB9、Isl、SMI-32、ChAT等のマーカー遺伝子の発現によって同定されうる。 Motor neurons can be identified by the expression of marker genes such as HB9, Isl, SMI-32, and ChAT.
４．ドパミン産生神経前駆細胞の分化誘導方法（方法４）
　本発明は、上記本発明の方法２により得られた神経幹細胞を、TGF-βファミリー阻害剤、GSK3β阻害剤、ソニックヘッジホッグ（蛋白質）、プルモルファミン及びFGF8の存在下で、2～10％の酸素分圧下、培養することを含む、ドパミン産生神経前駆細胞を分化誘導する方法（即ち、ドパミン産生神経前駆細胞の製造方法）を提供するものである。 4). Method for inducing differentiation of dopaminergic neural progenitor cells (Method 4)
The present invention relates to neural stem cells obtained by the above-described method 2 of the present invention in the presence of 2-10% in the presence of TGF-β family inhibitor, GSK3β inhibitor, sonic hedgehog (protein), purmorphamine and FGF8. A method for inducing differentiation of dopaminergic neural progenitor cells (ie, a method for producing dopaminergic neural progenitor cells), which comprises culturing under a partial pressure of oxygen.
　本発明の方法４における培養条件は、培地にソニックヘッジホッグ、プルモルファミン及びFGF8が添加されることを除き、本発明の方法２と同一である。 The culture conditions in the method 4 of the present invention are the same as those of the method 2 of the present invention except that sonic hedgehog, purmorphamine and FGF8 are added to the medium.
　培地中のソニックヘッジホッグ濃度は、ドパミン産生神経前駆細胞を分化誘導する範囲で特に限定されないが、通常1 ng/ml～5 μg/ml、好ましくは10～500 ng/ml、最も好ましくは50～300 ng/mlである。培地中のプルモルファミン濃度は、ドパミン産生神経前駆細胞を分化誘導する範囲で特に限定されないが、通常10 nM～100 μM、好ましくは100 nM～10 μM、より好ましくは0.3～3 μMである。培地中のFGF8濃度は、ドパミン産生神経前駆細胞を分化誘導する範囲で特に限定されないが、通常1 ng/ml～5 μg/ml、好ましくは10～500 ng/ml、最も好ましくは50～300 ng/mlである。 The concentration of sonic hedgehog in the medium is not particularly limited as long as it induces differentiation of dopaminergic neural progenitor cells, but is usually 1 ng / ml to 5 μg / ml, preferably 10 to 500 ng / ml, most preferably 50 to 300 ng / ml. The concentration of purmorphamine in the medium is not particularly limited as long as it induces differentiation of dopaminergic neural progenitor cells, but it is usually 10 to 100 μM, preferably 100 to 10 μM, more preferably 0.3 to 3 μM. The concentration of FGF8 in the medium is not particularly limited as long as it induces differentiation of dopaminergic neural progenitor cells, but is usually 1 ng / ml to 5 μg / ml, preferably 10 to 500 ng / ml, most preferably 50 to 300 ng. / ml.
　本発明の方法２において、神経幹細胞への分化が開始した段階（例えば、培養開始から２日以降）で、培地にソニックヘッジホッグ、プルモルファミン及びFGF8を添加して更に培養を続けることにより、自己再生可能なドパミン産生神経前駆細胞を分化誘導することができる。更なる培養期間は、通常、4日以上である。 In the method 2 of the present invention, at the stage where differentiation into neural stem cells has started (for example, after 2 days from the start of culture), sonic hedgehog, purmorphamine and FGF8 are added to the medium, and further culturing is continued. Self-renewable dopaminergic neural progenitor cells can be induced to differentiate. The further culture period is usually 4 days or longer.
　一態様において、上述の本発明の方法１において神経分化能が亢進した多能性幹細胞（好ましくは、iPS細胞）が、本発明の方法２において培養され、更に本発明の方法４においてドパミン産生神経前駆細胞へ分化誘導される。このように、本発明の方法１、２及び４を組み合わせることにより、極めて短期間でドパミン産生神経前駆細胞を分化誘導することができる。また、リンパ球（T細胞、B細胞等）由来のiPS細胞等の神経分化しにくい多能性幹細胞からも、効率的にドパミン産生神経前駆細胞を分化誘導することができる。 In one embodiment, the pluripotent stem cells (preferably iPS cells) whose neuronal differentiation ability has been enhanced in the above-described method 1 of the present invention are cultured in the method 2 of the present invention, and further in the method 4 of the present invention, Differentiation is induced into progenitor cells. Thus, by combining the methods 1, 2 and 4 of the present invention, it is possible to induce differentiation of dopaminergic neural progenitor cells in a very short period of time. In addition, dopaminergic neural progenitor cells can be efficiently induced to differentiate from pluripotent stem cells that are difficult to differentiate, such as iPS cells derived from lymphocytes (T cells, B cells, etc.).
　本発明は、本発明の方法４によって作製されたドパミン産生神経前駆細胞も提供する。 The present invention also provides a dopaminergic neural progenitor cell produced by the method 4 of the present invention.
　ドパミン産生神経前駆細胞は、細胞内に含有されるドパミンによって同定されうる。 Dopaminergic neural progenitor cells can be identified by the dopamine contained in the cells.
　ドパミン産生神経前駆細胞は、作製後、そのまま生体へ移植されてもよいし、あるいはドパミン産生神経細胞に完全にまたは部分的に分化させた後で生体に移植されてもよい。 The dopaminergic neuron progenitor cells may be transplanted to the living body as they are after preparation, or may be transplanted to the living body after being completely or partially differentiated into dopaminergic neuron cells.
　例えば、ドパミン産生神経前駆細胞を、Ｂ27、BDNF、GDNF、アスコルビン酸、TGF-β、cAMP及びDAPTを含む培地中で培養することにより、ドパミン産生神経細胞を分化誘導することができる。本発明は、このようなドパミン産生神経細胞の分化誘導方法（即ち、ドパミン産生神経細胞の製造方法）、及び当該方法により作製されたドパミン産生神経細胞も提供する。 For example, dopaminergic neuron progenitor cells can be induced to differentiate by culturing dopaminergic neuron progenitor cells in a medium containing B27, BDNF, GDNF, ascorbic acid, TGF-β, cAMP and DAPT. The present invention also provides a method for inducing differentiation of such a dopaminergic neuron (ie, a method for producing a dopaminergic neuron) and a dopaminergic neuron produced by the method.
　ドパミン産生神経細胞は、細胞内に含有されるドパミンによって同定されうる。 Dopaminergic neurons can be identified by dopamine contained in the cells.
＜神経疾患治療剤のスクリーニングへの利用＞
　本発明の神経系未分化細胞（神経幹細胞等）、運動神経前駆細胞、運動神経細胞、ドパミン産生神経前駆細胞及びドパミン産生神経細胞は、神経疾患治療用化合物(例えば医薬化合物、溶媒、小分子、ペプチド、またはポリヌクレオチド)のスクリーニングに用いることもできる。例えば、単独でまたは他の薬剤と組み合わせて、候補医薬化合物を、神経系未分化細胞（神経幹細胞等）、運動神経前駆細胞、運動神経細胞、ドパミン産生神経前駆細胞又はドパミン産生神経細胞に加えることによる、当該細胞の形態または機能的な変化により、評価を行うことができる。機能的な変化の例として、当該細胞から産生されるドパミンの量を計測することで行うことができる。ここで、神経系未分化細胞（神経幹細胞等）、運動神経前駆細胞、運動神経細胞、ドパミン産生神経前駆細胞及びドパミン産生神経細胞は、治療対象となる神経疾患と同様の表現型を呈する細胞が好ましく、特に好ましくは、疾患の罹患由来の体細胞から作製された人工多能性幹細胞もしくは疾患由来の体細胞の核を移植されたntES細胞を分化誘導した人工神経系未分化細胞、人工運動神経前駆細胞、人工運動神経細胞、人工ドパミン産生神経前駆細胞及び人工ドパミン産生神経細胞である。 <Use for screening of therapeutic agents for neurological diseases>
Neural undifferentiated cells (neural stem cells and the like), motor neural progenitor cells, motor neuron cells, dopaminergic neuron progenitor cells and dopaminergic neuron cells of the present invention are compounds for treating neurological diseases (for example, pharmaceutical compounds, solvents, small molecules, It can also be used for screening of peptides or polynucleotides. For example, adding a candidate pharmaceutical compound alone or in combination with other drugs to neural undifferentiated cells (such as neural stem cells), motor neural progenitor cells, motor neuron cells, dopaminergic neural progenitor cells or dopaminergic neuron cells The evaluation can be performed based on the morphological or functional change of the cell. As an example of the functional change, it can be performed by measuring the amount of dopamine produced from the cell. Here, neural undifferentiated cells (neural stem cells and the like), motor neural progenitor cells, motor neuron cells, dopaminergic neuron progenitor cells and dopaminergic neuron cells are cells that exhibit the same phenotype as the neurological disease to be treated. Preferably, particularly preferably, artificial pluripotent stem cells prepared from somatic cells derived from disease or artificially differentiated cells of the artificial nervous system induced by differentiation of ntES cells transplanted with nuclei of somatic cells derived from diseases, artificial motor neurons A progenitor cell, an artificial motor neuron, an artificial dopamine-producing neural progenitor cell, and an artificial dopamine-producing neuron.
　上記方法３および４で示した方法においては、プルモルファミンの代わりにＳＡＧ（Ｎ－メチル－Ｎ′－（３－ピリジニルベンジル）－Ｎ′－（３－クロロベンゾ［ｂ］チオフェン－２－カルボニル）－１，４－ジアミノシクロヘキサン）を用いることもできる。ＳＡＧを用いる場合の、培地中のＳＡＧ濃度は、運動神経前駆細胞またはドパミン産生神経前駆細胞を分化誘導する範囲で特に限定されないが、通常１０ｎＭ～１００μＭ、好ましくは１００ｎＭ～１０μＭ、より好ましくは１００ｎＭ～２μＭである。 In the methods shown in the above methods 3 and 4, SAG (N-methyl-N ′-(3-pyridinylbenzyl) -N ′-(3-chlorobenzo [b] thiophene-2- Carbonyl) -1,4-diaminocyclohexane) can also be used. The concentration of SAG in the medium when SAG is used is not particularly limited as long as it induces differentiation of motor neuron progenitor cells or dopaminergic neuron progenitor cells, but is usually 10 nM to 100 μM, preferably 100 nM to 10 μM, more preferably 100 nM to 2 μM.
＜再生医療への応用＞
　本発明の神経系未分化細胞（神経幹細胞等）、運動神経前駆細胞、運動神経細胞、ドパミン産生神経前駆細胞及びドパミン産生神経細胞は、損傷した神経系組織の正常化のために再生医療の分野で有効に使用し得る。それゆえ、この細胞は、種々の神経系細胞の障害に関係する疾患の治療用細胞になり得る。 <Application to regenerative medicine>
The undifferentiated nervous system cells (neural stem cells and the like), motor neural progenitor cells, motor neuron cells, dopaminergic neuron progenitor cells and dopaminergic neuron cells of the present invention are used in the field of regenerative medicine for normalizing damaged nervous system tissues Can be used effectively. Therefore, this cell can be a therapeutic cell for diseases related to various nervous system cell disorders.
　疾患の例としては、虚血性脳疾患(脳卒中など)、脳外傷、脊髄損傷、運動神経疾患、神経変性疾患、網膜色素変性症、加齢黄斑変性症、内耳性難聴、多発性硬化症、筋萎縮性側索硬化症、脊髄小脳変性症、ハンチントン舞踏病、アルツハイマー病、パーキンソン病、てんかん、および統合失調症などが挙げられる。 Examples of diseases include ischemic brain diseases (such as stroke), brain trauma, spinal cord injury, motor neuropathy, neurodegenerative diseases, retinitis pigmentosa, age-related macular degeneration, inner ear deafness, multiple sclerosis, muscle Examples include amyotrophic lateral sclerosis, spinocerebellar degeneration, Huntington's chorea, Alzheimer's disease, Parkinson's disease, epilepsy, and schizophrenia.
　また、細胞を治療薬として用いる場合、細胞の純度を高めることが望ましい。このための方法には、目的の細胞を選別する方法、例えばフローサイトメトリー法、抗癌剤含有培地での処理などが挙げられる。フローサイトメトリー法は、非常に細い流液中に細胞粒子を高速度で流し、レーザー光を照射して、粒子が発生する蛍光(細胞が予め蛍光標識された場合)、散乱光などの光を測定するものであり、セルソーターを備えると、目的の細胞を選別・分離することができる。細胞の蛍光標識は、神経系未分化細胞、運動神経前駆細胞、運動神経細胞、ドパミン産生神経前駆細胞及びドパミン産生神経細胞にそれぞれ特異的な抗体(蛍光標識化)、（神経幹細胞については、例えば抗Nestin抗体）によって行うことができる。また、抗癌剤含有培地での処理によって、未分化細胞を除去することができる。抗癌剤の例は、マイトマイシンC、5－フルオロウラシル、アドリアマイシン、メトトレキセートなどである。 Also, when cells are used as therapeutic agents, it is desirable to increase the purity of the cells. Examples of the method for this purpose include a method for selecting a target cell, for example, a flow cytometry method, treatment with an anticancer agent-containing medium, and the like. In the flow cytometry method, cell particles are flowed at a high speed in a very thin flow, irradiated with laser light, and light such as fluorescence (when cells are pre-fluorescently labeled) and scattered light are emitted. If a cell sorter is provided for measurement, target cells can be selected and separated. Fluorescent labeling of cells includes antibodies (fluorescent labeling) specific for neural undifferentiated cells, motor neural progenitor cells, motor neuron cells, dopaminergic neuron progenitor cells and dopaminergic neuron cells, respectively ( Anti-Nestin antibody). In addition, undifferentiated cells can be removed by treatment with an anticancer agent-containing medium. Examples of anticancer agents are mitomycin C, 5-fluorouracil, adriamycin, methotrexate and the like.
　神経系未分化細胞、運動神経前駆細胞、運動神経細胞、ドパミン産生神経前駆細胞及びドパミン産生神経細胞の疾患部位への移植は、例えば、Nature Neuroscience，2，1137(1999)もしくはN Engl J Med. ;344:710-9(2001)に記載されるような手法によって行うことができる。 For example, Nature Neuroscience, 2, 1137 (1999) or N Engl J Med. ; 344: 710-9 (2001).
　本明細書中で挙げられた特許及び特許出願明細書を含む全ての刊行物に記載された内容は、本明細書での引用により、その全てが明示されたと同程度に本明細書に組み込まれるものである。 The contents of all publications, including patents and patent application specifications cited in this specification, are hereby incorporated by reference herein to the same extent as if all were explicitly stated. Is.
　以下、実施例を示して本発明をより具体的に説明するが、本発明は以下に示す実施例によって何ら限定されるものではない。 Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the examples shown below.
［実施例１］
3D(3 Drugs)-iPSの誘導
　ヒトiPS細胞としては、ヒト線維芽細胞へ、レトロウイルスによりOct3/4、Sox2、Klf4及びc-Mycを導入することにより得られたWD39を用いた。また、ヒトT細胞へ、センダイウイルスによりOct3/4、Sox2、Klf4及びc-Mycを導入することにより得られたaTKA4も併せて用いた。ヒトiPS細胞の継代培地としては、20% KSR、1% NEAA、0.1%メルカプトエタノール、penicillin(50u/ml)/streptomycin(50μg/ml)、2mM glutamine及び4ng/ml bFGFを含有するDMEM/F12を用いた。
　3μM SB431542、3μM CHIR99021及び3μM dorsomorphinを加えた上記継代培地中でヒトiPS細胞を5日以上培養することにより、3D-iPS細胞を得た。3D-iPS細胞のコロニーは、通常の継代培地で培養したヒトiPS細胞と比較してより立体的な形態を呈していたことから（図１）、SB431542、CHIR99021及びdorsomorphin処理によりヒトiPS細胞はより分化した多能性細胞となったと考えられた。
　尚、3D-iPS細胞は、２回継代後も、多能性マーカー(Oct3/4、SSEA4、Tra-1-60及びTra-1-81)を発現することが免疫組織染色により確認された。従って、3D-iPS細胞は、多能性と、良好な神経分化能を維持したまま、継代可能であることが示唆された。 [Example 1]
WD39 obtained by introducing Oct3 / 4, Sox2, Klf4, and c-Myc into human fibroblasts by retrovirus was used as human iPS cells inducing 3D (3 Drugs) -iPS . In addition, aTKA4 obtained by introducing Oct3 / 4, Sox2, Klf4 and c-Myc into human T cells by Sendai virus was also used. As a passage medium for human iPS cells, DMEM / F12 containing 20% KSR, 1% NEAA, 0.1% mercaptoethanol, penicillin (50 u / ml) / streptomycin (50 μg / ml), 2 mM glutamine and 4 ng / ml bFGF. Was used.
3D-iPS cells were obtained by culturing human iPS cells in the above passage medium supplemented with 3 μM SB431542, 3 μM CHIR99021 and 3 μM dorsomorphin for 5 days or more. The colony of 3D-iPS cells exhibited a more three-dimensional morphology compared to human iPS cells cultured in a normal passage medium (Fig. 1). Therefore, human iPS cells were treated with SB431542, CHIR99021 and dorsomorphin. It was thought that it became a more differentiated pluripotent cell.
It was confirmed by immunohistochemical staining that 3D-iPS cells expressed pluripotency markers (Oct3 / 4, SSEA4, Tra-1-60 and Tra-1-81) even after two passages. . Therefore, it was suggested that 3D-iPS cells can be passaged while maintaining pluripotency and good nerve differentiation ability.
［実施例２］
ヒトiPS細胞からのニューロスフェアの形成
　ヒトiPS細胞としては、線維芽細胞由来のWD39及びT細胞由来のaTKA4を用いた。
　ConfluentになったヒトiPS細胞の培養上清を吸い取り、6mlのPBSで洗浄した。解離液Tを1ml加えたのち、すぐに吸い取り、5分程度インキュベートした。顕微鏡ではがれ具合を確認し、6mlのhES-medium（20% KSR、1% NEAA、0.1% メルカプトエタノール、penicillin(50u/ml)/streptomycin(50μg/ml)、2mM glutamine及び4ng/ml bFGFを含有するDMEM/F12）を加えて細胞を50mlチューブに回収した。iPS細胞を1000rpmで5分遠心をし、上清を吸い取った。10mlのhES-medium (ROCK-inhibitor Y27632 (+))を加え、ピペッティングをしてゼラチンコートディッシュに蒔いて、1～2時間インキュベートした。顕微鏡でSTO細胞の張り付き具合を確認し、ディッシュを回して、浮遊している細胞を中心に集め、50mlチューブに回収した。細胞を1000rpmで5分遠心し、上清を吸い取った。細胞のペレットに、Tryple Selectを1ml加え、37℃ウォーターバスで5分間インキュベートし、時々チューブを回して撹拌した。このインキュベーションの間、Trypsin Inhibitor 2mlを15mlチューブに入れておき、セルストレイナー (70μｍ)を用意しておいた。Tryple Select消化産物をP1000のピペットマンで10回程度ピペッティングした後、Trypsin Inhibitorに加え、MHMでボリュームアップしてCell Strainerで濾過した。細胞を5分遠心し、上清を吸い取り、3～5mlのMHMを加えた。細胞数をカウントし、MHM培地を加えて104 cells/mlの濃度に調整した。
　回収したヒトiPS細胞を、B27、20ng/ml bFGF、10ng/ml hLIF、及び10μM Y27632を含むMHM培地中で14日間浮遊培養することによりニューロスフェアを形成した（ここではこの方法をSingle Cell Floating Neural Differentiation (SND) 法という。）。このSND法における培地に、更に3μM CHIR99021及び2μM SB431542を添加した条件（GSKi+TGF-βi条件）でも、ニューロスフェアの形成を実施した。形成されたニューロスフェアの神経分化能は、PO/fibronectin コートplate上でＢ27を含有するMHM培地中、１０日間培養することにより確認した。
　その結果、GSKi+TGF-βi条件では、従来のSND法よりも早く、培養開始6日の時点で、神経分化可能なニューロスフェアが形成された（図２）。この結果から、GSKi+TGF-βi条件（CHIR99021及びSB431542の添加）により、浮遊培養においてもiPS細胞からの神経系未分化細胞（神経幹細胞等）の分化誘導が促進されることが示唆された。 [Example 2]
Formation of neurospheres from human iPS cells As human iPS cells, WD39 derived from fibroblasts and aTKA4 derived from T cells were used.
The culture supernatant of human iPS cells that became confluent was aspirated and washed with 6 ml of PBS. After adding 1 ml of the dissociation solution T, it was immediately sucked out and incubated for about 5 minutes. Confirmed with a microscope, 6ml of hES-medium (20% KSR, 1% NEAA, 0.1% mercaptoethanol, penicillin (50u / ml) / streptomycin (50μg / ml), 2mM glutamine and 4ng / ml bFGF DMEM / F12) was added and the cells were collected in 50 ml tubes. iPS cells were centrifuged at 1000 rpm for 5 minutes, and the supernatant was aspirated. 10 ml of hES-medium (ROCK-inhibitor Y27632 (+)) was added, pipetted and spread onto gelatin coated dishes and incubated for 1-2 hours. After confirming the sticking state of the STO cells with a microscope, the dish was rotated to collect the floating cells at the center and collected in a 50 ml tube. The cells were centrifuged at 1000 rpm for 5 minutes and the supernatant was aspirated. To the cell pellet, 1 ml of Tryple Select was added, incubated in a 37 ° C. water bath for 5 minutes, and occasionally stirred by turning the tube. During this incubation, 2 ml of Trypsin Inhibitor was placed in a 15 ml tube and a cell strainer (70 μm) was prepared. The Tryple Select digested product was pipetted about 10 times with a P1000 pipetteman, then added to Trypsin Inhibitor, volumed up with MHM, and filtered with Cell Strainer. The cells were centrifuged for 5 minutes, the supernatant was aspirated and 3-5 ml of MHM was added. The number of cells was counted and adjusted to a concentration of 10 4 cells / ml by adding MHM medium.
The collected human iPS cells were suspended in MHM medium containing B27, 20 ng / ml bFGF, 10 ng / ml hLIF, and 10 μM Y27632 for 14 days to form neurospheres (here, this method was used as a single cell floating neural network). Differentiation (SND) method). Neurospheres were also formed under conditions (GSKi + TGF-βi conditions) in which 3 μM CHIR99021 and 2 μM SB431542 were further added to the medium in this SND method. The neurosphere differentiation ability of the formed neurosphere was confirmed by culturing for 10 days in an MHM medium containing B27 on a PO / fibronectin-coated plate.
As a result, under the GSKi + TGF-βi condition, neurospheres capable of neuronal differentiation were formed 6 days after the start of culture, faster than the conventional SND method (FIG. 2). From this result, it was suggested that GSKi + TGF-βi conditions (addition of CHIR99021 and SB431542) promote differentiation induction of undifferentiated neural cells (neural stem cells, etc.) from iPS cells even in suspension culture.
［実施例３］
CHIR99021及びSB431542の添加タイミングの検討
　実施例２と同様に、3μM CHIR99021及び2μM SB431542を添加した条件で、SND法によりヒトiPS細胞を６日間培養することにより、神経幹細胞を含むニューロスフェアを形成した。その後、形成されたニューロスフェアの神経分化能を、実施例２と同様に評価した。ここで、SND法によるニューロスフェアの誘導が既に始まった後で、CHIR99021及びSB431542を添加し、添加のタイミングと神経幹細胞／神経系分化細胞の誘導効率との関係を調べた。
　その結果、CHIR99021及びSB431542をニューロスフェア誘導開始２日目に添加した場合よりも、これらをニューロスフェア誘導開始４日目に添加した場合のほうが、神経幹細胞／神経系分化細胞の誘導効率が低かった（図３）。
　これらの結果から、CHIR99021及びSB431542は、ニューロスフェア誘導培養のなるべく早い時点（好ましくはニューロスフェア誘導培養開始時）から培地に添加するほうが、神経幹細胞／神経系分化細胞の誘導効率が高いことが示唆された。 [Example 3]
Examination of addition timing of CHIR99021 and SB431542 As in Example 2, neurospheres containing neural stem cells were formed by culturing human iPS cells by SND method for 6 days under the condition of adding 3 μM CHIR99021 and 2 μM SB431542. Then, the neurodifferentiation ability of the formed neurosphere was evaluated in the same manner as in Example 2. Here, after induction of neurospheres by the SND method has already started, CHIR99021 and SB431542 were added, and the relationship between the timing of addition and the induction efficiency of neural stem cells / differentiated cells of the nervous system was examined.
As a result, the induction efficiency of neural stem cells / differentiated cells was lower when CHIR99021 and SB431542 were added on day 4 of neurosphere induction than when CHIR99021 and SB431542 were added on day 4 of neurosphere induction. (Figure 3).
From these results, it is suggested that CHIR99021 and SB431542 have higher induction efficiency of neural stem cells / neural system differentiated cells when added to the medium from the earliest possible time of neurosphere-induced culture (preferably at the start of neurosphere-induced culture). It was done.
［実施例４］
低酸素及びY27632の神経幹細胞分化に与える影響
　実施例２と同様に、3μM CHIR99021及び2μM SB431542を培養開始時から添加した条件で、SND法によりヒトiPS細胞を６日間培養することにより、神経幹細胞を含むニューロスフェアを形成した。その後、形成されたニューロスフェアの神経分化能を、実施例２と同様に評価した。ここで、この方法による神経幹細胞分化に与える低酸素及びROCK阻害剤（Y27632）の影響を評価した。
　その結果、ROCK阻害剤（Y27632）の添加により、神経幹細胞からの神経分化に有意な変化は認められなかった。一方、低酸素条件下では、GFAP陽性細胞の誘導が促進された（図４）。
　これらの結果からROCK阻害は、神経幹細胞への分化誘導に影響しないこと、低酸素はアストログリアへの分化誘導を促進することが示唆された。 [Example 4]
Effects of hypoxia and Y27632 on neural stem cell differentiation As in Example 2, under the condition that 3 μM CHIR99021 and 2 μM SB431542 were added from the beginning of the culture, human iPS cells were cultured for 6 days by the SND method. A neurosphere containing was formed. Then, the neurodifferentiation ability of the formed neurosphere was evaluated in the same manner as in Example 2. Here, the influence of hypoxia and ROCK inhibitor (Y27632) on neural stem cell differentiation by this method was evaluated.
As a result, the addition of the ROCK inhibitor (Y27632) showed no significant change in neural differentiation from neural stem cells. On the other hand, induction of GFAP positive cells was promoted under hypoxic conditions (FIG. 4).
These results suggest that ROCK inhibition does not affect differentiation induction into neural stem cells, and that hypoxia promotes differentiation induction into astroglia.
［実施例５］
3D-iPS細胞からのニューロスフェアの形成
　実施例１に記載の方法によりヒト3D-iPS細胞を調製した。
　Confluentになったヒト3D-iPS細胞の培養上清を吸い取り、6mlのPBSで洗浄した。解離液Tを1ml加えたのち、すぐに吸い取り、5分程度インキュベートした。顕微鏡ではがれ具合を確認し、6mlのhES-medium（20% KSR、1% NEAA、0.1% メルカプトエタノール、penicillin(50u/ml)/streptomycin(50μg/ml)、2mM glutamine及び4ng/ml bFGFを含有するDMEM/F12）を加えて細胞を50mlチューブに回収した。iPS細胞を1000rpmで5分遠心をし、上清を吸い取った。10mlのhES-medium (ROCK-inhibitor Y27632 (+))を加え、ピペッティングをしてゼラチンコートディッシュに蒔いて、1～2時間インキュベートした。顕微鏡でSTO細胞の張り付き具合を確認し、ディッシュを回して、浮遊している細胞を中心に集め、50mlチューブに回収した。細胞を1000rpmで5分遠心し、上清を吸い取った。細胞のペレットに、Tryple Selectを1ml加え、37℃ウォーターバスで5分間インキュベートし、時々チューブを回して撹拌した。このインキュベーションの間、Trypsin Inhibitor 2mlを15mlチューブに入れておき、セルストレイナー (70μｍ)を用意しておいた。Tryple Select消化産物をP1000のピペットマンで10回程度ピペッティングした後、Trypsin Inhibitorに加え、MHMでボリュームアップしてCell Strainerで濾過した。細胞を5分遠心し、上清を吸い取り、3～5mlのMHMを加えた。細胞数をカウントし、MHM培地を加えて104 cells/mlの濃度に調整した。
　回収したヒト3D-iPS細胞を、B27、20ng/ml bFGF、10ng/ml hLIF、10μM Y27632、3μM CHIR99021、及び2μM SB431542を含むMHM培地中で、低酸素（3% O2）条件下、培養した（SND法を改変）。
　その結果、培養開始３日で、神経分化可能なニューロスフェアを誘導することに成功した。また、培養開始４日でニューロスフェアからアストロサイトを誘導することができた（図５）。通常のヒトiPS細胞を用いたときにはニューロスフェア形成に6日を要したことから、3D- iPS細胞を用いたときの方が、より短期間でニューロスフェアを形成できることが分かった。 [Example 5]
Formation of neurospheres from 3D-iPS cells Human 3D-iPS cells were prepared by the method described in Example 1.
The culture supernatant of human 3D-iPS cells that became confluent was aspirated and washed with 6 ml of PBS. After adding 1 ml of the dissociation solution T, it was immediately sucked out and incubated for about 5 minutes. Confirmed with a microscope, 6ml hES-medium (20% KSR, 1% NEAA, 0.1% mercaptoethanol, penicillin (50u / ml) / streptomycin (50μg / ml), 2mM glutamine and 4ng / ml bFGF DMEM / F12) was added and the cells were collected in 50 ml tubes. iPS cells were centrifuged at 1000 rpm for 5 minutes, and the supernatant was aspirated. 10 ml of hES-medium (ROCK-inhibitor Y27632 (+)) was added, pipetted and spread onto gelatin coated dishes and incubated for 1-2 hours. After confirming the sticking state of the STO cells with a microscope, the dish was rotated to collect the floating cells at the center and collected in a 50 ml tube. The cells were centrifuged at 1000 rpm for 5 minutes and the supernatant was aspirated. To the cell pellet, 1 ml of Tryple Select was added, incubated in a 37 ° C. water bath for 5 minutes, and occasionally stirred by turning the tube. During this incubation, 2 ml of Trypsin Inhibitor was placed in a 15 ml tube and a cell strainer (70 μm) was prepared. The Tryple Select digested product was pipetted about 10 times with a P1000 pipetteman, then added to Trypsin Inhibitor, volumed up with MHM, and filtered with Cell Strainer. The cells were centrifuged for 5 minutes, the supernatant was aspirated and 3-5 ml of MHM was added. The number of cells was counted and adjusted to a concentration of 10 4 cells / ml by adding MHM medium.
The collected human 3D-iPS cells were cultured under low oxygen (3% O 2 ) conditions in MHM medium containing B27, 20 ng / ml bFGF, 10 ng / ml hLIF, 10 μM Y27632, 3 μM CHIR99021, and 2 μM SB431542. (SND method modified).
As a result, neurospheres capable of neuronal differentiation were successfully induced 3 days after the start of culture. In addition, astrocytes could be induced from the neurosphere 4 days after the start of culture (FIG. 5). When normal human iPS cells were used, neurosphere formation took 6 days. Therefore, it was found that neurospheres can be formed in a shorter period of time when 3D-iPS cells are used.
［実施例６］
運動ニューロンの誘導
　実施例５と同様に、実施例１に記載の方法で作成したヒト3D-iPS細胞を、B27、20ng/ml bFGF、10ng/ml hLIF、10μM Y27632、3μM CHIR99021、及び2μM SB431542を含むMHM培地中で、低酸素（3% O2）条件下、培養した。培養開始３日目に、1μM レチノイン酸、及び1μM purmorphamineを培地に加え、更に培養することにより、自己複製可能な、運動ニューロン前駆細胞（ＭＰＣ）を含むニューロスフェアが形成された。 [Example 6]
Induction of motor neurons Like Example 5, human 3D-iPS cells prepared by the method described in Example 1 were treated with B27, 20 ng / ml bFGF, 10 ng / ml hLIF, 10 μM Y27632, 3 μM CHIR99021, and 2 μM SB431542. Culturing was carried out under low oxygen (3% O 2 ) conditions in the MHM medium containing. On the third day from the start of culture, 1 μM retinoic acid and 1 μM purmorphamine were added to the medium, and further cultured to form self-replicating neurospheres including motor neuron progenitor cells (MPC).
［実施例７］
運動ニューロンの誘導２
　実施例５と同様に、実施例１に記載の方法で作成したヒト3D iPS細胞を、B27、20ng/ml bFGF、10ng/ml hLIF、10μM Y27632、3μM CHIR99021、及び2μM SB431542を含むMHM培地中で、低酸素（3% O2）条件下、培養した。培養開始３日目に、1μM レチノイン酸、及び1μM purmorphamineを培地に加え、更に4日間培養することにより、自己複製可能な、運動ニューロン前駆細胞（ＭＰＣ）を含むニューロスフェアが形成された。
　MPCを含むニューロスフィアをN2, Glutamax, 10ng/ml IGF-1, 10ng/ml GDNF, 10ng/ml BDNF, 200ng/ml アスコルビン酸、50nM レチノイン酸、50ng/ml Shh、1μM cAMPを含むDMEM/F12へ培地を変更し、PO/lamininコートplate上で更に10日間培養を継続した。
　その結果、高い効率で運動ニューロンが誘導された（図６）。 [Example 7]
Induction of motor neurons 2
As in Example 5, human 3D iPS cells prepared by the method described in Example 1 were cultured in MHM medium containing B27, 20 ng / ml bFGF, 10 ng / ml hLIF, 10 μM Y27632, 3 μM CHIR99021, and 2 μM SB431542. The cells were cultured under hypoxic (3% O 2 ) conditions. On the 3rd day from the start of the culture, 1 μM retinoic acid and 1 μM purmorphamine were added to the medium, and further cultured for 4 days to form neurospheres including motor neuron progenitor cells (MPC) capable of self-replication.
Neurospheres containing MPC are transferred to DMEM / F12 containing N2, Glutamax, 10ng / ml IGF-1, 10ng / ml GDNF, 10ng / ml BDNF, 200ng / ml ascorbic acid, 50nM retinoic acid, 50ng / ml Shh, 1μM cAMP The medium was changed, and the culture was continued on the PO / laminin-coated plate for another 10 days.
As a result, motor neurons were induced with high efficiency (FIG. 6).
［実施例８］
ヒト3D iPS細胞の遺伝子発現パターン解析
　ヒト線維芽細胞由来iPS細胞２ライン（WD39、B7）を実施例１に準じて3μM SB431542、3μM CHIR99021及び3μM dorsomorphinの存在下で５又は６日間培養することにより3D-iPS細胞を作成し、その性質をqRT-PCRによるマーカー遺伝子の発現により確認した。
　結果を図７に示す。SB431542、CHIR99021及びdorsomorphinによる処理により、nanogの発現が減少し、Sox2（神経系マーカー）、nestin（神経幹細胞マーカー）、Sox1（神経系マーカー）、brachyury（中胚葉マーカー）及びSox17（内胚葉マーカー）の発現が亢進した。一方、Oct3/4の発現はほぼ不変であった。この結果から、3D-iPS細胞は、ナイーブというよりはむしろ分化傾向にあること、分化の方向は神経系特異的ではなく、３胚葉全てのマーカーの発現が上昇していることが示された。また3D-iPS細胞は、この発現パターンを維持しながら継代可能であった。 [Example 8]
Gene expression pattern analysis of human 3D iPS cells By culturing 2 lines of human fibroblast-derived iPS cells (WD39, B7) in the presence of 3 μM SB431542, 3 μM CHIR99021 and 3 μM dorsomorphin according to Example 1 for 5 or 6 days. 3D-iPS cells were prepared and their properties were confirmed by expression of marker genes by qRT-PCR.
The results are shown in FIG. Treatment with SB431542, CHIR99021, and dorsomorphin reduced nanog expression, Sox2 (nervous system marker), nestin (neural stem cell marker), Sox1 (neural system marker), brachyury (mesoderm marker) and Sox17 (endoderm marker) Expression was increased. On the other hand, Oct3 / 4 expression was almost unchanged. From this result, it was shown that 3D-iPS cells tend to differentiate rather than naive, and the direction of differentiation is not nervous system specific, and expression of all three germ layers is increased. 3D-iPS cells could be passaged while maintaining this expression pattern.
［実施例９］
T細胞由来iPS細胞からの神経幹細胞の誘導
　Seki et al. Cell Stem Cell, Volume 7, Issue 1, 11-14, 2 July(2010)およびNishimura　K et al. JBC 2011 11;286 4760-71に記載された方法に準じて、センダイウイルスを用いてヒトT細胞から誘導したiPS細胞（TiPS細胞）、及びNature Methods 8,409-412 (2011)に記載された方法に準じて、プラスミドベクターを用いてヒトT細胞から誘導したTiPS細胞を使用した。 [Example 9]
Induction of neural stem cells from T cell-derived iPS cells Seki et al. Cell Stem Cell, Volume 7, Issue 1, 11-14, 2 July (2010) and Nishimura K et al. JBC 2011 11; 286 4760-71 IPS cells (TiPS cells) derived from human T cells using Sendai virus according to the methods described above, and human T cells using plasmid vectors according to the methods described in Nature Methods 8,409-412 (2011) TiPS cells derived from the cells were used.
　ヒトTiPS細胞２ラインを、実施例１に準じて3μM SB431542、3μM CHIR99021及び3μM dorsomorphinの存在下で5日間培養することにより3D-TiPS細胞を作成した。 3D-TiPS cells were prepared by culturing 2 human TiPS cell lines in the presence of 3 μM SB431542, 3 μM CHIR99021, and 3 μM dorsomorphin according to Example 1 for 5 days.
　更に、3D-TiPS細胞を実施例５と同様に、B27、20ng/ml bFGF、10ng/ml hLIF、10μM Y27632、3μM CHIR99021、及び2μM SB431542を含むMHM培地中で、低酸素（3% O2）条件下で、３日間培養することにより、神経幹細胞を含むニューロスフェアが形成された。即ち、3日間で、TiPS細胞から、神経幹細胞が分化誘導された。 Further, in the same manner as in Example 5, 3D-TiPS cells were hypoxic (3% O 2 ) in MHM medium containing B27, 20 ng / ml bFGF, 10 ng / ml hLIF, 10 μM Y27632, 3 μM CHIR99021, and 2 μM SB431542. Neurospheres containing neural stem cells were formed by culturing under conditions for 3 days. That is, neural stem cells were induced to differentiate from TiPS cells in 3 days.
　一方、ヒトTiPS細胞を、直接SND法により培養すると、神経幹細胞を含むニューロスフェアが誘導されるまでに14日間を要した。 On the other hand, when human TiPS cells were directly cultured by the SND method, it took 14 days to induce neurospheres containing neural stem cells.
［実施例１０］
ドパミン産生神経前駆細胞及びドパミン産生神経細胞の誘導
　実施例５と同様に、実施例１に記載の方法で作成したヒト3D-iPS細胞（線維芽細胞由来及びＴ細胞由来）を、B27、20ng/ml bFGF、10ng/ml hLIF、10μM Y27632、3μM CHIR99021、及び2μM SB431542を含むMHM培地中で、低酸素（3% O2）条件下、培養した。培養開始２日目に、2μM プルモルファミン、100ng/ml Sonic hedgehog及び100ng/ml FGF8を培地に加え、更に4日間培養することにより、自己複製可能な、ドパミン産生ニューロン前駆細胞を含むニューロスフェアが形成された。 [Example 10]
Induction of dopaminergic neural progenitor cells and dopaminergic neuron cells As in Example 5, human 3D-iPS cells (derived from fibroblasts and T cells) prepared by the method described in Example 1 were B27, 20 ng / The cells were cultured under low oxygen (3% O 2 ) conditions in MHM medium containing ml bFGF, 10 ng / ml hLIF, 10 μM Y27632, 3 μM CHIR99021, and 2 μM SB431542. On the second day from the start of culture, 2 μM purmorphamine, 100 ng / ml Sonic hedgehog and 100 ng / ml FGF8 were added to the medium, and further cultured for 4 days. Been formed.
　ドパミン産生ニューロン前駆細胞を含むニューロスフィアを2mM グルタミン、B27、20ng/ml BDNF、20ng/ml GDNF、0.2mM アスコルビン酸、1ng/ml TGF-β3、0.5mM dibutyryl cAMP及び10nM DAPTを含むNeurobasal培地中、PO/lamininコートplate上で更に８～１０日間培養を継続した。
　その結果、高い効率でドパミン産生ニューロンが誘導された（図８）。 Neurospheres containing dopaminergic neuron progenitor cells in Neurobasal medium containing 2 mM glutamine, B27, 20 ng / ml BDNF, 20 ng / ml GDNF, 0.2 mM ascorbic acid, 1 ng / ml TGF-β3, 0.5 mM dibutyryl cAMP and 10 nM DAPT, Culturing was continued on PO / laminin-coated plates for an additional 8-10 days.
As a result, dopaminergic neurons were induced with high efficiency (FIG. 8).
［実施例１１］
ヒトＢ細胞からの3D-iPSの誘導
　ヒトiPS細胞としては、ヒトＢリンパ芽球へ、レトロウイルスによりOct3/4、Sox2、Klf4及びc-Mycを導入することにより得られたiPS細胞（LiPS細胞）を用いる。ヒトLiPS細胞の継代培地としては、20% KSR、1% NEAA、0.1%メルカプトエタノール、penicillin(50u/ml)/streptomycin(50μg/ml)、2mM glutamine及び4ng/ml bFGFを含有するDMEM/F12を用いる。
　3μM SB431542、3μM CHIR99021及び3μM dorsomorphinを加えた上記継代培地中でヒトiPS細胞を5日以上培養することにより、3D-LiPS細胞を得る。3D-LiPS細胞は、多能性と、良好な神経分化能を維持したまま、継代可能であり得る。 [Example 11]
Induction of 3D-iPS from human B cells As human iPS cells, iPS cells (LiPS cells) obtained by introducing Oct3 / 4, Sox2, Klf4 and c-Myc by retrovirus into human B lymphoblasts. ) Is used. As a passage medium for human LiPS cells, DMEM / F12 containing 20% KSR, 1% NEAA, 0.1% mercaptoethanol, penicillin (50 u / ml) / streptomycin (50 μg / ml), 2 mM glutamine and 4 ng / ml bFGF. Is used.
By culturing human iPS cells in the above passage medium supplemented with 3 μM SB431542, 3 μM CHIR99021 and 3 μM dorsomorphin for 5 days or longer, 3D-LiPS cells are obtained. 3D-LiPS cells can be passaged while maintaining pluripotency and good neuronal differentiation potential.
［実施例１２］
ヒトLiPS細胞からのニューロスフェアの形成
　実施例１１で得られるヒト3D-LiPS細胞を用いる。
　Confluentになったヒト3D-LiPS細胞の培養上清を吸い取り、6mlのPBSで洗浄する。解離液Tを1ml加えたのち、すぐに吸い取り、5分程度インキュベートする。顕微鏡ではがれ具合を確認し、6mlのhES-medium（20% KSR、1% NEAA、0.1% メルカプトエタノール、penicillin(50u/ml)/streptomycin(50μg/ml)、2mM glutamine及び4ng/ml bFGFを含有するDMEM/F12）を加えて細胞を50mlチューブに回収する。3D-LiPS細胞を1000rpmで5分遠心をし、上清を吸い取った。10mlのhES-medium (ROCK-inhibitor Y27632 (+))を加え、ピペッティングをしてゼラチンコートディッシュに蒔いて、1～2時間インキュベートする。顕微鏡でSTO細胞の張り付き具合を確認し、ディッシュを回して、浮遊している細胞を中心に集め、50mlチューブに回収する。細胞を1000rpmで5分遠心し、上清を吸い取る。細胞のペレットに、Tryple Selectを1ml加え、37℃ウォーターバスで5分間インキュベートし、時々チューブを回して撹拌した。このインキュベーションの間、Trypsin Inhibitor 2mlを15mlチューブに入れておき、セルストレイナー (70μｍ)を用意しておいた。Tryple Select消化産物をP1000のピペットマンで10回程度ピペッティングした後、Trypsin Inhibitorに加え、MHMでボリュームアップしてCell Strainerで濾過する。細胞を5分遠心し、上清を吸い取り、3～5mlのMHMを加えた。細胞数をカウントし、MHM培地を加えて104 cells/mlの濃度に調整する。
　回収したヒトiPS細胞を、B27、20ng/ml bFGF、10ng/ml hLIF、10μM Y27632、3μM CHIR99021及び2μM SB431542を含むMHM培地中で10日間浮遊培養することにより神経幹細胞を含むニューロスフェアを得る。 [Example 12]
Formation of neurospheres from human LiPS cells The human 3D-LiPS cells obtained in Example 11 are used.
Aspirate the culture supernatant of human 3D-LiPS cells that have become Confluent and wash with 6 ml of PBS. After adding 1 ml of dissociation solution T, immediately absorb and incubate for about 5 minutes. Confirmed with a microscope, 6ml of hES-medium (20% KSR, 1% NEAA, 0.1% mercaptoethanol, penicillin (50u / ml) / streptomycin (50μg / ml), 2mM glutamine and 4ng / ml bFGF Add DMEM / F12) to collect the cells in a 50 ml tube. 3D-LiPS cells were centrifuged at 1000 rpm for 5 minutes, and the supernatant was aspirated. Add 10 ml of hES-medium (ROCK-inhibitor Y27632 (+)), pipet and spread on gelatin coated dish and incubate for 1-2 hours. Use a microscope to check the adhesion of STO cells, rotate the dish, collect floating cells at the center, and collect them in a 50 ml tube. Centrifuge the cells for 5 minutes at 1000 rpm and aspirate the supernatant. To the cell pellet, 1 ml of Tryple Select was added, incubated in a 37 ° C. water bath for 5 minutes, and occasionally stirred by turning the tube. During this incubation, 2 ml of Trypsin Inhibitor was placed in a 15 ml tube and a cell strainer (70 μm) was prepared. Pipette the Tryple Select digested product about 10 times with a P1000 pipetteman, add to Trypsin Inhibitor, volume up with MHM, and filter with Cell Strainer. The cells were centrifuged for 5 minutes, the supernatant was aspirated and 3-5 ml of MHM was added. Count the number of cells and add MHM medium to adjust the concentration to 10 4 cells / ml.
The collected human iPS cells are subjected to suspension culture in MHM medium containing B27, 20 ng / ml bFGF, 10 ng / ml hLIF, 10 μM Y27632, 3 μM CHIR99021 and 2 μM SB431542 for 10 days to obtain neurospheres containing neural stem cells.
［実施例１３］
ヒトLiPS細胞からの運動ニューロン前駆細胞及び運動ニューロンの誘導
　実施例５と同様に、実施例１１に記載の方法で作成したヒト3D-LiPS細胞を、B27、20ng/ml bFGF、10ng/ml hLIF、10μM Y27632、3μM CHIR99021、及び2μM SB431542を含むMHM培地中で、低酸素（3% O2）条件下、培養する。培養開始３日目に、1μM レチノイン酸、及び1μM purmorphamineを培地に加え、更に4日間培養することにより、自己複製可能な、運動ニューロン前駆細胞（ＭＰＣ）を含むニューロスフェアが形成される。
　MPCを含むニューロスフィアをN2, Glutamax, 10ng/ml IGF-1, 10ng/ml GDNF, 10ng/ml BDNF, 200ng/ml アスコルビン酸、50nM レチノイン酸、50ng/ml Shh、1μM cAMPを含むDMEM/F12へ培地を変更し、PO/lamininコートplate上で更に10日間培養を継続することにより、運動ニューロンが誘導される。 [Example 13]
Induction of motor neuron progenitor cells and motor neurons from human LiPS cells As in Example 5, human 3D-LiPS cells prepared by the method described in Example 11 were treated with B27, 20 ng / ml bFGF, 10 ng / ml hLIF, The cells are cultured under low oxygen (3% O 2 ) conditions in MHM medium containing 10 μM Y27632, 3 μM CHIR99021, and 2 μM SB431542. On the third day from the start of culture, 1 μM retinoic acid and 1 μM purmorphamine are added to the medium, and further cultured for 4 days to form neurospheres including motor neuron progenitor cells (MPCs) capable of self-replication.
Neurospheres containing MPC are transferred to DMEM / F12 containing N2, Glutamax, 10ng / ml IGF-1, 10ng / ml GDNF, 10ng / ml BDNF, 200ng / ml ascorbic acid, 50nM retinoic acid, 50ng / ml Shh, 1μM cAMP Motor neurons are induced by changing the medium and continuing the culture on PO / laminin-coated plates for another 10 days.
［実施例１４］
ヒトLiPS細胞からのドパミン産生神経前駆細胞及びドパミン産生神経細胞の誘導
　実施例５と同様に、実施例１１に記載の方法で作成したヒト3D-LiPS細胞（を、B27、20ng/ml bFGF、10ng/ml hLIF、10μM Y27632、3μM CHIR99021、及び2μM SB431542を含むMHM培地中で、低酸素（3% O2）条件下、培養する。培養開始２日目に、2μM プルモルファミン、100ng/ml Sonic hedgehog及び100ng/ml FGF8を培地に加え、更に4日間培養することにより、自己複製可能な、ドパミン産生ニューロン前駆細胞を含むニューロスフェアが形成される。 [Example 14]
Induction of dopaminergic neural progenitor cells and dopaminergic neurons from human LiPS cells In the same manner as in Example 5, human 3D-LiPS cells prepared by the method described in Example 11 (B27, 20 ng / ml bFGF, 10 ng / ml hLIF, 10 μM Y27632, 3 μM CHIR99021, and 2 μM SB431542 in MHM medium under hypoxia (3% O 2 ) condition 2 μM purmorphamine, 100 ng / ml Sonic By adding hedgehog and 100 ng / ml FGF8 to the medium and further culturing for 4 days, a neurosphere containing dopaminergic neuron progenitor cells capable of self-replication is formed.
　ドパミン産生ニューロン前駆細胞を含むニューロスフェアを2mM グルタミン、B27、20ng/ml BDNF、20ng/ml GDNF、0.2mM アスコルビン酸、1ng/ml TGF-β3、0.5mM dibutyryl cAMP及び10nM DAPTを含むNeurobasal培地中、PO/lamininコートplate上で更に８～１０日間培養を継続する。その結果、ドパミン産生ニューロンが誘導される。 Neurospheres containing dopaminergic neuron progenitor cells in Neurobasal medium containing 2 mM glutamine, B27, 20 ng / ml NFNF, 20 ng / ml NFNF, 0.2 mM ascorbic acid, 1 ng / ml TGF-β3, 0.5 mM dibutyryl cAMP and 10 nM DAPT, Continue culturing for an additional 8-10 days on PO / laminin coated plates. As a result, dopaminergic neurons are induced.
［参考例１］
　ヒトiPS細胞としては、PARK2患者由来のＢリンパ芽球へ、プラスミドベクターpCXLE-hOCT3/4-shp53, pCXLE-hSK, pCXLE-hUL (Okita et al. Nature Methods 8, 409-412 (2012)によりOct3/4、Sox2、Klf4, shp53, Lin28及びL-Mycを導入することにより得られたiPS細胞（LiPS細胞）を用いた。ヒトLiPS細胞の継代培地としては、20% KSR、1% NEAA、0.1%メルカプトエタノール、penicillin(50u/ml)/streptomycin(50μg/ml)、2mM glutamine及び4ng/ml bFGFを含有するDMEM/F12を用いた。 [Reference Example 1]
As human iPS cells, B lymphoblasts derived from PARK2 patients were transferred to plasmid vectors pCXLE-hOCT3 / 4-shp53, pCXLE-hSK, pCXLE-hUL (Okita et al. Nature Methods 8, 409-412 (2012). / 4, iPS cells (LiPS cells) obtained by introducing Sox2, Klf4, shp53, Lin28 and L-Myc were used as the passage medium for human LiPS cells, 20% KSR, 1% NEAA, DMEM / F12 containing 0.1% mercaptoethanol, penicillin (50 u / ml) / streptomycin (50 μg / ml), 2 mM glutamine and 4 ng / ml bFGF was used.
　ConfluentになったPARK2患者由来のLiPS細胞を含む10cmディッシュからメディウムを吸い取り、6mlのPBSでiPS細胞を洗浄した。解離液Tを1ml加えたのちすぐに吸い取り、5分程度インキュベートした。顕微鏡ではがれ具合を確認し、6mlのhES-mediumを加えて細胞を50ml tubeに回収した。1000rpm×5分遠心をし、上澄みを吸い取った。LiPS細胞へ10mlのhES-medium(ROCK-inhibitor(+))を加え、ピペッティングをし、ゼラチンコート10cm dishに蒔いて、1～2時間インキュベートした。顕微鏡でSTOの張り付き具合を確認し、dishを回して浮遊している細胞を中心に集めてから50ml tubeに回収した。1000rpm×5分遠心し、上澄みを吸い取った。LiPS細胞のペレットへ、Tryple Selectを1.5ml加え、37℃ウォーターバスで5分間incubateし、時々tubeを回して撹拌することにより、該細胞を分散した。P1000のピペットマンで10回程度ピペッティングした後、細胞懸濁液をTrypsin Inhibitor 3mlが入った15ml tubeに加え、MHMでボリュームアップし、Cell Strainer (70μｍ)で濾過した。通過した細胞懸濁液を5分遠心し、上澄みを吸い取り、3～5mlのMHMを加えた。細胞数をカウントし、MHM培地を加えて10 cells/μlに調整し、試薬(下表１)を加えてフラスコで低酸素条件 (4% O2)下で2週間培養することにより、神経幹細胞を含むニューロスフェアを形成した。 Medium was sucked from a 10 cm dish containing LiPS cells derived from PARK2 patients who became Confluent, and iPS cells were washed with 6 ml of PBS. Immediately after adding 1 ml of dissociation solution T, it was sucked and incubated for about 5 minutes. The condition of peeling was confirmed with a microscope, 6 ml of hES-medium was added, and the cells were collected in a 50 ml tube. Centrifugation was performed at 1000 rpm × 5 minutes, and the supernatant was sucked off. 10 ml of hES-medium (ROCK-inhibitor (+)) was added to LiPS cells, pipetted, placed in a gelatin-coated 10 cm dish, and incubated for 1-2 hours. The STO sticking condition was confirmed with a microscope, and the dish was turned to collect floating cells in the center, and then collected in a 50 ml tube. Centrifugation was performed at 1000 rpm for 5 minutes, and the supernatant was sucked off. 1.5 ml of Tryple Select was added to the LiPS cell pellet, incubated for 5 minutes in a 37 ° C water bath, and the cells were dispersed by occasionally rotating the tube and stirring. After pipetting about 10 times with a P1000 pipetman, the cell suspension was added to a 15 ml tube containing 3 ml of Trypsin Inhibitor, and the volume was increased with MHM, followed by filtration with a Cell Strainer (70 μm). The cell suspension that passed through was centrifuged for 5 minutes, the supernatant was aspirated, and 3-5 ml of MHM was added. Count the number of cells, add MHM medium to adjust to 10 cells / μl, add reagents (Table 1 below), and culture in a flask under hypoxic conditions (4% O 2 ) for 2 weeks. A neurosphere containing was formed.
Figure JPOXMLDOC01-appb-T000001
  
Figure JPOXMLDOC01-appb-T000001
  
　培養開始１週間後にメディウムチェンジを行った。メディウムチェンジは、以下の通りに行った。Sphereを50mlチューブに回収し、1000rpmで5分間遠心した。3～5mlくらいメディウムを残すように、上澄みを吸い取り、軽くタッピングすることにより細胞をほぐした。細胞へ培地（MHM＋B27+FGF+LIF+Y27632）を加えて、T75フラスコにて培養した。 Medium change was performed one week after the start of culture. Medium changes were performed as follows. The Sphere was collected in a 50 ml tube and centrifuged at 1000 rpm for 5 minutes. The cells were loosened by sucking the supernatant and gently tapping to leave about 3-5 ml of medium. A medium (MHM + B27 + FGF + LIF + Y27632) was added to the cells, and the cells were cultured in a T75 flask.
　細胞の継代は以下の通りに行った。Sphereを50mlチューブに回収し、1000rpmで５分間遠心した。上澄みを吸い取り、細胞ペレットへTryple selectを1.5ml加えて、37.0℃ウォーターバスで10分間インキュベートした。チューブをウォーターバスから取り出し、P1000ピペットで白いもやもやがなくなるまでピペッティングした。細胞懸濁液をTIを3ml入れた15mlチューブに加え、MHMで10mlくらいまでボリュームアップして70μmのフィルターに通した。通過した細胞懸濁液を1000rpmで５分間遠心した。上澄みを吸い取り、細胞ペレットを軽くタッピングすることによりほぐし、MHMを3～5ml加え、細胞数をカウントした。細胞懸濁液を50 cells/μlになるようにMHMで希釈し、45ml / T75フラスコに分注し、試薬(上表１)を加えた。低酸素(4% O2)インキュベーターにて2週間培養した。培養開始１週間後に上記と同様にメディウムチェンジを行った。 Cell passage was performed as follows. The Sphere was collected in a 50 ml tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was blotted out, 1.5 ml of Tryple select was added to the cell pellet and incubated in a 37.0 ° C. water bath for 10 minutes. The tube was removed from the water bath and pipetted with a P1000 pipette until there was no white haze. The cell suspension was added to a 15 ml tube containing 3 ml of TI, and the volume was increased to about 10 ml with MHM and passed through a 70 μm filter. The passed cell suspension was centrifuged at 1000 rpm for 5 minutes. The supernatant was blotted out and loosened by lightly tapping the cell pellet, 3-5 ml of MHM was added, and the number of cells was counted. The cell suspension was diluted with MHM to 50 cells / μl, dispensed into a 45 ml / T75 flask, and reagents (table 1 above) were added. The cells were cultured for 2 weeks in a hypoxic (4% O 2 ) incubator. One week after the start of culture, medium change was performed in the same manner as described above.
　形成されたニューロスフェアを3x105cells/mlの濃度に懸濁して、PO/fibronectin コートした48 well plateに500μl/wellで播種し、B27を含有するMHM培地で培養することにより、その神経分化能を確認した（図９）。 The neurospheres formed were suspended at a concentration of 3x10 5 cells / ml, seeded at 500 μl / well in a 48-well plate coated with PO / fibronectin, and cultured in MHM medium containing B27. Was confirmed (FIG. 9).
　本発明によれば、多能性幹細胞から、神経幹細胞や運動神経前駆細胞等の神経前駆細胞を短期間で、効率的に分化誘導することができる。特に、神経分化しにくいT細胞由来iPS細胞およびBリンパ球由来iPS細胞から、効率的に神経幹細胞や運動神経前駆細胞等の神経系未分化細胞を分化誘導することができる。
　本出願は日本で出願された特願２０１２－１３３２９９（出願日：２０１２年６月１２日）を基礎としており、その内容は本明細書に全て包含されるものである。 According to the present invention, neural progenitor cells such as neural stem cells and motor neural progenitor cells can be efficiently induced to differentiate from pluripotent stem cells in a short period of time. In particular, it is possible to efficiently induce differentiation of neural undifferentiated cells such as neural stem cells and motor neural progenitor cells from T cell-derived iPS cells and B lymphocyte-derived iPS cells that are difficult to differentiate.
This application is based on Japanese Patent Application No. 2012-133299 filed in Japan (filing date: June 12, 2012), the contents of which are incorporated in full herein.

Claims (15)

  1. 　TGF-βファミリー阻害剤、GSK3β阻害剤及びBMP阻害剤の存在下で、多能性幹細胞を培養することを含む、多能性幹細胞の神経分化能の亢進方法。 A method for enhancing the neuronal differentiation ability of pluripotent stem cells, comprising culturing pluripotent stem cells in the presence of a TGF-β family inhibitor, a GSK3β inhibitor, and a BMP inhibitor.
  2. 　LIFの不在下で、多能性幹細胞を培養する、請求項１記載の方法。 The method according to claim 1, wherein pluripotent stem cells are cultured in the absence of LIF.
  3. 　多能性幹細胞が人工多能性幹細胞である、請求項１又は２記載の方法。 The method according to claim 1 or 2, wherein the pluripotent stem cell is an induced pluripotent stem cell.
  4. 　人工多能性幹細胞がT細胞又はB細胞由来である、請求項３記載の方法。 The method according to claim 3, wherein the induced pluripotent stem cells are derived from T cells or B cells.
  5. 　TGF-βファミリー阻害剤が４－［４－（１，３－ベンゾジオキソール－５－イル）－５－（２－ピリジニル）－１Ｈ－イミダゾール－２－イル］－ベンズアミド又はその水和物である、請求項１～４のいずれか記載の方法。 TGF-β family inhibitor is 4- [4- (1,3-benzodioxol-5-yl) -5- (2-pyridinyl) -1H-imidazol-2-yl] -benzamide or a hydrate thereof The method according to any one of claims 1 to 4, wherein
  6. 　GSK3β阻害剤が６－［［２－［［４－（２，４－ジクロロフェニル）－５－（４－メチル－１Ｈ－イミダゾール－２－イル）－２－ピリミジニル］アミノ］エチル］アミノ］ニコチノニトリルである、請求項１～５のいずれか記載の方法。 The GSK3β inhibitor is 6-[[2-[[4- (2,4-dichlorophenyl) -5- (4-methyl-1H-imidazol-2-yl) -2-pyrimidinyl] amino] ethyl] amino] nicotino The method according to any one of claims 1 to 5, which is a nitrile.
  7. 　BMP阻害剤が６－［４－（２－ピペリジン－１－イルエトキシ）フェニル］－３－ピリジン－４－イルピラゾロ［１，５－ａ］ピリミジンである、請求項１～６のいずれか記載の方法。 The method according to any one of claims 1 to 6, wherein the BMP inhibitor is 6- [4- (2-piperidin-1-ylethoxy) phenyl] -3-pyridin-4-ylpyrazolo [1,5-a] pyrimidine. .
  8. 　多能性幹細胞を、TGF-βファミリー阻害剤及びGSK3β阻害剤の存在下で、2～10％の酸素分圧下、培養することを含む、神経幹細胞の製造方法。 A method for producing neural stem cells, comprising culturing pluripotent stem cells in the presence of a TGF-β family inhibitor and a GSK3β inhibitor under an oxygen partial pressure of 2 to 10%.
  9. 　多能性幹細胞が人工多能性幹細胞である、請求項８記載の方法。 The method according to claim 8, wherein the pluripotent stem cell is an induced pluripotent stem cell.
  10. 　人工多能性幹細胞がT細胞又はB細胞由来である、請求項９記載の方法。 The method according to claim 9, wherein the induced pluripotent stem cells are derived from T cells or B cells.
  11. 　TGF-βファミリー阻害剤が４－［４－（１，３－ベンゾジオキソール－５－イル）－５－（２－ピリジニル）－１Ｈ－イミダゾール－２－イル］－ベンズアミド又はその水和物である、請求項８～１０のいずれか記載の方法。 TGF-β family inhibitor is 4- [4- (1,3-benzodioxol-5-yl) -5- (2-pyridinyl) -1H-imidazol-2-yl] -benzamide or a hydrate thereof The method according to any one of claims 8 to 10, wherein
  12. 　GSK3β阻害剤が６－［［２－［［４－（２，４－ジクロロフェニル）－５－（４－メチル－１Ｈ－イミダゾール－２－イル）－２－ピリミジニル］アミノ］エチル］アミノ］ニコチノニトリルである、請求項８～１１のいずれか記載の方法。 The GSK3β inhibitor is 6-[[2-[[4- (2,4-dichlorophenyl) -5- (4-methyl-1H-imidazol-2-yl) -2-pyrimidinyl] amino] ethyl] amino] nicotino The method according to any one of claims 8 to 11, which is a nitrile.
  13. 　以下の工程を含む、神経幹細胞の製造方法：
    （１）TGF-βファミリー阻害剤、GSK3β阻害剤及びBMP阻害剤の存在下で、多能性幹細胞を培養することにより、当該多能性幹細胞の神経分化能を亢進すること、及び
    （２）工程（１）で得られた、神経分化能が亢進した多能性幹細胞を、TGF-βファミリー阻害剤及びGSK3β阻害剤の存在下で、２～１０％の酸素分圧下、培養すること。     A method for producing neural stem cells, comprising the following steps:
    (1) culturing pluripotent stem cells in the presence of a TGF-β family inhibitor, a GSK3β inhibitor, and a BMP inhibitor to enhance the neuronal differentiation ability of the pluripotent stem cells; and (2) Culturing the pluripotent stem cells with enhanced neuronal differentiation ability obtained in step (1) in the presence of a TGF-β family inhibitor and a GSK3β inhibitor under 2 to 10% oxygen partial pressure.
  14. 　以下の工程を含む、運動神経前駆細胞の製造方法：
    （１）多能性幹細胞を、TGF-βファミリー阻害剤及びGSK3β阻害剤の存在下で、2～10％の酸素分圧下、培養することにより、神経幹細胞を得ること、及び
    （２）工程（１）で得られた神経幹細胞を、レチノイン酸、９－シクロヘキシル－Ｎ－［４－（４－モルホリニル）フェニル］－２－（１－ナフタレニルオキシ）－９Ｈ－プリン－６－アミン、TGF-βファミリー阻害剤及びGSK3β阻害剤の存在下で、2～10％の酸素分圧下、培養することにより、運動神経前駆細胞を分化誘導すること。     A method for producing motor neural progenitor cells, comprising the following steps:
    (1) obtaining neural stem cells by culturing pluripotent stem cells in the presence of a TGF-β family inhibitor and a GSK3β inhibitor under 2 to 10% oxygen partial pressure, and (2) step ( The neural stem cells obtained in 1) were mixed with retinoic acid, 9-cyclohexyl-N- [4- (4-morpholinyl) phenyl] -2- (1-naphthalenyloxy) -9H-purin-6-amine, TGF -Inducing differentiation of motor progenitor cells by culturing in the presence of β family inhibitor and GSK3β inhibitor under 2-10% oxygen partial pressure.
  15. 　以下の工程を含む、ドパミン産生神経前駆細胞の製造方法：
    （１）多能性幹細胞を、TGF-βファミリー阻害剤及びGSK3β阻害剤の存在下で、2～10％の酸素分圧下、培養することにより、神経幹細胞を得ること、及び
    （２）工程（１）で得られた神経幹細胞を、TGF-βファミリー阻害剤、GSK3β阻害剤、ソニックヘッジホッグ、９－シクロヘキシル－Ｎ－［４－（４－モルホリニル）フェニル］－２－（１－ナフタレニルオキシ）－９Ｈ－プリン－６－アミン及びFGF8の存在下で、2～10％の酸素分圧下、培養することにより、ドパミン産生神経前駆細胞を分化誘導すること。     A method for producing dopaminergic neural progenitor cells, comprising the following steps:
    (1) obtaining neural stem cells by culturing pluripotent stem cells in the presence of a TGF-β family inhibitor and a GSK3β inhibitor under 2 to 10% oxygen partial pressure, and (2) step ( The neural stem cells obtained in 1) were transformed into TGF-β family inhibitors, GSK3β inhibitors, Sonic hedgehog, 9-cyclohexyl-N- [4- (4-morpholinyl) phenyl] -2- (1-naphthalenyl) Differentiation induction of dopaminergic neural progenitor cells by culturing in the presence of 2-10% oxygen partial pressure in the presence of (oxy) -9H-purin-6-amine and FGF8.
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