WO2013187416A1 - PROCÉDÉ D'AMPLIFICATION D'UNE CELLULE iPS APPROPRIÉE POUR UNE DIFFÉRENCIATION NEURALE ET PROCÉDÉ DESTINÉ À L'INDUCTION D'UNE CELLULE SOUCHE NEURALE - Google Patents

PROCÉDÉ D'AMPLIFICATION D'UNE CELLULE iPS APPROPRIÉE POUR UNE DIFFÉRENCIATION NEURALE ET PROCÉDÉ DESTINÉ À L'INDUCTION D'UNE CELLULE SOUCHE NEURALE 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.

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Abstract

La présente invention concerne un procédé destiné à augmenter l'aptitude à la différenciation neurale d'une cellule souche pluripotente, qui comprend la culture de la cellule souche pluripotente en présence d'un inhibiteur de la famille de TGF-β, d'un inhibiteur de GSK3β et d'un inhibiteur de BMP. La présente invention concerne également un procédé destiné à induire la différenciation d'une cellule souche pluripotente en une cellule neurale indifférenciée, qui comprend la culture de la cellule souche pluripotente en présence d'un inhibiteur de la famille de TGF-β et d'un inhibiteur de GSK3β sous une pression partielle d'oxygène de 2 à 10 %.
PCT/JP2013/066102 2012-06-12 2013-06-11 PROCÉDÉ D'AMPLIFICATION D'UNE CELLULE iPS APPROPRIÉE POUR UNE DIFFÉRENCIATION NEURALE ET PROCÉDÉ DESTINÉ À L'INDUCTION D'UNE CELLULE SOUCHE NEURALE WO2013187416A1 (fr)

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