WO2012115120A1 - Préparation de neurones 5-ht - Google Patents

Préparation de neurones 5-ht Download PDF

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WO2012115120A1
WO2012115120A1 PCT/JP2012/054175 JP2012054175W WO2012115120A1 WO 2012115120 A1 WO2012115120 A1 WO 2012115120A1 JP 2012054175 W JP2012054175 W JP 2012054175W WO 2012115120 A1 WO2012115120 A1 WO 2012115120A1
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cells
receptor
tgf
nerve
kinase inhibitor
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橋本　均
明道 馬場
淳史 山▲崎▼
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国立大学法人 大阪大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/15—Transforming growth factor beta (TGF-β)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases (EC 2.)
    • C12N2501/727—Kinases (EC 2.7.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/80—Neurotransmitters; Neurohormones
    • C12N2501/825—Serotonine (5-HT); Melatonine

Definitions

  • the present invention relates to a method for producing 5-HT nerve cells.
  • Serotonin (5-HT) nervous system in the brain uses the raphe nucleus of the brainstem as the nucleus of origin, and has a wide range of central nervous systems such as the limbic cortex, basal ganglia, hypothalamus, lower brainstem and spinal cord Projecting. It is functionally diverse, such as controlling aggression, feeding behavior, and memory learning, and some psychiatric treatments regulate the function of the 5-HT nervous system. Is considered to be closely related.
  • the 5-HT nervous system is recognized from the early stage of the embryonic period and has been shown to regulate cell division / differentiation, migration of neural cells, synapse formation, and the like. Moreover, it has been shown that abnormal development of the brain occurs along with abnormalities in the 5-HT nervous system during development.
  • Non-patent Documents 1 and 2 disclose the neurotransmitter 5-HT itself has a function of promoting differentiation of 5-HT nerves.
  • Many subtypes are known to exist in 5-HT receptors, but their role in 5-HT neuronal differentiation is unknown.
  • the 5-HT 7 receptor has been pointed out to be possibly involved in depression, anxiety, pain, schizophrenia and the like.
  • Non-patent Documents 3-5 As a useful tool for studying the developmental stage of nerve cells, attention has been focused on a nerve induction system from embryonic stem cells (ES cells) (Non-patent Documents 3-5).
  • the present inventors have found that neuronal cells are induced by culturing ES cells on Matrigel, which is a basement membrane preparation (Matrigel method), and further directly binds to BMP (bone morphogenic protein).
  • BMP bone morphogenic protein
  • An object of the present invention is to provide a 5-HT nerve cell production method using the novel 5-HT nerve cell induction factor obtained by a 5-HT nerve differentiation induction system from ES cells and the production kit.
  • the differentiation induction mechanism was examined using the Matrigel-Noggin method. As a result, it was found that the action of Noggin is caused by blocking of the BMP signal, although it is not caused by other BMP antagonists. In addition, the inventors have succeeded in clarifying that differentiation from ES cells into 5-HT nerves can be promoted by the action via 5-HT 7 receptor. Furthermore, in the present invention, an inhibitor of BMP receptor kinase can promote differentiation into 5-HT nerves, and Noggin's 5-HT neuronal differentiation-inducing action is inhibited by type I TGF- ⁇ receptor kinase inhibitors. It was found that type I TGF- ⁇ receptor signal is involved in differentiation into 5-HT nerves. As a result of further studies based on these findings, the present inventors have completed the present invention.
  • the present invention [1] A 5-HT neuron culturing a stem cell on a polymerized extracellular matrix protein in the presence of a BMP receptor kinase inhibitor, a 5-HT 7 receptor agonist, or a type I TGF- ⁇ receptor ligand Production method; [2]
  • the BMP receptor kinase inhibitor is 4- (6- (4- (piperazin-1-yl) phenyl) pyrazolo [1,5-a] pyrimidin-3-yl) quinoline or 4- [6-- 4- (1-Methylethoxy) phenyl] pyrazolo [1,5-a] pyrimidin-3-yl] -quinoline, the production method according to [1], [3]
  • the BMP receptor kinase inhibitor is 4- (6- (4- (piperazin-1-yl) phenyl) pyrazolo [1,5-a] pyrimidin-3-yl) quinoline, [2] Manufacturing method of [4] A 5-HT 7
  • the 5-HT nerve cell production kit [14] The 5-HT neuron according to [9], wherein the type I TGF- ⁇ receptor ligand is selected from at least one of TGF- ⁇ , Activin, Nodal, GDF-1, GDF-8, or GDF-11 Production kit; [15] The 5-HT nerve cell production kit according to any one of [9] to [14], further comprising noggin; [16] The 5-HT neuronal cell production kit according to any one of [9] to [15], wherein the stem cells are ES cells or iPS cells; [17] A 5-HT nerve cell regenerative agent comprising a BMP receptor kinase inhibitor, a 5-HT 7 receptor agonist, or a type I TGF- ⁇ receptor ligand; [18] A 5-HT nerve cell regeneration method comprising administering to a patient an effective amount of a BMP receptor kinase inhibitor, a 5-HT 7 receptor agonist, or a type I TGF- ⁇ receptor ligand; [19] BMP receptor kinase inhibitor
  • a BMP receptor kinase inhibitor, a 5-HT 7 receptor agonist, or a type I TGF- ⁇ receptor ligand newly found as a 5-HT nerve cell inducer Can be used to efficiently differentiate stem cells into 5-HT neurons.
  • FIG. 1 shows the results of culturing ES cells on Matrigel for 14 days in the presence of various concentrations of LDN-193189.
  • Luminescence intensity is the activity of the enhancer region of ETS (Pet1), which is an index of 5-HT neuronal differentiation, and is shown as 100% when no drug is added. Data were expressed as mean ⁇ standard error of the mean. * P ⁇ 0.05 by Dunnett test.
  • FIG. 2 shows the results of culturing ES cells on Matrigel for 14 days in the presence of various concentrations of 5-HT agonist, antagonist or SSRI. Data were expressed as mean ⁇ standard error of the mean. * P ⁇ 0.05 by Dunnett test.
  • FIG. 1 shows the results of culturing ES cells on Matrigel for 14 days in the presence of various concentrations of 5-HT agonist, antagonist or SSRI. Data were expressed as mean ⁇ standard error of the mean. * P ⁇ 0.05 by Dunnett test.
  • FIG. 3A shows the results of culturing ES cells for 14 days on Matrigel when Noggin was added in the presence of various concentrations of 5-HT 7 agonists or antagonists. Data were expressed as mean ⁇ standard error of the mean.
  • FIG. 3B shows nuclei by Hoechst 33258 and 5-HT by anti-5-HT polyclonal antibody in cells cultured on Matrigel for 14 days in the presence of Noggin (left) and Noggin + 5-HT 7 antagonist SB269970 10 ⁇ M (right); It is a figure which shows a co-stained image. Scale bar; 200 ⁇ m FIG.
  • FIG. 4 shows the results of measuring dopamine (DA) and serotonin (5-HT) concentrations when ES cells were cultured on Matrigel for 18 days in the presence or absence of a 5-HT 7 agonist (AS-19). Indicates. Data were expressed as mean ⁇ standard error of the mean.
  • FIG. 5 shows the result of measuring the mRNA expression level of 5-HT 1A receptor when ES cells were cultured on Matrigel in the presence or absence of Noggin. Data were expressed as mean ⁇ standard error of the mean. ** p ⁇ 0.01 by two-way repeated ANOVA. , #P ⁇ 0.05, ## p ⁇ 0.01 by Turkey-Kramer test.
  • FIG. 6 shows the results of measuring the mRNA expression level of 5-HT 7 receptor when ES cells were cultured on Matrigel in the presence or absence of Noggin. Data were expressed as mean ⁇ standard error of the mean. ** p ⁇ 0.01 by two-way repeated ANOVA. , #P ⁇ 0.05, ## p ⁇ 0.01 by Turkey-Kramer test.
  • FIG. 7 shows the results when ES cells were cultured for 14 days on Matrigel when Noggin was added in the presence of various concentrations of SB431542. Luminescence intensity is the activity of the enhancer region of ETS (Pet1), which is an index of 5-HT neuronal differentiation, and is shown as 100% when no drug is added. Data were expressed as mean ⁇ standard error of the mean.
  • FIG. 8 shows the ratio of 5-HT cells to total neurons when ES cells were cultured for 14 days on Matrigel when Noggin was added in the presence of various concentrations of SB431542. Data were expressed as mean ⁇ standard error of the mean.
  • the left figure shows the ratio of 5-HT cells to neurons by anti-Tuj1 monoclonal antibody which is a neuronal differentiation marker and anti-5-HT polyclonal antibody in the presence of Noggin and Noggin + type I TGF- ⁇ receptor antagonist SB431542 .
  • FIG. 9 shows the results of measuring the mRNA expression level of each downstream gene of BMP receptor and type I TGF- ⁇ receptor when ES cells were cultured on Matrigel in the presence or absence of Noggin. Data were expressed as mean ⁇ standard error of the mean. ** p ⁇ 0.01 by Student's test.
  • the present invention comprises culturing stem cells on polymerized extracellular matrix protein in the presence of a BMP receptor kinase inhibitor, a 5-HT 7 receptor agonist, or a type I TGF- ⁇ receptor ligand.
  • a BMP receptor kinase inhibitor for producing HT neurons.
  • 5-HT neurons produced by the method of the present invention is not particularly limited, and model cells used for all tests and research on 5-HT neurons, schizophrenia, depression, autism,
  • the present invention provides a use as a cell for transplantation used for treatment / amelioration of mental disorders such as eating disorders and sleep disorders, motor dysfunction after spinal cord injury, and symptoms accompanied by pain.
  • the stem cell used in the 5-HT nerve cell production method of the present invention refers to a cell that can be cultured in vitro and can differentiate into a plurality of cells constituting a living body.
  • ES cells fetal primordial germ cell-derived pluripotent stem cells (EG cells: Proc Natl Acad Sci US A. 1998, 95: 13726-31), testis-derived pluripotent stem cells (GS cells: Nature, 2008, 456: 344-9), somatic cell-derived induced pluripotent stem cells (iPS cells), human somatic stem cells (tissue stem cells), preferably iPS cells, ES cells It is.
  • ES cells ES cells derived from any warm-blooded animal, preferably a mammal, can be used.
  • mammals include mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses, goats, monkeys, and humans.
  • Preferable examples of ES cells include ES cells derived from humans or mice.
  • ES cells such as mammals established by culturing early embryos before implantation, ES established by culturing early embryos produced by nuclear transfer of somatic cell nuclei, etc.
  • ES cells obtained by modifying cells and genes on the chromosomes of these ES cells using genetic engineering techniques.
  • Each ES cell can be prepared according to a method commonly practiced in the art or a known literature.
  • Mouse ES cells were obtained in 1981 from Evans et al. (1981, Nature 292: 154-6) and Martin et al. (Martin GR. Et al., 1981, Proc Natl Acad Sci 78: 7634-8). And can be purchased from, for example, Dainippon Sumitomo Pharma Co., Ltd. (Osaka, Japan).
  • Human ES cells were established in 1998 by Thomson et al. (Science, 1998, 282: 1145-7) and are available from the WiCell Research Institute (WiCell Research Institute, website: http: // www. available from Wisell.org/, Madison, Wisconsin, USA, National Institute of Health, Kyoto University, etc., for example, Cellaritis (website: http://www.cellaritis.com). /, Sweden).
  • an iPS cell an iPS cell derived from any warm-blooded animal, preferably a mammal can be used.
  • the mammal include mouse, rat, guinea pig, hamster, rabbit, cat, dog, sheep, pig, cow, horse, goat, monkey and human.
  • Preferable examples of iPS cells include iPS cells derived from humans or mice.
  • Specific examples of iPS cells include cells obtained by introducing a plurality of genes into somatic cells such as skin cells and having acquired multipotency similar to ES cells.
  • Oct3 / 4 gene, Klf4 gene, C- Examples include iPS cells obtained by introducing Myc gene and Sox2 gene, iPS cells obtained by introducing Oct3 / 4 gene, Klf4 gene and Sox2 gene (Nat Biotechnol 2008; 26: 101-106).
  • a method in which a transgene is further reduced (Nature. 2008 Jul 31; 454 (7204): 646-50), a method using a low molecular weight compound (Cell Stem Cell. 2009 Jan 9; 4 (1): 16 -9, Cell Stem Cell. 2009 Nov 6; 5 (5): 491-503), a method using a transcription factor protein instead of a gene (Cell Stem Cell.
  • iPS cell lines include the 253G1 strain (iPS cell line prepared by expressing OCT4 / SOX2 / KLF4 in skin fibroblasts of a 36-year-old female), 201B7 strain (skin fiber of a 36-year-old female).
  • IPS cell line prepared by expressing OCT4 / SOX2 / KLF4 / c-MYC in blast cells
  • 1503-iPS (297A1) (skinned fibroblasts of a 73 year old female with OCT4 / SOX2 / KLF4 / c-MYC IPS cell line prepared by expression)
  • 1392-iPS iPS cell line prepared by expressing OCT4 / SOX2 / KLF4 / c-MYC in 56-year-old male skin fibroblasts
  • NHDF- iPS (297L1) i created by expressing OCT4 / SOX2 / KLF4 / c-MYC in newborn male skin fibroblasts S cell line
  • the stem cells are cultured on a polymerized extracellular matrix protein.
  • an extracellular matrix protein is a protein that constitutes a space outside a cell and has a cell adhesion action, and the cells cultured therein perform normal cell function maintenance, self-proliferation and differentiation.
  • the type is not particularly limited, but an extracellular matrix protein constituting the basement membrane is preferable.
  • extracellular matrix proteins include laminin, collagen, entactin, proteoglycan, glycosaminoglycan, nidogen, fibronectin, gelatin, vitronectin, elastin, and tenascin.
  • extracellular matrix proteins constituting the basement membrane.
  • extracellular matrix proteins can be used.
  • Matrigel composed of basement membrane components extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma It can be preferably used in the invention.
  • EHS Engelbreth-Holm-Swarm
  • the extracellular matrix protein is polymerized in advance on a cell culture vessel when used in the method for producing 5-HT nerve cells of the present invention.
  • Polymerization can be easily carried out by dissolving the extracellular matrix protein in advance at a low temperature and incubating at 22 to 35 ° C.
  • the cell culture vessel coated with the extracellular matrix protein to be polymerized include, for example, a flask, a flask for tissue culture, a dish, a petri dish, a tissue culture dish, a multi-dish, a microplate, a microwell plate, a multiplate, Examples include multi-well plates, chamber slides, petri dishes, tubes, trays, culture bags, and roller bottles. Preferred are a dish, a petri dish, a tissue culture dish, a multi-dish, a microplate, a microwell plate, a multiplate, a multiwell plate, and the like.
  • the stem cells may be seeded at a density of 1.0 ⁇ 10 3 on the polymerized extracellular matrix protein by suspending the cells suspended as monocells in a medium.
  • the culture is usually carried out in an incubator at 5% CO 2 /95% air at 37 ° C. for 7 to 20 days, preferably 12 to 20 days to differentiate stem cells into nervous system cells.
  • the medium to be used may be a medium usually used for culturing stem cells (hereinafter referred to as a basal medium).
  • the medium used in the 5-HT nerve cell production method of the present invention may be a serum-containing medium, but is preferably a serum-free medium.
  • the serum-free medium means a basal medium that does not contain unconditioned or unpurified serum, and there is no medium in which purified blood-derived components or animal tissue-derived components (for example, growth factors) are mixed. It shall correspond to serum medium.
  • the medium used in the 5-HT nerve cell production method of the present invention may also contain a serum replacement.
  • Serum substitutes include, for example, albumin (eg, lipid-rich albumin), transferrin, fatty acid, collagen precursor, trace elements (eg, zinc, selenium), B-27 supplement, N2 supplement, knockout sealum replacement, 2-mercapto Ethanol, 3 ′ thiol glycerol, or the equivalent thereof.
  • concentration in these media is, for example, usually 0.01 to 20% by weight, preferably 0.1 to 10% by weight.
  • Knockout sealum replacements can be purchased from Life Technologies.
  • Other serum substitutes can be purchased from Life Technologies, SIGMA, Wako Pure Chemical Industries, Dainippon Sumitomo Pharma Co., Ltd., etc. Regardless.
  • the medium used in the 5-HT nerve cell production method of the present invention also includes lipids, amino acids (eg, non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, Inorganic salts, antibiotics (for example, penicillin and streptomycin), antibacterial agents (for example, amphotericin B), and the like may be contained.
  • concentrations in these media are the same as those in the serum replacement medium described above.
  • stem cell culture is carried out using a BMP receptor kinase inhibitor, a 5-HT 7 receptor agonist, or a type I TGF- ⁇ receptor ligand as a 5-HT nerve cell inducer. It is performed by adding to the medium.
  • BMP receptors are receptor-type serine / threonine kinases of BMP (Bone Morphogenic Protein), a protein secreted extracellularly, and are roughly classified into type I receptors and type II receptors.
  • ALK Activate like kinase is known as a BMP receptor.
  • BMP receptor kinase inhibitors include 4- (6- (4- (piperazin-1-yl) phenyl) pyrazolo [1,5-a] pyrimidin-3-yl) quinoline (LDN-193189), 4- [ 6- [4- (1-Methylethoxy) phenyl] pyrazolo [1,5-a] pyrimidin-3-yl] -quinoline (DMH-1) and the like.
  • 4- (6- (4- (piperazin-1-yl) phenyl) pyrazolo [1,5-a] pyrimidin-3-yl) quinoline is preferably used.
  • 5-HT 7 receptor is one of serotonin receptor subtypes with serotonin synthesized from tryptophan as a ligand, and is a G protein-coupled receptor.
  • 5-HT 7 receptor agonists (2S)-(+)-5- (1,3,5-Trimethylpyrazol-4-yl) -2- (dimethylamino) tetralin (AS-19), 4- (2 -Diphenyl) -N- (1,2,3,4-tetrahydronaphthalen-1-yl) -1-piperazine hexanamide hydrochloride (LP12), 4- [2- (Methylthio) phenyl] -N- (1,2,3,3) 4-tetrahydro-1-naphthalenyl) -1-piperazine hexaneamide hydrochloride (LP44) and the like.
  • the TGF- ⁇ receptor is a receptor type serine / threonine kinase of TGF- ⁇ that forms a family with BMP, and, like the BMP receptor, is roughly divided into a type I receptor and a type II receptor.
  • Examples of the type I TGF- ⁇ receptor ligand include TGF- ⁇ , Activin, Nodal, GDF-1, GDF-8, and GDF-11.
  • TGF- ⁇ , Activin, Nodal, GDF-1, GDF-8 or GDF-11 can be purchased, for example, from R & D Systems, Inc.
  • the ligand is an accession number (GenBank), for example, NC in the case of TGF- ⁇ 1. 0000199.9 (NP 000651.3) (human), NC 000073.5 (NP 03577.1) (mouse), in the case of TGF- ⁇ 2, NC 00000.10 (NP 001129071.1) (human), NC 000067.5 (NP 03393.2) (mouse), in the case of TGF- ⁇ 3, NC 000001.8 (NP003230.1) (human), NC 0000785.5 (NP 03394.2) (mouse), Activin is NC 000007.13 (NP 002183.1) (human), NC 000001.5 (NP 03406.1) (mouse), Nodal is NC 000010.10 (NP 060525.3) (human), NC 000076.5 (NP 03839.3) (mouse), GDF-1 is 0000199.9 (NP 001483.3) (human), NC 000074.5 (NP 001156754.1) (mouse), GDF-8 is NC 000002.11 (NP 00 00
  • the BMP receptor kinase inhibitor, 5-HT 7 receptor agonist, or type I TGF- ⁇ receptor ligand may be added at any point in the stem cell culture, but for BMP receptor kinase inhibitors, It is preferable to add to the medium from 0 to 5 days after the start of the culture, and the 5-HT 7 receptor agonist is preferably added 6 to 14 days after the start of the culture. In the production method of the present invention, Since mature neurons are induced after the 8th day of culture, in the case of AS-19 among 5-HT 7 receptor agonists, it is preferable to add them after the 6th to 7th days after the start of the culture.
  • the type I TGF- ⁇ receptor ligand is preferably added 0 to 5 days after the start of culture.
  • the BMP receptor kinase inhibitor, 5-HT 7 receptor agonist, or type I TGF- ⁇ receptor ligand may be added alone or at the same time. Furthermore, two or more agents and ligands may be added simultaneously to the BMP receptor kinase inhibitor, 5-HT 7 receptor agonist, or type I TGF- ⁇ receptor ligand, respectively.
  • the amount of the BMP receptor kinase inhibitor, 5-HT 7 receptor agonist, or type I TGF- ⁇ receptor ligand added to the medium can be appropriately determined by those skilled in the art.
  • 4- (6- (4- (piperazin-1-yl) phenyl) pyrazo [1,5-a] pyrimidin-3-yl) quinoline is 1 nM to 1000 nM, preferably 10 nM to 300 nM. Is added. If (2S)-(+)-5- (1,3,5-Trimethylpyrazol-4-yl) -2- (dimethylamino) tetralin is added, 0.1 ⁇ M to 100 ⁇ M, preferably 1 ⁇ M to 10 ⁇ M is added. Is exemplified. Those skilled in the art can also use the above-mentioned ranges by changing them.
  • noggin may further coexist.
  • Noggin can be purchased from, for example, R & D Systems, Inc., like the type I TGF- ⁇ receptor ligand.
  • noggin can easily obtain the amino acid sequence and base sequence of a protein as an accession number (GenBank), for example, Np_032737 (mouse) or AAH34027 (human), those skilled in the art can use well-known recombinant proteins.
  • GenBank for example, Np_032737 (mouse) or AAH34027 (human)
  • those skilled in the art can use well-known recombinant proteins.
  • Noggin can be easily produced using technology.
  • 5-HT nerve cells induced to differentiate from stem cells are confirmed as a result of the culture. Confirmation that differentiation was induced in the 5-HT neuron, This can be confirmed by measuring at least one of (1) measuring the amount of 5-HT and (2) immunostaining a marker of 5-HT neuronal cells, but it is limited to these methods. First, it is only necessary to verify that 5-HT neurons are present.
  • Measurement of the 5-HT amount is not limited to this, but can be performed using an existing method for measuring the amount of biogenic amine (Matsuda et al., European Journal of Pharmacology 1989, 170: 75-82). That is, it can be carried out by adding a solution containing K + at a high concentration (for example, 56 mM) to the cultured ES cells and incubating, and measuring the solution by HPLC / ECD or the like.
  • a buffer salt solution such as Hanks solution can be used as the solution containing K + , but is not limited thereto.
  • 5-HT or 5-HT synthase can be used as a marker for 5-HT neurons, but is not limited thereto.
  • a known marker that is specifically expressed in the serotonin nerve may be used.
  • 5-HT and 5-HT synthase are not expressed on the cell surface, so the cells to be stained are immobilized by a well-known method. After that, it is preferable to allow the antibody molecule to reach the inside of the cell by increasing the permeability of the cell membrane with a surfactant or the like.
  • 5-HT neuronal cells since there is no appropriate marker expressed on the cell surface of 5-HT neuronal cells, for example, when concentrated using FACS, GFP (green fluorescent protein) or the like is expressed specifically in 5-HT neurons. It is necessary to use a method for transformation in advance. However, if a 5-HT neuronal cell preparation prepared using the method according to the present invention is used, there is a possibility that an appropriate cell surface marker can be found.
  • the present invention also provides a kit for producing 5-HT neuron as an extracellular matrix protein, a stem cell, a BMP receptor kinase inhibitor as a 5-HT neuron inducer, a 5-HT 7 receptor agonist, or type I
  • a kit for producing a 5-HT neuron cell comprising a TGF- ⁇ receptor ligand is provided.
  • the 5-HT nerve cell production kit of the present invention is not particularly limited as long as it contains reagents, instruments, devices and the like. Extracellular matrix proteins, BMP receptor kinase inhibitors as 5-HT neuron inducers, 5-HT 7 receptor agonists, or type I TGF- ⁇ receptor ligands are described in the method for producing 5-HT neurons.
  • the 5-HT nerve cell inducer may further contain noggin as in the 5-HT nerve cell production method. Moreover, they may be provided in a dry state or a solution state. By using the kit and carrying out the method for producing 5-HT neurons, 5-HT neurons can be prepared.
  • BMP receptor kinase inhibitors include 4- (6- (4- (piperazin-1-yl) phenyl) pyrazolo [1,5-a] pyrimidin-3-yl) quinoline, 4- [6-- 4- (1-Methylethoxy) phenyl] pyrazolo [1,5-a] pyrimidin-3-yl] -quinoline and the like may be preferably included, and among them 4- (6- (4- (piperazin-1-yl) ) Phenyl) pyrazolo [1,5-a] pyrimidin-3-yl) quinoline is more preferred.
  • the 5-HT 7 receptor agonists include (2S)-(+)-5- (1,3,5-Trimethylpyrazol-4-yl) -2- (dimethylamino) tetralin, 4- (2-Diphenyl) -N -(1,2,3,4-tetrahydronaphthalen-1-yl) -1-piperazynehexanamide hydrochloride, 4- [2- (Methylthio) phenyl] -N- (1,2,3,4-tetrahydr-1-yl) -1-piperazine hexanamide hydrochloride and the like may be preferably included, among which (2S)-(+)-5- (1,3,5-Trimethylpyrazole-4-yl) 2- (dimethylamino) tetralin is more preferable.
  • the type I TGF- ⁇ receptor ligand preferably includes TGF- ⁇ , Activin, Nodal, GDF-1, GDF-8, GDF-11, and the like. Two or more BMP receptor kinase inhibitors, 5-HT 7 receptor agonists, or type I TGF- ⁇ receptor ligands may be included. Furthermore, the present kit can use the same stem cells as those described in the above-mentioned 5-HT neuron production method, and may preferably be included as ES cells or iPS cells.
  • 5-HT nerve dysfunction is known to contribute to increased aggression and impulsivity, eating disorders, and other mental disorders (eg, schizophrenia, depression, autism) Disease, eating disorder, sleep disorder, etc.), motor dysfunction after spinal cord injury, and pain.
  • BMP receptor signaling the 5-HT 7 receptor signaling or type I TGF-beta receptor signaling
  • the present invention provides a 5-HT nerve cell regenerative agent comprising a BMP receptor kinase inhibitor, a 5-HT 7 receptor agonist, or a type I TGF- ⁇ receptor ligand.
  • the administration form and dosage form of the 5-HT nerve cell regenerative agent of the present invention may be either oral or parenteral administration.
  • oral administration agent include solids such as powders, granules, capsules, tablets and chewable agents.
  • Liquids such as pills, solutions, syrups, and parenteral agents include injections, ointments, sprays, and the like.
  • parenteral administration more preferably an injection.
  • the component added as necessary in the preparation of the 5-HT nerve cell regenerating agent of the present invention includes an appropriate pharmaceutically acceptable carrier such as an excipient, a binder, a lubricant, a solvent, Disintegrants, solubilizers, suspending agents, emulsifiers, tonicity agents, stabilizers, soothing agents, preservatives, antioxidants, flavoring agents, coloring agents and the like can be mentioned.
  • excipients include sugars such as lactose, glucose, D-mannitol, organic excipients such as starches and celluloses such as crystalline cellulose, and inorganic excipients such as calcium carbonate and kaolin.
  • pregelatinized starch gelatin, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, D-mannitol, trehalose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, etc.
  • Is stearic acid, fatty acid salts such as stearate, talc, silicates, etc., solvent is purified water, physiological saline, etc.
  • disintegrator is low substituted hydroxypropyl cellulose, chemically modified Cellulose, starch, etc., as solubilizers, polyethylene glycol, propylene glycol, trehalose, benzyl benzoate, ethanol, sodium carbonate, sodium citrate, sodium salicylate, sodium acetate, etc. are used as suspending agents or emulsifiers.
  • isotonic agents examples include sodium lauryl sulfate, gum arabic, gelatin, lecithin, glyceryl monostearate, polyvinyl alcohol, polyvinyl pyrrolidone, sodium carboxymethyl cellulose, polysorbates, polyoxyethylene hydrogenated castor oil, etc.
  • Sodium chloride, potassium chloride, saccharides, glycerin, urea, etc., stabilizers include polyethylene glycol, sodium dextran sulfate, and other amino acids
  • a soothing agent glucose, calcium gluconate, procaine hydrochloride, etc.
  • preservatives paraoxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, etc. are antioxidants.
  • As sulfites, ascorbic acid and the like, as flavoring agents, sweeteners and fragrances commonly used in the pharmaceutical field, and as coloring agents, colorants commonly used in the pharmaceutical field are mentioned. .
  • aqueous and non-aqueous isotonic sterile injection solutions may contain antioxidants, buffers, antibacterial agents, isotonic agents and the like.
  • aqueous and non-aqueous sterile suspensions can be mentioned, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like.
  • Antioxidants, isotonic agents, suspending agents, stabilizers, solubilizers and preservatives include those described above.
  • TE buffer solution 10 mM Tris, 1 mM EDTA [pH 8.0]
  • PBS phosphate buffer solution
  • a buffer solution having a pH of 6 to 9 is preferable.
  • Antibacterial agents include oxophosphate, olmetoprim, trimethoprim, sulfa, fosfomycin, penicillin antibacterial, cephalosporin antibacterial, vancomycin, tetracycline antibacterial, rifampicin, fluoroquinone antibacterial, etc.
  • the agent include gum arabic, lipiodol, sodium polyacrylate, sodium hyaluronate, sodium carboxymethyl cellulose and the like.
  • the 5-HT nerve cell regenerative agent of the present invention can be enclosed in a container in unit doses or multiple doses like ampoules and vials.
  • the active ingredient and a pharmaceutically acceptable carrier can be lyophilized and stored in a state that may be dissolved or suspended in a suitable sterile vehicle immediately before use.
  • the dose and frequency of administration of the 5-HT nerve cell regenerative agent of the present invention vary depending on the age, body weight, disease state, administration method and the like of the administration subject, but usually, for example, a BMP receptor kinase inhibitor per dosage unit dosage form
  • the 5-HT 7 receptor agonist or type I TGF- ⁇ receptor ligand is usually contained in an amount of usually 5 to 500 mg, particularly 5 to 100 mg for injections and 10 to 250 mg for other dosage forms. Administration may be a single dose or multiple doses.
  • the 5-HT nerve cell regenerating agent of the present invention may be directly administered into the brain.
  • the 5-HT nerve cell regenerating agent of the present invention suspended in an appropriate buffer for example, phosphate buffered saline (PBS) or the like
  • PBS phosphate buffered saline
  • the present invention is also obtained by culturing stem cells on polymerized extracellular matrix protein in the presence of a BMP receptor kinase inhibitor, a 5-HT 7 receptor agonist, or a type I TGF- ⁇ receptor ligand.
  • Transplant compositions for the treatment of mental disorders eg, schizophrenia, depression, autism, eating disorders, sleep disorders, etc.
  • motor dysfunction after spinal cord injury including 5-HT neurons Offer things. It is effective when in vivo differentiation induction of neural stem cells cannot be achieved by the regenerative agent by transplanting and establishing 5-HT nerve cells themselves.
  • the composition for transplantation containing 5-HT nerve cells of the present invention has low toxicity.
  • the method for producing 5-HT nerve cells of the present invention does not require the use of feeder cells or serum. This is preferable in that there is no concern of introducing an exogenous factor to the ent.
  • iPS cells derived from the patient itself are used as stem cells, there is no problem of HLA mismatch and rejection due to immune response can be avoided. Therefore, the 5-HT nerve cells obtained by the production method of the present invention may be transplanted as they are, or may be frozen and stored and thawed before use. Examples of patients (subjects) to be transplanted include humans or other mammals (eg, mice, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.).
  • the transplant composition containing 5-HT nerve cells of the present invention can be prepared by suspending in a sterile aqueous solution suitable for transplantation in addition to 5-HT neurons.
  • a sterile aqueous liquid suitable for transplantation in addition to 5-HT neurons.
  • examples of the sterile aqueous liquid include isotonic solutions containing physiological saline, glucose and other adjuvants.
  • the number of cells contained in the transplant composition containing 5-HT nerve cells of the present invention varies depending on the transplant subject, transplant range, target disease, severity thereof, etc., for example, in the case of an adult psychiatric patient,
  • the transplant composition containing 5-HT nerve cells of the present invention can be transplanted in a number of about 10 to 100,000 cells, preferably about 100 to 10,000 cells, as a single transplant.
  • transplantation may be performed multiple times over a number of days in view of the degree of establishment.
  • ES cells Mouse ES cells (E14 strain) were cultured in 10 cm tissue culture dish coated with 0.1% gelatin in the presence of 1000 U / mL LIF (Leukemia Inhibitory Factor), 10% fetal bovine serum, Seed using ESM [GMEM (Glasgow) containing 0.1 mM NEAA (non-essential amino acid; Sigma), 1 mM sodium pyruvate (Sigma), 0.1 mM 2-mercaptoethanol (Sigma)], 5% CO 2 / 95% air at 37 ° C. incubator. Passaging was performed every other day using trypsin-EDTA (0.25% trypsin, 1 mM EDTA ⁇ 4Na) (GIBCO BRL).
  • Noggin prepared by the inventors
  • BMP receptor kinase inhibitor LDN-193189 SEMGENT
  • 5-HT agonist as serotonin hydrochloride Sigma-Aldrich
  • GR 127935 Hydrochloride hydrate Sigma-Aldrich
  • cisapride monohydrate Sigma-Aldrich
  • AS 19 Tocris
  • SB 269970 hydrochloride Tocris
  • fluoxetine hydrochloride (LKT laboratories)
  • type I TGF- ⁇ receptor SB431542 was used as a kinase inhibitor.
  • the cells were washed twice with PBS and then fixed with 4% paraformaldehyde at 4 ° C. for 10 minutes. This was washed once with PBS and subjected to blocking treatment with PBST (0.1 mM PBS with 0.3% Triton) / 10% FBS.
  • Mouse anti-GFP monoclonal antibody 500-fold dilution, Clontech
  • rabbit anti-serotonin polyclonal antibody 1000-fold dilution, DiaSorin
  • mouse anti-Tuj1 monoclonal antibody 500-fold dilution, Covance
  • rabbit anti-Map2 antibody 500-fold dilution
  • the plate was washed twice with PBST at 37 ° C. for 10 minutes. Then, as secondary antibodies, Alexa594-conjugated anti-mouse IgG (1000-fold diluted, Molecular Probes), Alexa488-conjugated anti-rabbit IgG (1000-fold diluted, Jackson ImmunoResearch Laboratories), Hoechst 33258 (1000-fold diluted with BIOCH33EM, Block-L diluted with BIOCH33EM) And reacted at 37 ° C. for 1 hour. Thereafter, the plate was washed 3 times with PBST at 37 ° C. for 10 minutes, mounted on a slide glass, and observed using a fluorescence microscope.
  • ES cells were cultured with addition of AS-19 from the 10th day of culturing on matrigel, washed twice with HBSS (Hanks balanced salt solution), and HBSS containing 56 mM K +. Was added at 500 ⁇ l / well, incubated at 37 ° C. for 15 minutes, and collected as a sample on the 18th day of culture. For the measurement, 10 ⁇ l of a sample was manually injected into a high performance liquid chromatography / electrochemical detector (HPLC / ECD) system to simultaneously detect dopamine and serotonin.
  • HPLC / ECD high performance liquid chromatography / electrochemical detector
  • Eicompak CA-50DS (2.1 mm id ⁇ 150 mm, Eicom) was used for the column, and 500 mg / l sodium decanesulfonate, 134 ⁇ M EDTA, 20% (v / v) was used for the mobile phase of HPLC.
  • Real-Time PCR RNA extraction was performed by the guanidine thiocyanate acidic phenol method.
  • reverse transcription was performed using reverse transcriptase M-MLV (GIBCO BRL) using 1.0 ⁇ g of total RNA extracted from ES cells or differentiation-induced cells as a template to prepare a cDNA pool.
  • a cDNA pool diluted 20-fold as a template, using primers specific to each gene, GoTaq (registered trademark) qPCR Master Mix (Promega), CFX 96 Real time System (BIO-RAD) Time PCR was performed.
  • Rat ⁇ -actin was used as an internal standard.
  • the reaction conditions were 95 ° C. for 15 seconds and 58 to 66 ° C. for 60 seconds for 40 cycles.
  • the sequences of the specific primers used and the annealing temperatures thereof are as follows.
  • ⁇ -actin 60 ° C 5′-ACCCACACTGTGCCCCATCTA-3 ′ (SEQ ID NO: 1) 5'-GCCACAGGATTCCATACCCA-3 '(SEQ ID NO: 2)
  • 5-HT 7 receptor 66 ° C 5′-TCCAGGTCAGTACCGGAATACAAAC-3 ′ (SEQ ID NO: 3) 5′-TACTCTTTCTCCCAGGGTTCCGCCTCT-3 ′ (SEQ ID NO: 4)
  • 5-HT 1A receptor 60 ° C.
  • Example 1 Induction of 5-HT Neuronal Differentiation by BMP Receptor Kinase Inhibitors
  • BMP Receptor Kinase Inhibitors Similar applied other major BMP antagonists, Chordin and Follistatin. It was clarified that no significant effect was observed. Therefore, in this example, a 5-HT neuronal differentiation-inducing action was examined using LDN-193189, which is a selective inhibitor of BMP receptor kinase, which causes BMP signal blockade from an action point different from that of the BMP antagonist.
  • LDN-193189 (STEMGENT) was added to the culture solution on the 0th to 5th days of culture, and ES cells were cultured on Matrigel.
  • the ES cells and culture method used were as described above, and 5-HT neuronal differentiation on the 14th day of culture was evaluated. As a result, it was revealed that LDN-193189 (3 to 100 nM) has an action of inducing differentiation of 5-HT nerve (FIG. 1).
  • Example 2 Effect of 5-HT on 5-HT nerve differentiation Reported by studies using primary cultured cells of rodent fetal brain that 5-HT can influence 5-HT nerve differentiation
  • 5-HT can influence 5-HT nerve differentiation
  • pluripotent undifferentiated stem cells such as ES cells and iPS cells
  • 5-HT receptor agonists and antagonists on 5-HT neuronal differentiation was examined.
  • the HT 7 receptor antagonist SB 269970 strongly inhibited the action by Noggin (FIG. 3A). Further, at this time, it was confirmed by immunostaining using an anti-5-HT antibody that SB 269970 decreased the number of 5-HT neurons in colonies containing 5-HT nerves induced by Noggin (Fig. 3B).
  • Example 3 Induction of 5-HT Neuronal Differentiation with 5-HT 7 Receptor Agonist (AS-19) ES cells were cultured on Matrigel for 18 days in the presence or absence of 5-HT 7 receptor agonist AS-19 Cultured and 5-HT neuronal differentiation was assessed by measuring dopamine and serotonin concentrations. AS-19 was added at 10 ⁇ M from the 10th day of Matrigel culture. As a result, it was clarified that the serotonin concentration was significantly higher when AS-19 was added than when AS-19 was not added (control) (FIG. 4).
  • Example 4 Induction of 5-HT neuronal differentiation by Noggin ES cells were cultured on Matrigel in the presence or absence of Noggin, and 5-HT neuronal differentiation was induced by mRNA of 5-HT 1A receptor and 5-HT 7 receptor. The expression level was evaluated by measuring. Noggin started to be added at an amount equivalent to 200 ng / mL from the start of Matrigel culture to the 5th day, and cells were collected on the 8th, 10th, 12th, and 14th days after the culture, and Real-Time was obtained using mRNA extracted from the cells as a template. PCR was performed. As a result, it was revealed that the mRNA expression levels of 5-HT 1A receptor and 5-HT 7 receptor were significantly higher when Noggin was added than when Noggin was not added (control). (FIGS. 5 and 6).
  • 5-HT 7 receptor agonist is influenced 5-HT 7 receptor agonists applied to the 5-HT neuronal differentiation, differentiation of ES cells into efficient 5-HT neurons AS-19 is on matrigel Induced and antagonists suppressed Noggin's effect of inducing 5-HT neurons, suggesting that 5-HT 7 receptors are involved in differentiation into 5-HT neurons. Therefore, it is examined whether ES cells can be efficiently differentiated into 5-HT neurons on Matrigel using various 5-HT 7 receptor agonists.
  • 5-HT 7 receptor agonists include 4- (2-Diphenyl) -N- (1,2,3,4-tetrahydronaphthalen-1-yl) -1-piperazine hexaneamide hydrochloride (LP12) , 4- [2- (Methylthio) phenyl] -N- (1,2,3,4-tetrahydro-1-naphthalenyl) -1-piperazine hexaneamide (LP44) or the like is used.
  • 5-HT neuronal differentiation is significantly promoted in the presence of the 5-HT 7 receptor agonist as compared to untreated cells, it can be selected as a 5-HT neuronal inducer.
  • a 5-HT 7 receptor agonist may be selected as a 5-HT nerve cell inducer by adding Noggin and further enhancing its 5-HT nerve cell induction effect. Such evaluation can also be performed by immunostaining for 5-HT or measuring the amount of 5-HT.
  • Example 6 Effect of SB431542 on serotonin neuronal differentiation (luciferase assay) It has been reported that the inhibitor of TGF- ⁇ signal SB431542 promotes differentiation from human ES cells to nerve cells alone or in combination with the BMP antagonist Noggin. Therefore, it was decided to examine what effect SB431542 would have on 5-HT neuronal differentiation. Noggin and SB431542 were added at 48 well plate from the beginning of the culture until the fifth day, Noggin was added in an amount equivalent to 200 ng / mL, and SB431542 was added at 0.1 to 10 ⁇ M. On the 14th day of culture, each culture solution was collected and luciferase assay was performed.
  • Example 7 Effect of SB431542 on serotonin neuronal differentiation (immunostaining)
  • Noggin was equivalent to 200 ng / mL
  • SB431542 was added at 1 to 10 ⁇ M from the start of the culture to the fifth day, and cultured for 14 days.
  • 2 units / mL Dispase (Gibco) was added at 400 ⁇ L / well, allowed to stand in an incubator at 5% CO 2 /95% air, 37 ° C. for 2 hours, and then 1.5 mL of detached colonies were obtained. It was collected in a tube and centrifuged (5000 rpm, 5 minutes), and the supernatant was removed.
  • Trypsin-EDTA (0.25% Trypsin, 1 mM EDTA ⁇ 4Na) was added at 100 ⁇ L / tube and pipetting was performed well. Three minutes later, 500 ⁇ L of ESM / 10% FBS was added to stop the reaction, and the mixture was centrifuged again (5000 rpm, 5 minutes), and the supernatant was removed. Then, suspension is performed with ESM / 10% knockout sealant replacement to 4 ⁇ 10 6 cells / mL, and 500 ⁇ L is placed on a 24-well plate with a cover glass coated with 0.075% poly-L-lysine. / Well.
  • Example 8 Effect of Noggin on downstream gene expression of TGF- ⁇ and BMP signal ES cell culture on Matrigel in the presence or absence of Noggin and the effect of TGF- ⁇ and BMP signal on downstream gene expression Were assessed by measuring mRNA expression levels.
  • Noggin was added in an amount equivalent to 200 ng / mL from the start of Matrigel culture to the second day, cells were collected on the second day after the culture, and Real-Time PCR was performed using mRNA extracted from the cells as a template.
  • the expression of the Id gene known as the downstream gene of the BMP signal was reduced by the addition of Noggin, and the expression of Lefty known as the downstream gene of TGF- ⁇ was increased.
  • 5-HT 7 receptor was first found to be involved in 5-HT neuronal differentiation, and 5-HT neuron was used in schizophrenia, depression, autism, eating disorders, sleep disorders. It is suspected that it may be involved in mental illness such as psychiatric disorders or dysfunction after spinal injury, pain-related symptoms, etc., so that BMP receptor, 5-HT 7 receptor, type I TGF- ⁇ receptor Elucidation of the relationship between the disease and symptoms can be used for the development of a therapeutic agent for the disease in the future.
  • This application is based on Japanese Patent Application No. 2011-035192 (filing date: February 21, 2011) filed in Japan, the contents of which are incorporated in full herein.

Abstract

La présente invention concerne un procédé de préparation de neurones 5-HT, par la culture de cellules souches sur des protéines de matrice extracellulaire polymérisées en présence d'un inhibiteur des récepteurs kinase des BMP, d'un agoniste du récepteur 5-HT7 ou d'un ligand du récepteur de TGF-β de type I.
PCT/JP2012/054175 2011-02-21 2012-02-21 Préparation de neurones 5-ht WO2012115120A1 (fr)

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JP2016515119A (ja) * 2013-03-14 2016-05-26 ザ ブリガム アンド ウィメンズ ホスピタル インコーポレイテッドＴｈｅ Ｂｒｉｇｈａｍ ａｎｄ Ｗｏｍｅｎ’ｓ Ｈｏｓｐｉｔａｌ， Ｉｎｃ． Ｂｍｐ阻害剤およびその使用方法
WO2016210292A1 (fr) 2015-06-25 2016-12-29 Children's Medical Center Corporation Procédés et compositions se rapportant à l'expansion, l'enrichissement et la conservation de cellules souches hématopoïétiques
WO2017161001A1 (fr) 2016-03-15 2017-09-21 Children's Medical Center Corporation Procédés et compositions concernant l'expansion de cellules souches hématopoïétiques
US11499138B2 (en) 2016-10-21 2022-11-15 National University Corporation Gunma University Method for manufacturing peripheral nerve cells

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016515119A (ja) * 2013-03-14 2016-05-26 ザ ブリガム アンド ウィメンズ ホスピタル インコーポレイテッドＴｈｅ Ｂｒｉｇｈａｍ ａｎｄ Ｗｏｍｅｎ’ｓ Ｈｏｓｐｉｔａｌ， Ｉｎｃ． Ｂｍｐ阻害剤およびその使用方法
WO2016210292A1 (fr) 2015-06-25 2016-12-29 Children's Medical Center Corporation Procédés et compositions se rapportant à l'expansion, l'enrichissement et la conservation de cellules souches hématopoïétiques
WO2017161001A1 (fr) 2016-03-15 2017-09-21 Children's Medical Center Corporation Procédés et compositions concernant l'expansion de cellules souches hématopoïétiques
EP4049665A1 (fr) 2016-03-15 2022-08-31 Children's Medical Center Corporation Procédés et compositions associées à l'expansion de cellules souches hématopoïétiques
US11499138B2 (en) 2016-10-21 2022-11-15 National University Corporation Gunma University Method for manufacturing peripheral nerve cells

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