WO2023010897A1 - 人诱导性多能干细胞向少突胶质细胞分化的方法，试剂盒以及应用 - Google Patents

人诱导性多能干细胞向少突胶质细胞分化的方法，试剂盒以及应用 Download PDF

Info

Publication number
WO2023010897A1
WO2023010897A1 PCT/CN2022/088517 CN2022088517W WO2023010897A1 WO 2023010897 A1 WO2023010897 A1 WO 2023010897A1 CN 2022088517 W CN2022088517 W CN 2022088517W WO 2023010897 A1 WO2023010897 A1 WO 2023010897A1
Authority
WO
WIPO (PCT)
Prior art keywords
medium
combination
supplement
small molecule
essential amino
Prior art date
Application number
PCT/CN2022/088517
Other languages
English (en)
French (fr)
Inventor
吴理达
顾雨春
Original Assignee
呈诺再生医学科技(珠海横琴新区)有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 呈诺再生医学科技(珠海横琴新区)有限公司 filed Critical 呈诺再生医学科技(珠海横琴新区)有限公司
Publication of WO2023010897A1 publication Critical patent/WO2023010897A1/zh
Priority to US18/521,956 priority Critical patent/US20240093148A1/en

Links

Images

Classifications

    • 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
    • C12N5/0622—Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/22—Anxiolytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/02—Non-specific cardiovascular stimulants, e.g. drugs for syncope, antihypotensives
    • 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
    • C12N5/0623—Stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/44—Thiols, e.g. mercaptoethanol
    • 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
    • C12N2501/01—Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
    • 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
    • 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
    • C12N2501/105—Insulin-like growth factors [IGF]
    • 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
    • C12N2501/12—Hepatocyte growth factor [HGF]
    • 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
    • C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
    • 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
    • C12N2501/135—Platelet-derived growth factor [PDGF]
    • 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
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • 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
    • C12N2501/30—Hormones
    • C12N2501/33—Insulin
    • 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
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/385—Hormones with nuclear receptors of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
    • 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
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/395—Thyroid hormones
    • 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
    • C12N2501/40—Regulators of development
    • C12N2501/41—Hedgehog proteins; Cyclopamine (inhibitor)
    • 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
    • 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
    • C12N2501/999—Small molecules not provided for elsewhere
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the field of biotechnology, in particular to a method, kit and application for human induced pluripotent stem cells to differentiate into oligodendrocytes.
  • Oligodendrocyte progenitor cells are central nervous system cells present in vertebrates, which form myelin sheaths to wrap around nerve fibers, and play a key role in the transmission of nerve signals and the nutrition and protection of nerve fibers .
  • Oligodendrocytes produce a lipid-rich lamellar myelin sheath that matures to form myelin, which coats neuronal axons and creates defined electrically insulating segments to maximize action potential conduction velocity.
  • Myelin is also important for axonal integrity and survival, and it has been shown that even small changes affecting oligodendrocyte metabolism can lead to neurodegeneration.
  • oligodendrocytes not only provide inert insulation, but that myelination is a dynamic process that affects cognitive function and even behavior.
  • MS Multiple sclerosis
  • adrenoleukodystrophy white matter ablative disease
  • Pelizaeus-Merzbacher disease and leukodystrophy are conditions of demyelination or dysmyelination instance of .
  • the critical role of oligodendrocytes is emerging in many other neurological and neurodegenerative conditions, including amyotrophic lateral sclerosis, Huntington's disease, Alzheimer's disease, and In schizophrenia.
  • iPSCs Human induced pluripotent stem cells
  • NSCs neural stem cells
  • OPCs oligodendrocyte precursor cells
  • astrocytes glial cells
  • iPSCs can also be directly induced into OPCs, but current techniques are time-consuming and inefficient.
  • the present invention provides an improved method for the differentiation of induced pluripotent stem cells into oligodendrocytes mediated by small molecules and other factors.
  • the present invention is based on the same improved method for inducing OPCs from iPSCs via NSCs, and the experiment It is confirmed that puerarin increases the induction efficiency and quantity of induced pluripotent stem cells differentiated into oligodendrocytes, and can increase the number of oligodendrocytes by 30% in the late stage of induction.
  • the present invention provides a method for inducing oligodendrocytes, the method comprising culturing stem cells using at least one of the following media:
  • the method is to sequentially use complete neural induction medium, N2 medium, B27 medium, and OPC maturation medium for stem cell culture.
  • the time for using the complete neural induction medium is 4-10 days; preferably, 7 days; more preferably, 0-7 days.
  • the time for using the N2 medium is 1-6 days; preferably, 3 days; more preferably, 8-11 days.
  • the time for using the B27 medium is 4-10 days; preferably, 7 days; more preferably, 12-19 days.
  • the OPC maturation medium is used for at least 7 days; preferably, at least 10 days; more preferably, 20-30 days.
  • the culturing is performed on any cell derived from humans, orangutans, monkeys, horses, cows, sheep, pigs, donkeys, camels, dogs, rabbits, cats, rats, mice, fish, birds or insects. cell culture.
  • the stem cells include one of ESC (embryonic stem cells), induced pluripotent stem cells (iPSC), embryoid bodies, hematopoietic stem cells, neural stem cells, mesenchymal stem cells, skin stem cells, fat stem cells, and umbilical cord blood stem cells. one or more species.
  • ESC embryonic stem cells
  • iPSC induced pluripotent stem cells
  • embryoid bodies hematopoietic stem cells
  • neural stem cells mesenchymal stem cells
  • skin stem cells skin stem cells
  • fat stem cells and umbilical cord blood stem cells.
  • umbilical cord blood stem cells one or more species.
  • said stem cells are iPSCs.
  • the iPSC cells can be commercialized cell lines, or can be induced from donor cells, including villi cells, skin (fibroblasts and keratinocytes), amniotic fluid, extraembryonic One or more of tissues (placenta and umbilical cord), umbilical cord blood, periosteum, dental tissue, adipose tissue, neural stem cells, liver cells, mesenchymal stem cells, peripheral blood cells, mammary gland epithelial cells, adipose stem cells, umbilical cord stroma, and placenta.
  • donor cells including villi cells, skin (fibroblasts and keratinocytes), amniotic fluid, extraembryonic One or more of tissues (placenta and umbilical cord), umbilical cord blood, periosteum, dental tissue, adipose tissue, neural stem cells, liver cells, mesenchymal stem cells, peripheral blood cells, mammary gland epithelial cells, adipose stem cells,
  • the complete medium for neural induction contains the first combination of small molecule compounds.
  • the complete medium for nerve induction is composed of a combination of the first basal medium, non-essential amino acids, glutamine, a reducing agent and the first small molecule compound.
  • the first basal medium, the second basal medium, the third basal medium, and the fourth basal medium of the present invention each independently include TeSR-E8, mTESR1, E8, Essential 8 TM Medium, Dalbe Gram modified Eagle's Medium (DMEM, Dulbecco's Modified Eagle's Medium), Minimal Essential Medium (MEM, Minimal Essential Medium), Eagle's Basic Medium (BME, Basal Medium Eagle), F-10, F-12, ⁇ -Minimal Essential Medium ( ⁇ -MEM, ⁇ -Minimal Essential Medium), G-Minimal Essential Medium (G-MEM, Glasgow's Minimal Essential Medium), IMPM (IMDM, Iscove's Modified Dulbecco's Medium), AmnioMax, new second generation Amniotic fluid medium (Amino MaxIIcomplete Medium, Gibco, Newyork, USA), Chang's medium, MesemCult-XF medium (STEMCELL Technologies, Vancouver, Canada), RPMI1640, Ham's F12, DMEM/
  • the first basal medium, the second basal medium, the third basal medium, and the fourth basal medium of the present invention are DMEM/F12.
  • the reducing agent of the present invention includes but not limited to ⁇ -mercaptoethanol (2-Mercaptoethanol), dithiothreitol, dithioerythritol, reduced glutathione, cysteine, thio Carbamate, sodium dithiosulfinate, ascorbate, tin dichloride, or sodium borohydride.
  • the glutamine in the present invention is GlutaMAX-I
  • the "GlutaMAX” is a cell culture additive that can directly replace L-glutamine in the cell culture medium.
  • the non-essential amino acids in the present invention include alanine, arginine, aspartic acid, cystine, proline, tyrosine.
  • the complete nerve induction medium is composed of 98% DMEM/F-12 medium, 1% non-essential amino acid, 1% GlutaMAX-I, 2-Mercaptoethanol and the first small molecule compound.
  • the complete medium for neural induction is composed of 98% DMEM/F-12 medium (Life Technologies, cat.no.11039021), 1% non-essential amino acids (Life Technologies, cat.no.11140-050), Composition of 1% GlutaMAX-I (Life Technologies, cat.no.35050061), 2-Mercaptoethanol (Gibco, 31350010) and the first small molecular compound.
  • the "brackets" represent the manufacturer and article number of the product.
  • the first combination of small molecule compounds includes at least one of insulin, TGF- ⁇ signaling pathway inhibitors, BMP signaling pathway inhibitors, and RAR nuclear receptor agonists.
  • the TGF- ⁇ signaling pathway inhibitors include SB431542, Dorsomorphin, A83-01, LDN193189, RepSox, SB 525334, DMH-1, SB-505124, BMP signaling agonist sb4, GW788388, ITD-1, LSKL, SD -208, LDN-212854, K02288, LDN-214117, R-268712, SM16, A77-01, ALK2-IN-2, PD-161570, pm26, TGF- ⁇ 1 peptide TFA, Isosaponarin, BIO-013077-01.
  • the TGF- ⁇ signaling pathway inhibitor is selected from SB431542; preferably, its product number is medchemexpress, HY-10431.
  • the working concentration of SB431542 is 10 ⁇ M.
  • the BMP signaling pathway inhibitors include Dorsomorphin, A83-01, LDN193189, RepSox, SB 525334, DMH-1, SB-505124, BMP signaling agonist sb4, GW788388, ITD-1, LSKL, SD-208, LDN -212854, K02288, LDN-214117, R-268712, SM16, A77-01, ALK2-IN-2, PD-161570, pm26, TGF- ⁇ 1 peptide TFA, Isosaponarin, BIO-013077-01.
  • the BMP signaling pathway inhibitor is LDN193189; preferably, its product number is medchemexpress, HY-12071.
  • the working concentration of LDN193189 is 0.25 ⁇ M.
  • the agonists of the RAR nuclear receptor include retinoic acid, Rapamycin, 3-Methyladenine, Acetylcysteine, 5-Fluorouracil, Hydrocortisone, Docetaxel, Rosiglitazone, Estradiol, Melatonin, GW9662, Nicotinamide, Cytarabine, Isoprenaline hydrochloride, Prostaglandin E2 , Acetaminophen, (-)-Epigallocatechin Gallate, ⁇ -Nicotinamide mononucleotide, Calcitriol, DHEA, Liothyronine, NAD + , Luteolin, Thymidine, Docosahexaenoic Acid, Kaempferol, Palmitic acid, Cyclopamine, Genistein, L-Glutathione reduced.
  • the agonist of the RAR nuclear receptor is retinoic acid; preferably, its product number is medchemexpress, HY-14649.
  • the working concentration of the vitamin A acid is 100 ⁇ M.
  • the N2 medium contains the second combination of small molecule compounds.
  • the N2 medium is composed of the second basal medium, non-essential amino acids, glutamine, reducing agent, N2 supplement and the second small molecule compound.
  • the "N2 supplement" of the present invention contains Human Transferrin Holo, Insulin Recombinant Full Chain, Progesterone, Putrescine, Selenite, It can be a commercial product or self-prepared.
  • the N2 medium is composed of 97% DMEM/F-12 medium, 1% non-essential amino acid, 1% GlutaMAX-I, 2-Mercaptoethanol, 1% N2 supplement and a combination of the second small molecule compound.
  • the N2 medium is composed of 97% DMEM/F-12 medium (Life Technologies, cat.no.11039021), 1% non-essential amino acids (Life Technologies, cat.no.11140-050), 1% GlutaMAX -I (Life Technologies, cat. no. 35050061), 2-Mercaptoethanol (Gibco, 31350010), 1% N2 supplement (ThermoFisher, cat. no. 17502001) and the second small molecule compound.
  • the "brackets" represent the manufacturer and article number of the product.
  • the second small molecule compound combination includes an agonist of RAR nuclear receptor and/or an activator of Hedgehog signaling pathway.
  • the agonist of the RAR nuclear receptor is the same as described above.
  • the Hedgehog signaling pathway activator includes SAG, Cyclopamine, Purmorphamine, 20(S)-Hydroxycholesterol, Halcinonide, Jervine, ALLO-2, IHR-Cy3.
  • the Hedgehog signaling pathway activator is SAG; preferably, its product number is medchemexpress, HY-12848.
  • the B27 medium contains a third combination of small molecule compounds.
  • the B27 medium is composed of a third basal medium, non-essential amino acids, glutamine, a reducing agent, N2 supplement, B27 supplement and a third small molecule compound.
  • the "B27 supplement” described in the present invention contains biotin (Biotin), DL- ⁇ -tocopherol acetate (DLAlpha Tocopherol Acetate), DL- ⁇ -tocopherol (DLAlpha-Tocopherol), BSA (fattyacidfreeFractionV), peroxide Catalase, Human Recombinant Insulin, Human Transferrin, Superoxide Dismutase, Corticosterone, D-Galactose, Ethanolamine HCl, Glutathione reduced, L-Carnitine HCl, Linoleic Acid, Linolenic Acid, Progesterone, Putrid Amine (Putrescine 2HCl), sodium selenite (Sodium Selenite), triiodothyronine (T3 triodo-I-thyronine), which can be commercial products or obtained by self-preparation.
  • Biotin biotin
  • DLAlpha Tocopherol Acetate DLAlpha Tocopherol Acetate
  • the B27 medium consists of 95% DMEM/F-12 medium, 1% non-essential amino acids, 1% GlutaMAX-I, 2-Mercaptoethanol, 1% N2 supplement, 2% B27 supplement and a third small molecule compound Composition.
  • the B27 medium is composed of 95% DMEM/F-12 medium (Life Technologies, cat.no.11039021), 1% non-essential amino acids (Life Technologies, cat.no.11140-050), 1% GlutaMAX -I (Life Technologies, cat.no.35050061), 2-Mercaptoethanol (Gibco, 31350010), 1% N2 supplement (ThermoFisher, cat.no.17502001), 2% B27supplement (ThermoFisher, cat.no.12587010) and Combination of three small molecule compounds.
  • the "brackets" represent the manufacturer and article number of the product.
  • the third small molecule compound combination includes at least one of insulin, an agonist of RAR nuclear receptor and an activator of Hedgehog signaling pathway.
  • the agonist of the RAR nuclear receptor and the activator of the Hedgehog signaling pathway are the same as described above.
  • the OPC maturation medium contains a fourth combination of small molecule compounds.
  • the OPC maturation medium also includes the fourth basal medium, non-essential amino acids, glutamine, reducing agent, N2 supplement, B27 supplement.
  • the OPC maturation medium also includes 95% DMEM/F-12 medium, 1% non-essential amino acids, 1% GlutaMAX-I, 2-Mercaptoethanol, 1% N2 supplement, 2% B27 supplement.
  • the OPC maturation medium also includes 95% DMEM/F-12 medium (Life Technologies, cat.no.11039021), 1% non-essential amino acids (Life Technologies, cat.no.11140-050), 1 % GlutaMAX-I (Life Technologies, cat.no.35050061), 2-Mercaptoethanol (Gibco, 31350010), 1% N2 supplement (ThermoFisher, cat.no.17502001), 2% B27 supplement (ThermoFisher, cat.no.12587010 ).
  • the "brackets" represent the manufacturer and article number of the product.
  • the fourth small molecule compound combination includes at least one of insulin, PDGF-AA, IGF-1, HGF, NT3, T3, Biotin, and cAMP.
  • the fourth small molecule compound combination includes insulin, PDGF-AA (PeproTech, cat.no.AF-100-13A), IGF-1 (PeproTech, cat.no.AF-100-11), HGF ( PeproTech, cat.no.100-39H), NT3 (PeproTech, cat.no.450-03), T3 (Sigma-Aldrich, cat.no.T2877), Biotin (Sigma-Aldrich, cat.no.B4639), cAMP (Sigma-Aldrich, cat. no. D0260) and 5-HT2C receptor antagonist.
  • the "brackets" represent the manufacturer and article number of the product.
  • the fourth small molecule compound combination also contains a 5-HT2C receptor antagonist
  • the 5-HT2C receptor antagonists include Puerarin (puerarin), Harmine, SCH-23390hydrochloride, Olanzapine, GTS-21dihydrochloride, Thioridazine hydrochloride, Risperidone, SB-269970hydrochloride, Dihydroeriptamine mesylate, Amitriptyline serhydrochloride, Aritriptyline hydrochloride, Aritriptyline hydrochloride, Aritriptyline hydrochloride, WAY-100635Maleate ⁇ Buspirone hydrochloride ⁇ Cisapride ⁇ Methiothepin mesylate ⁇ Trazodone hydrochloride ⁇ Pindolol ⁇ Quetiapine ⁇ Lumateperone tosylate ⁇ Perphenazine ⁇ 8-OH-DPAT ⁇ Levomepromazine ⁇ SB-224289hydrochloride ⁇ Volinanserin ⁇ Ziprasidone ⁇ Paliperidone ⁇ Ser
  • the 5-HT2C receptor antagonist is Puerarin; preferably, its product number is medchemexpress, HY-N0145.
  • the present invention provides a method for inducing neural stem cells, the method comprising the aforementioned complete neural induction medium and/or the aforementioned N2 medium for cell culture.
  • the method includes the aforementioned complete nerve induction medium and the aforementioned N2 medium for cell culture.
  • the present invention also provides a combination of small molecule compounds comprising a 5-HT2C receptor antagonist.
  • the combination of small molecule compounds is the aforementioned fourth combination of small molecule compounds.
  • the present invention also provides a kit for inducing oligodendrocytes, which includes the aforementioned insulin, TGF- ⁇ signaling pathway inhibitors, BMP signaling pathway inhibitors, agonists of RAR nuclear receptors, Hedgehog One or more of signal pathway activators and 5-HT2C receptor antagonists.
  • the kit includes reagents for configuring at least one of the following culture media:
  • the present invention provides any one of the following medium and its application in inducing oligodendrocytes:
  • the present invention provides the application of the combination of small molecule compounds comprising 5-HT2C receptor antagonists in inducing oligodendrocytes.
  • the present invention provides the application of Puerarin in inducing oligodendrocytes.
  • the present invention provides the application of the aforementioned kit in inducing oligodendrocytes.
  • the present invention provides any one of insulin, TGF- ⁇ signaling pathway inhibitor, BMP signaling pathway inhibitor, RAR nuclear receptor agonist, Hedgehog signaling pathway activator, 5-HT2C receptor antagonist inducing Applications in oligodendrocytes.
  • the present invention provides the application of insulin, TGF- ⁇ signal pathway inhibitor, BMP signal pathway inhibitor, RAR nuclear receptor agonist and Hedgehog signal pathway activator in inducing neural stem cells.
  • the present invention provides a cell population, characterized in that the cell population is selected from the following one:
  • a cell population prepared by the aforementioned method for inducing neural stem cells includes cells expressing at least one of NKX2.2+, Nestin+, and PAX6+.
  • Olig2+ expressing cells are present in said population of cells.
  • the cell population prepared by the aforementioned method of inducing oligodendrocytes in which the oligodendrocytes account for at least 20% of all cells; preferably, at least 21%, 25%, 30%, 35%, 36%.
  • the oligodendrocytes are cells expressing at least one of Olig2, Nkx2.2, O4 and MBP.
  • the oligodendrocytes are cells that co-express Olig2 and Nkx2.2; or, the oligodendrocytes are cells that co-express O4 and MBP.
  • the present invention provides the use of the cells prepared by the aforementioned method of inducing oligodendrocytes in the treatment of diseases of the central nervous system.
  • the cells are oligodendrocytes.
  • the diseases of the central nervous system include but are not limited to neurodegenerative diseases, demyelinating diseases, epilepsy, traumatic brain injury, shock, dementia, glaucoma, regeneration after nervous system damage, and mental diseases.
  • the neurodegenerative diseases include Alzheimer's disease, cerebellar atrophy, primary lateral sclerosis, spinal muscular atrophy, Parkinson's disease, Huntington's disease, Creutzfeldt-Jakob disease, bovine spongiform Encephalopathy, ataxia-telangiectasia, amyotrophic lateral sclerosis, mental illness.
  • the "demyelinating disease” mentioned in the present invention refers to the damage of myelin sheath after the formation of myelin sheath. group of diseases.
  • the demyelinating diseases include two categories: hereditary and acquired.
  • the demyelinating diseases include stroke, multiple sclerosis, neuromyelitis optica, acute disseminated encephalomyelitis, diffuse sclerosis, concentric sclerosis, leukodystrophy, central pontine myelolysis, acute Inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyneuropathy, leukoencephalopathy caused by ischemic-hypoxic diseases, subacute combined degeneration caused by nutritional deficiency diseases, subacute sclerosis caused by viral infection Panencephalitis or progressive multifocal leukoencephalopathy, diabetic neuropathy, neuropathy in systemic lupus erythematosus, adrenoleukodystrophy, leukoblastic disease, Pelizaeus-Merzbacher disease disease).
  • said mental illness includes schizophrenia, depression, paranoia, anxiety, obsessive-compulsive disorder, phobia,
  • the present invention provides a method of treating the aforementioned neurological disease, the method comprising inducing oligodendrocytes by using the aforementioned method.
  • Figure 1 is the image of immunofluorescence detection on day 16; A: olig2+, Nkx2.2+, B: olig2+, Nestin+, C: olig2+, PAX6+.
  • Figure 2 is the image of immunofluorescence detection on the 33rd day.
  • Fig. 3 is a graph showing the statistical results of the percentage of oligodendrocytes obtained by comparing the OPC maturation medium with or without puerarin.
  • Fig. 4 is the immunofluorescence detection diagram of the comparison of adding or not adding puerarin in the OPC maturation medium.
  • Example 1 Inducing iPSC cell differentiation and verifying the induction effect
  • Complete medium for neural induction 98% DMEM/F-12 medium, 1% non-essential amino acids, 1% GlutaMAX-I, 0.1mM 2-Mercaptoethanol, 10 ⁇ M SB431542, 0.25 ⁇ M LDN193189 and 100 ⁇ M tretinoin, 25 ⁇ g/ml insulin .
  • N2 medium 97% DMEM/F-12 medium, 1% non-essential amino acid, 1% GlutaMAX-I, 0.1mM 2-Mercaptoethano, 1% N2 supplement, 1 ⁇ M SAG and 100 ⁇ M vitamin A acid.
  • B27 medium 95% DMEM/F-12 medium, 1% non-essential amino acid, 1% GlutaMAX-I, 0.1mM 2-Mercaptoethanol, 1% N2 supplement, 2% B27supplement and 1 ⁇ M SAG and 100 ⁇ M vitamin A acid, 25 ⁇ g /ml insulin.
  • OPC maturation medium - without puerarin 95% DMEM/F-12 medium, 1% non-essential amino acids, 1% GlutaMAX-I, 0.1mM 2-Mercaptoethanol, 1% N2 supplement, 2% B27supplement, 10ng/mL PDGF-AA, 10ng/mL IGF-1, 5ng/mL HGF, 10ng/mL NT3, 60ng/mL T3, 100ng/mL Biotin, 1 ⁇ M cAMP, 25 ⁇ g/ml insulin.
  • OPC maturation medium-with puerarin 95% DMEM/F-12 medium, 1% non-essential amino acids, 1% GlutaMAX-I, 0.1mM2-Mercaptoethanol, 1% N2 supplement, 2% B27supplement, 10ng/mL PDGF- AA, 10ng/mL IGF-1, 5ng/mL HGF, 10ng/mL NT3, 60ng/mL T3, 100ng/mL Biotin, 1 ⁇ M cAMP, 25 ⁇ M Puerarin, 25 ⁇ g/ml insulin.
  • iPSCs were replaced with E8 complete medium (STEMCELL, 05991) for neural induction complete medium.
  • the B27 medium was replaced with OPC maturation medium - without puerarin, and the cells were converted from adherent culture to suspension culture.
  • Olig2+ green, OPC marker
  • NKX2.2+ red, neural stem cell marker
  • Olig2+ green, OPC marker
  • Nestin+ red, neural stem cell marker
  • Olig2+ green, OPC marker
  • PAX6+ red, neural stem cell marker
  • Embodiment 2 the effect of puerarin in OPC maturation medium on inducing oligodendrocytes
  • the OPC maturation medium without puerarin was used as the control group, and about 21% oligodendrocytes could be generated in the control group, and about 36% oligodendrocytes could be generated after adding puerarin. It proved that the addition of puerarin in OPC maturation medium was beneficial to the production of oligodendrocytes.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Developmental Biology & Embryology (AREA)
  • Psychiatry (AREA)
  • Ophthalmology & Optometry (AREA)
  • Reproductive Health (AREA)
  • Pain & Pain Management (AREA)
  • Psychology (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Gynecology & Obstetrics (AREA)
  • Immunology (AREA)
  • Hospice & Palliative Care (AREA)
  • Epidemiology (AREA)
  • Virology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了人诱导性多能干细胞向少突胶质细胞分化的方法，试剂盒以及应用。所述方法包括使用以下至少一种培养基培养干细胞：神经诱导完全培养基、N2培养基、B27培养基、OPC成熟培养基；更优选的，使用含有葛根素的OPC成熟培养基诱导少突胶质细胞相较于不使用葛根素可以提高30％少突胶质细胞的数量。

Description

人诱导性多能干细胞向少突胶质细胞分化的方法，试剂盒以及应用 技术领域
本发明涉及生物技术领域，具体涉及人诱导性多能干细胞向少突胶质细胞分化的方法，试剂盒以及应用。
背景技术
少突胶质前体细胞(Oligodendrocyte progenitor cells，OPC)是存在于脊椎动物中的中枢神经系统细胞，它形成髓鞘包绕神经纤维，为神经信号的传递和神经纤维的营养及保护起关键作用。少突胶质细胞产生富含脂质的层状髓鞘，分化成熟形成髓鞘，其覆盖神经元轴突并产生限定的电绝缘区段以使动作电位传导速度最大化。髓磷脂对于轴突完整性和存活也是重要的，并且已显示即使影响少突胶质细胞代谢的小变化也可导致神经退化。髓鞘化过程在人类中特别重要，因为人脑具有较高含量的有髓鞘神经元(白质)，并且髓鞘化在出生之后持续并贯穿一生。这表明少突胶质细胞不只提供惰性绝缘，而且髓鞘化是动态过程，从而影响认知功能乃至行为。多发性硬化症(MS)、肾上腺脑白质营养不良、白质消融性疾病、佩利措伊斯-梅茨巴赫病(Pelizaeus-Merzbacher disease)和脑白质营养不良是脱髓鞘或髓鞘形成障碍病症的实例。此外，少突胶质细胞的关键作用正显现在许多其它神经病症和神经退化性病状，包括肌萎缩性侧索硬化、亨廷顿氏病(Huntington’s disease)、阿尔茨海默氏病(Alzheimer's disease)和精神分裂症中。
神经干细胞生物学和临床应用的进展为难以治愈的神经系统疾病的治疗提供了可能性。同时，提高被植入的OPC的存活率以及改善它们的微环境对于利用神经干细胞治疗神经系统疾病也具有至关重要的作用。来源于人的OPC不仅有助于更好的了解少突胶质细胞的功能及轴突-神经元的相互作用，更为髓鞘的修复研究及药物治疗提供了必不可少的工具。
人诱导性多能干细胞(iPSC)可被体外诱导成神经干细胞(NSC)，进而产生不同谱系的神经元、胶质细胞，如少突胶质前体细胞(OPCs)，或星形胶质细胞。iPSC也可被直接诱导成OPCs，但目前技术所需的时间长，且效率低。
发明内容
本发明提供了一种由小分子和其他因子介导的，经诱导性多能干细胞向少突胶质细胞分化的改良方法，本发明基于相同的iPSC经NSC诱导OPCs的改良方法，并且，实验中证实了葛根素增加经诱导性多能干细胞向少突胶质细胞分化的诱导效率和数量，在诱导后期可增加30％少突胶质细胞的数量。
方法
第一方面，本发明提供了一种诱导少突胶质细胞的方法，所述方法包括使用以下培养基至少一种培养干细胞：
1)神经诱导完全培养基；
2)N2培养基；
3)B27培养基；
4)OPC成熟培养基。
优选地，所述方法是依次使用神经诱导完全培养基、N2培养基、B27培养基、OPC成熟培养基进行干细胞培养。
优选地，使用所述神经诱导完全培养基的时间是4-10天；优选地，7天；更优选地，第0-7天。
优选地，使用所述N2培养基的时间是1-6天；优选地，3天；更优选地，第8-11天。
优选地，使用所述B27培养基的时间是4-10天；优选地，7天；更优选地，第12-19天。
优选地，使用所述OPC成熟培养基的时间是至少7天；优选地，至少10天；更优选地，第20-30天。
优选地，所述的培养是对来源于人、猩猩、猴、马、牛、羊、猪、驴、骆驼、狗、兔、猫、大鼠、小鼠、鱼、鸟或昆虫的任意细胞进行的细胞培养。
优选地，所述干细胞包括ESC(胚胎干细胞)、诱导性多能干细胞(iPSC)、拟胚体、细胞造血干细胞、神经干细胞、间充质干细胞、皮肤干细胞、脂肪干细胞、脐血干细胞中的一种或多种。
更优选地，所述干细胞是iPSC。
优选地，所述iPSC细胞可以是商品化的细胞系，也可以是由供体细胞诱导而来，所述供体细胞包括绒毛细胞、皮肤(成纤维细胞和角质形成细胞)、羊水、胚外组织(胎盘和脐带)、脐带血、骨膜、牙组织、脂肪组织、神经干细胞、肝细胞、间质干细胞、外周血细胞、乳腺上皮细胞、脂肪干细胞，脐带基质和胎盘中的一种或多种。
在一种实施方式中，所述神经诱导完全培养基中含有第一小分子化合物组合。
在一种实施方式中，所述神经诱导完全培养基是由第一基础培养基、非必须氨基酸、谷氨酰胺、还原剂和第一小分子化合物组合组成。
优选地，本发明所述的第一基础培养基、第二基础培养基、第三基础培养基、第四基础培养基各自独立地包括TeSR-E8、mTESR1、E8、Essential 8 TMMedium、达尔伯克改良伊格尔培养基(DMEM，Dulbecco’s Modified Eagle’s Medium)、最小必需培养基(MEM，Minimal Essential Medium)、伊格尔基本培养基(BME，Basal Medium Eagle)、F-10、F-12、α-最小必需培养基(α-MEM，α-Minimal Essential Medium)、G-最小必需培养基(G-MEM、Glasgow’s Minimal Essential Medium)、IMPM(IMDM，Iscove’s Modified Dulbecco’s Medium)、AmnioMax、新型二代羊水培养基(Amino MaxⅡcomplete Medium，Gibco，Newyork，USA)、Chang’s培养基、MesemCult-XF培养基(STEMCELL Technologies，Vancouver，Canada)、RPMI1640、Ham’s F12、DMEM/F12、Ham's F-12K Medium、Hepato ZYME-SFM、William’s EMedium、Waymouth’s Medium或Hepatocyte Culture Medium。
优选地，本发明所述第一基础培养基、第二基础培养基、第三基础培养基、第四基础培养基是DMEM/F12。
优选地，本发明所述还原剂包括但不限于β-巯基乙醇(2-Mercaptoethanol)、二硫苏糖醇、二硫赤藓糖醇、还原型谷胱甘肽、半胱氨酸、硫代胺甲酸盐、二硫亚磺酸钠、抗坏血酸盐、二氯化锡或硼氢化钠。
优选地，本发明所述谷氨酰胺选用GlutaMAX-I，所述“GlutaMAX”是一种细胞培养添加剂，可直接替代细胞培养基中的L-谷氨酰胺。
优选地，本发明所述非必须氨基酸包括丙氨酸、精氨酸、天门冬氨酸、胱氨酸、脯氨酸、酪氨酸。
优选地，所述神经诱导完全培养基是由98％DMEM/F-12培养基、1％非必须氨基酸、1％GlutaMAX-I、2-Mercaptoethanol和第一小分子化合物组合组成。
优选地，所述神经诱导完全培养基是由98％DMEM/F-12培养基(Life Technologies，cat.no.11039021)、1％非必须氨基酸(Life Technologies，cat.no.11140-050)、1％GlutaMAX-I(Life Technologies，cat.no.35050061)、2-Mercaptoethanol(Gibco，31350010)和第一小分子化合物组合组成。“括号”内代表所述产品的厂家及货号。
优选地，所述第一小分子化合物组合包括胰岛素、TGF-β信号通路抑制剂、BMP信号通路抑制剂、RAR核受体的激动剂中的至少一种。
优选地，所述TGF-β信号通路抑制剂包括SB431542、Dorsomorphin、A83-01、LDN193189、RepSox、SB 525334、DMH-1、SB-505124、BMP signaling agonist sb4、GW788388、ITD-1、LSKL，SD-208、LDN-212854、K02288、LDN-214117、R-268712、SM16、A 77-01、ALK2-IN-2、PD-161570、pm26、TGF-β1peptide TFA、Isosaponarin、BIO-013077-01。
优选地，所述TGF-β信号通路抑制剂选用SB431542；优选地，其货号为medchemexpress，HY-10431。
优选地，所述SB431542的工作浓度为10μM。
优选地，所述BMP信号通路抑制剂包括Dorsomorphin、A83-01、LDN193189、RepSox、SB 525334、DMH-1、SB-505124、BMP signaling agonist sb4、GW788388、ITD-1、LSKL、SD-208、LDN-212854、K02288、LDN-214117、R-268712、SM16、A 77-01、ALK2-IN-2、PD-161570、pm26、TGF-β1peptide TFA、Isosaponarin、BIO-013077-01。
优选地，所述BMP信号通路抑制剂是LDN193189；优选地，其货号为medchemexpress，HY-12071。
优选地，所述LDN193189的工作浓度为0.25μM。
优选地，所述RAR核受体的激动剂包括维生素A酸、Rapamycin、3-Methyladenine、Acetylcysteine、5-Fluorouracil，Hydrocortisone、Docetaxel、Rosiglitazone、Estradiol、Melatonin、GW9662、Nicotinamide、Cytarabine、Isoprenaline hydrochloride、Prostaglandin E2、Acetaminophen、(-)-Epigallocatechin Gallate、β-Nicotinamide mononucleotide、Calcitriol、DHEA、Liothyronine、NAD +、Luteolin、Thymidine、Docosahexaenoic Acid、Kaempferol、Palmitic acid、Cyclopamine、Genistein、L-Glutathione reduced。
优选地，所述RAR核受体的激动剂是维生素A酸；优选地，其货号为medchemexpress，HY-14649。
优选地，所述维生素A酸的工作浓度为100μM。
在一种实施方式中，所述N2培养基中包含第二小分子化合物组合。
在一种实施方式中，所述N2培养基是由第二基础培养基、非必须氨基酸、谷氨酰胺、还原剂、N2 supplement和第二小分子化合物组合组成。
本发明所述的“N2 supplement”中含有人全铁转铁蛋白(HumanTransferrin Holo)、重组胰岛素全链(InsulinRecombinantFullChain)、孕酮(Progesterone)、腐胺(Putrescine)、亚硒酸盐(Selenite)，其可以是商品化的产品或自行配制获得。
优选地，所述N2培养基由97％DMEM/F-12培养基、1％非必须氨基酸、1％GlutaMAX-I、2-Mercaptoethanol、1％N2 supplement和第二小分子化合物组合组成。
优选地，所述N2培养基由97％DMEM/F-12培养基(Life Technologies，cat.no.11039021)、1％非必须氨基酸(Life Technologies，cat.no.11140-050)、1％GlutaMAX-I(Life Technologies，cat.no.35050061)、2-Mercaptoethanol(Gibco，31350010)、1％N2 supplement(ThermoFisher，cat.no.17502001)和第二小分子化合物组合组成。“括号”内代表所述产品的厂家及货号。
优选地，所述第二小分子化合物组合包括RAR核受体的激动剂和/或Hedgehog信号通路激活剂。
优选地，所述RAR核受体的激动剂与前述一致。
优选地，所述Hedgehog信号通路激活剂包括SAG、Cyclopamine、Purmorphamine、20(S)-Hydroxycholesterol、Halcinonide、Jervine、ALLO-2、IHR-Cy3。
优选地，所述Hedgehog信号通路激活剂是SAG；优选地，其货号为medchemexpress，HY-12848。
在一种实施方式中，所述B27培养基中包含第三小分子化合物组合。
在一种实施方式中，所述B27培养基是由第三基础培养基、非必须氨基酸、谷氨酰胺、还原剂、N2 supplement、B27 supplement和第三小分子化合物组合组成。
本发明所述“B27 supplement”中含有生物素(Biotin)、DL-α-生育酚乙酸酯(DLAlpha Tocopherol Acetate)、DL-α-生育酚(DLAlpha-Tocopherol)、BSA(fattyacidfreeFractionV)、过氧化氢酶(Catalase)、人重组胰岛素(Human Recombinant Insulin)、人转铁蛋白(Human Transferrin)、超氧化物歧化酶(Superoxide Dismutase)、皮质酮(Corticosterone)、D-半乳糖(D-Galactose)、乙醇胺盐酸(EthanolamineHCl)、还原型谷胱甘肽(Glutathione reduced)、左旋肉碱盐酸(L-Carnitine HCl)、亚油酸(Linoleic Acid)、亚麻酸(Linolenic Acid)、孕酮(Progesterone)、腐胺(Putrescine 2HCl)、亚硒酸钠(Sodium Selenite)、三碘甲状腺原氨酸(T3 triodo-I-thyronine)，其可以是商品化的产品或自行配制获得。
优选地，所述B27培养基由95％DMEM/F-12培养基、1％非必须氨基酸、1％GlutaMAX-I、2-Mercaptoethanol、1％N2 supplement、2％B27 supplement和第三小分子化合物组合组成。
优选地，所述B27培养基由95％DMEM/F-12培养基(Life Technologies，cat.no.11039021)、1％非必须氨基酸(Life Technologies，cat.no.11140-050)、1％GlutaMAX-I(Life Technologies，cat.no.35050061)、2-Mercaptoethanol(Gibco，31350010)、1％N2 supplement(ThermoFisher，cat.no.17502001)、2％B27supplement(ThermoFisher，cat.no.12587010)和第三小分子化合物组合组成。“括号”内代表所述产品的厂家及货号。
优选地，所述第三小分子化合物组合包括胰岛素、RAR核受体的激动剂和Hedgehog 信号通路激活剂中的至少一种。
优选地，所述RAR核受体的激动剂和Hedgehog信号通路激活剂与前述一致。
在一种实施方式中，所述OPC成熟培养基中包含第四小分子化合物组合。
在一种实施方式中，所述OPC成熟培养基还包括第四基础培养基、非必须氨基酸、谷氨酰胺、还原剂、N2 supplement、B27 supplement。
优选地，所述OPC成熟培养基还包括95％DMEM/F-12培养基、1％非必须氨基酸、1％GlutaMAX-I、2-Mercaptoethanol、1％N2 supplement、2％B27supplement。
优选地，所述OPC成熟培养基还包括95％DMEM/F-12培养基(Life Technologies，cat.no.11039021)、1％非必须氨基酸(Life Technologies，cat.no.11140-050)、1％GlutaMAX-I(Life Technologies，cat.no.35050061)、2-Mercaptoethanol(Gibco，31350010)、1％N2 supplement(ThermoFisher，cat.no.17502001)、2％B27 supplement(ThermoFisher，cat.no.12587010)。“括号”内代表所述产品的厂家及货号。
优选地，所述第四小分子化合物组合包括胰岛素、PDGF-AA、IGF-1、HGF、NT3、T3、Biotin、cAMP中的至少一种。
优选地，所述第四小分子化合物组合包括胰岛素、PDGF-AA(PeproTech，cat.no.AF-100-13A)、IGF-1(PeproTech，cat.no.AF-100-11)、HGF(PeproTech，cat.no.100-39H)、NT3(PeproTech，cat.no.450-03)、T3(Sigma-Aldrich，cat.no.T2877)、Biotin(Sigma-Aldrich，cat.no.B4639)、cAMP(Sigma-Aldrich，cat.no.D0260)和5-HT2C受体拮抗剂。“括号”内代表所述产品的厂家及货号。
优选地，所述第四小分子化合物组合中还含有5-HT2C受体拮抗剂；
优选地，所述5-HT2C受体拮抗剂包括Puerarin(葛根素)、Harmine、SCH-23390hydrochloride、Olanzapine、GTS-21dihydrochloride、Thioridazine hydrochloride、Risperidone、SB-269970hydrochloride、Dihydroergotamine mesylate、Amitriptyline hydrochloride、Aripiprazole、Ketanserin、WAY-100635Maleate、Buspirone hydrochloride、Cisapride、Methiothepin mesylate、Trazodone hydrochloride、Pindolol、Quetiapine、Lumateperone tosylate、Perphenazine、8-OH-DPAT、Levomepromazine、SB-224289hydrochloride、Volinanserin、Ziprasidone、Paliperidone、Sertindole、Alprenolol hydrochloride、Asenapine maleate。
优选地，所述5-HT2C受体拮抗剂是Puerarin；优选地，其货号为medchemexpress，HY-N0145。
另一方面本发明提供了一种诱导神经干细胞的方法，所述方法包括前述神经诱导完全培养基和/或前述N2培养基进行细胞培养。
所述方法包括前述神经诱导完全培养基和前述N2培养基进行细胞培养。
小分子化合物组合
另一方面本发明还提供了一种包含5-HT2C受体拮抗剂的小分子化合物组合。
优选地，所述小分子化合物组合是前述第四小分子化合物组合。
试剂盒
另一方面本发明还提供了诱导少突胶质细胞的试剂盒，所述试剂盒中包括前述胰岛素、TGF-β信号通路抑制剂、BMP信号通路抑制剂、RAR核受体的激动剂、Hedgehog信号通路 激活剂、5-HT2C受体拮抗剂中的一种或多种。
优选地，所述试剂盒中包括配置以下至少一种培养基的试剂：
1)神经诱导完全培养基；
2)N2培养基；
3)B27培养基；
4)OPC成熟培养基。
应用
另一方面，本发明提供了以下任意一种培养基及其在诱导少突胶质细胞中的应用：
1)神经诱导完全培养基；
2)N2培养基；
3)B27培养基；
4)OPC成熟培养基。
另一方面，本发明提供了包含5-HT2C受体拮抗剂的小分子化合物组合在诱导少突胶质细胞中的应用。
另一方面，本发明提供了Puerarin在诱导少突胶质细胞中的应用。
另一方面，本发明提供了前述试剂盒在诱导少突胶质细胞中的应用。
另一方面，本发明提供了胰岛素、TGF-β信号通路抑制剂、BMP信号通路抑制剂、RAR核受体的激动剂、Hedgehog信号通路激活剂、5-HT2C受体拮抗剂任意一种在诱导少突胶质细胞中的应用。
另一方面，本发明提供了胰岛素、TGF-β信号通路抑制剂、BMP信号通路抑制剂、RAR核受体的激动剂、Hedgehog信号通路激活剂在诱导神经干细胞中的应用。
细胞及其应用
另一方面，本发明提供了一种细胞群，其特征在于，所述细胞群选自以下一种：
1)由前述诱导神经干细胞的方法制备得到的细胞群，所述细胞群中包括表达NKX2.2+、Nestin+、PAX6+中至少一种的细胞。
优选地，所述细胞群中存在表达Olig2+的细胞。
2)由前述诱导少突胶质细胞的方法制备得到的细胞群，所述细胞群中少突胶质细胞占全部细胞比例的至少20％；优选地，至少21％、25％、30％、35％、36％。
优选地，所述少突胶质细胞是表达Olig2、Nkx2.2、O4和MBP中至少一种的细胞。
优选地，所述少突胶质细胞是Olig2和Nkx2.2共表达的细胞；或者，所述少突胶质细胞是O4和MBP共表达的细胞。
另一方面，本发明提供了经过前述诱导少突胶质细胞的方法制得的细胞在治疗中枢神经系统疾病中的用途。
优选地，所述细胞是少突胶质细胞。
优选地，所述中枢神经系统疾病包括但不限于神经退行性疾病、脱髓鞘疾病、癫痫、脑外伤、休克、痴呆、青光眼、神经系统损伤后再生以及精神类疾病。
优选地，所述神经退行性疾病包括阿尔兹海默症、小脑萎缩症、原发性侧索硬化、脊髓性肌萎缩症、帕金森病、亨廷顿氏病、克雅二氏病、牛海绵状脑病、共济失调毛细血管扩 张症、肌肉萎缩性侧索硬化症、精神类疾病。
本发明所述“脱髓鞘疾病”是指髓鞘形成后发生的髓鞘损坏，脱髓鞘疾病是以神经髓鞘脱失为主，神经元胞体及轴突相对受累较轻为特征的一组疾病。
优选地，所述脱髓鞘疾病包括遗传性和获得性两大类。
优选地，所述脱髓鞘疾病包括脑卒中、多发性硬化、视神经脊髓炎、急性播散性脑脊髓炎、弥漫性硬化、同心圆硬化、脑白质营养不良、脑桥中央髓鞘溶解症、急性炎症性脱髓鞘性多发性神经病、慢性炎症性脱髓鞘性多发性神经病、缺血-缺氧性病引起的白质脑病、营养缺乏性疾病引起的亚急性联合变性、病毒感染引起的亚急性硬化性全脑炎或进行性多灶性白质脑病、糖尿病性神经病、系统性红斑狼疮的神经病变、肾上腺脑白质营养不良、白质消融性疾病、佩利措伊斯-梅茨巴赫病(Pelizaeus-Merzbacher disease)。
优选地，所述精神性疾病包括精神分裂症，抑郁症，偏执狂，焦虑症，强迫症，恐惧症，
另一方面，本发明提供了治疗前述神经疾病的方法，所述方法包括通过使用前述方法诱导少突胶质细胞。
附图说明
图1是第16天免疫荧光检测图；A：olig2+，Nkx2.2+，B：olig2+，Nestin+，C：olig2+，PAX6+。
图2是第33天免疫荧光检测图。
图3是OPC成熟培养基中添加或不添加葛根素对比得到的少突胶质细胞百分比统计结果图。
图4是OPC成熟培养基中添加或不添加葛根素对比的免疫荧光检测图。
具体实施方式
下面结合实施例对本发明做进一步的说明，以下所述，仅是对本发明的较佳实施例而已，并非对本发明做其他形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更为同等变化的等效实施例。凡是未脱离本发明方案内容，依据本发明的技术实质对以下实施例所做的任何简单修改或等同变化，均落在本发明的保护范围内。
本发明所使用的试剂
Figure PCTCN2022088517-appb-000001
*二抗：与一抗对应的Alexa Fluor荧光-标记抗体
通用方法：免疫荧光染色鉴定
工作液的配置：
1.配置10ml封闭血清稀释液(5％BSA+0.5％Triton X-100+DPBS液，以配置10ml为例。即在9.4ml DPBS中加入500μl正常5％BSA和100μl 30％Triton X-100)。
2.配置一抗工作液：封闭血清稀释液加入合适一抗滴度(见一抗使用说明书中具体滴度值)。
3.配置二抗工作液：封闭血清稀释液加入合适二抗滴度(见二抗使用说明书中具体滴度值)。
4.配置90％甘油：用DPBS稀释。
免疫荧光染色具体步骤如下：
DPBS清洗三遍，3min/次，4％PFA室温固定40min。DPBS清洗三遍，3min/次。0.5％TritonX-100，穿孔15min。5％BSA+0.15％TritonX-100，室温封闭1h。配PBST：DPBS+1％BSA+0.15％TritonX-100。上一抗，4度过夜。回收一抗溶液，PBST清洗三遍，每次10min。上二抗，1：500，4度过夜，避光。PBST清洗三遍，每次10min。5μg/ml DAPI for 2-3min，避光。用DPBS清洗一遍，加入90％的甘油。
实施例1、诱导iPSC细胞分化，并验证诱导效果
配制以下培养基备用：
神经诱导完全培养基：98％DMEM/F-12培养基、1％非必须氨基酸、1％GlutaMAX-I、0.1mM 2-Mercaptoethanol、10μM SB431542、0.25μM LDN193189和100μM维生素A酸、25μg/ml胰岛素。
N2培养基：97％DMEM/F-12培养基、1％非必须氨基酸、1％GlutaMAX-I、0.1mM 2-Mercaptoethano、1％N2 supplement、1μM SAG和100μM维生素A酸。
B27培养基：95％DMEM/F-12培养基、1％非必须氨基酸、1％GlutaMAX-I、0.1mM 2-Mercaptoethanol、1％N2 supplement、2％B27supplement和1μM SAG和100μM维生素A酸、25μg/ml胰岛素。
OPC成熟培养基-不含葛根素：95％DMEM/F-12培养基、1％非必须氨基酸、1％GlutaMAX-I、0.1mM 2-Mercaptoethanol、1％N2 supplement、2％B27supplement、10ng/mL PDGF-AA、10ng/mL IGF-1、5ng/mL HGF、10ng/mL NT3、60ng/mL T3、100ng/mL Biotin、1μM cAMP、25μg/ml胰岛素。
OPC成熟培养基-含葛根素：95％DMEM/F-12培养基、1％非必须氨基酸、1％GlutaMAX-I、0.1mM2-Mercaptoethanol、1％N2 supplement、2％B27supplement、10ng/mL PDGF-AA、10ng/mL IGF-1、5ng/mL HGF、10ng/mL NT3、60ng/mL T3、100ng/mL Biotin、1μM cAMP、25μM Puerarin(葛根素)、25μg/ml胰岛素。
按照以下步骤诱导神经干细胞，并验证是否高效分化出少突胶质细胞：
1.从第0天起，iPSC由E8完全培养基(STEMCELL，05991)更换为神经诱导完全培养基。
2.置于37℃，5％CO 2培养箱。
3.之后第1-7天，每天换液。
4.每天仔细观察细胞形态改变。
5.从第8天起，神经诱导完全培养基更换为N2培养基。
6.置于37℃，5％CO 2培养箱。
7.之后第8-11天，每天换液。
8.每天仔细观察细胞形态改变。
9.从第12天起，将N2培养基更换为B27培养基，细胞由贴壁培养转换为悬浮培养。
10.在第12天，将旧的培养基吸掉，每孔加入B27培养基；
11.用灭菌的刀片刮细胞，至少刮20下，然后分别将孔旋转90°和45°，同样各刮至少20下；
12.用细胞刮将整个孔沿着刮线将细胞刮下；
13.用1ml枪头轻轻吹打3-5次，然后将1孔细胞转移到低吸附的6孔板的两个孔中。然后每个孔分别补加B27培养基，使每孔的终体积为3ml；置于37℃，5％CO 2培养箱。
14.之后第12-19天，隔天换一次液。
15.每天仔细观察细胞形态改变。
16.从第20天起，将B27培养基更换为OPC成熟培养基-不含葛根素，细胞由贴壁培养转换为悬浮培养。
17.在第20天，用1ml枪头将球形聚集体转移到15ml离心管中，静置3min使球形聚 集体沉到离心管底部，吸掉2/3的旧的培养基，然后重新补加同样体积的OPC成熟培养基，然后将球形聚集体重新转回到原来的低吸附6孔板中。
18.之后第20-30天，隔天换一次液。
在第16天按通用方法进行免疫荧光检测，结果如图1所示：
A：细胞中同时存在Olig2+(绿色，OPC marker)的细胞和NKX2.2+(红色，神经干细胞marker)的细胞，但是没有同时表达Olig2+(绿色)和NKX2.2+(红色)的细胞，说明还没有成熟的OPC细胞；
B：细胞中同时存在Olig2+(绿色，OPC marker)的细胞和Nestin+(红色，神经干细胞marker)阳性的细胞，但是没有同时表达Olig2+(绿色)和Nestin+(红色)的细胞，说明还没有成熟的OPC细胞；
C：细胞中同时存在Olig2+(绿色，OPC marker)的细胞和PAX6+(红色，神经干细胞marker)的细胞，但是没有同时表达Olig2+(绿色)和PAX6+(红色)的细胞，说明还没有成熟的OPC细胞。
在第33天按通用方法进行免疫荧光检测；结果如图2所示诱导day33之后Olig2和Nkx2.2共表达于细胞中，表明诱导OPC取得成功。
实施例2、OPC成熟培养基中葛根素对诱导少突胶质细胞的影响
按照实施例1的方法配置神经诱导完全培养基、N2培养基、B27培养基、OPC成熟培养基；并且配置不含有葛根素的OPC成熟培养基；以含有葛根素或不含有葛根素的OPC成熟培养基为对照，探索葛根素在诱导少突胶质细胞形成中的影响。
在培养的第37天，定量统计少突胶质细胞占全部细胞的百分比，统计如图3所示，进行免疫荧光分析，结果如图4所示。
使用不含葛根素的OPC成熟培养基的为对照组，对照组能够产生约21％少突胶质细胞，添加葛根素后可以产生约36％少突胶质细胞。证明OPC成熟培养基中添加葛根素有利于少突胶质细胞的产生。

Claims (11)

  1. 一种培养基组合，所述培养基组合由神经诱导完全培养基、N2培养基、B27培养基、OPC成熟培养基组成；
    所述神经诱导完全培养基是由第一基础培养基、非必须氨基酸、谷氨酰胺、还原剂和第一小分子化合物组合组成；所述第一小分子化合物组合由10μM SB431542、0.25μM LDN193189和100μM维生素A酸、25μg/ml胰岛素组成；
    所述N2培养基是由第二基础培养基、非必须氨基酸、谷氨酰胺、还原剂、N2 supplement和第二小分子化合物组合组成；所述第二小分子化合物组合由1μM SAG和100μM维生素A组成；
    所述B27培养基是由第三基础培养基、非必须氨基酸、谷氨酰胺、还原剂、N2 supplement、B27 supplement和第三小分子化合物组合组成；所述第三小分子化合物由1μM SAG、100μM维生素A酸和25μg/ml胰岛素组成；
    所述OPC成熟培养基由第四基础培养基、非必须氨基酸、谷氨酰胺、还原剂、N2 supplement、B27 supplement、第四小分子化合物组合组成；所述第四小分子化合物组合是由10ng/mL PDGF-AA、10ng/mL IGF-1、5ng/mL HGF、10ng/mL NT3、60ng/mL T3、100ng/mL Biotin、1μM cAMP、25μM Puerarin、25μg/ml胰岛素组成。
  2. 如权利要求1所述的培养基组合，所述第一基础培养基、第二基础培养基、第三基础培养基、第四基础培养基各自独立地是DMEM/F-12培养基。
  3. 如权利要求1所述的培养基组合，所述非必须氨基酸包括丙氨酸、精氨酸、天门冬氨酸、胱氨酸、脯氨酸、酪氨酸。
  4. 如权利要求1所述的培养基组合，所述谷氨酰胺使用的是GlutaMAX-I。
  5. 如权利要求1所述的培养基组合，所述还原剂是β-巯基乙醇。
  6. 如权利要求1所述的培养基组合，所述神经诱导完全培养基是由98％ DMEM/F-12培养基、1％ 非必须氨基酸、1％ GlutaMAX-I、0.1mM β-巯基乙醇、10μM SB431542、0.25μM LDN193189和100μM维生素A酸、25μg/ml胰岛素组成。
  7. 如权利要求1所述的培养基组合，所述N2培养基是由97％ DMEM/F-12培养基、1％非必须氨基酸、1％ GlutaMAX-I、0.1mM β-巯基乙醇、1％ N2 supplement、1μM SAG和100μM维生素A酸组成。
  8. 如权利要求1所述的培养基组合，所述B27培养基是由95％ DMEM/F-12培养基、1％非必须氨基酸、1％ GlutaMAX-I、0.1mM β-巯基乙醇、1％ N2 supplement、2％ B27supplement和1μM SAG和100μM维生素A酸、25μg/ml胰岛素组成。
  9. 如权利要求1所述的培养基组合，所述OPC成熟培养基是由95％ DMEM/F-12培养基、1％非必须氨基酸、1％ GlutaMAX-I、0.1mM β-巯基乙醇、1％ N2 supplement、2％B27supplement、10ng/mL PDGF-AA、10ng/mL IGF-1、5ng/mL HGF、10ng/mL NT3、60ng/mL T3、100ng/mL Biotin、1μM cAMP、25μM Puerarin、25μg/ml胰岛素组成。
  10. 一种诱导人诱导性多能干细胞形成少突胶质细胞的方法，所述方法包括在第0-7天使用权利要求1所述神经诱导完全培养基进行培养、第8-11天使用权利要求1所述N2培养基进行培养、第12-19天使用权利要求1所述B27培养基进行培养、第20-30天使用权利要求1所述OPC成熟培养基进行培养。
  11. 权利要求1-9所述任意一种培养基组合在诱导人诱导性多能干细胞形成少突胶质 细胞中的应用。
PCT/CN2022/088517 2021-08-05 2022-04-22 人诱导性多能干细胞向少突胶质细胞分化的方法，试剂盒以及应用 WO2023010897A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US18/521,956 US20240093148A1 (en) 2021-08-05 2023-11-28 Method for differentiating human induced pluripotent stem cells into oligodendrocytes, and kit and use

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110894940.X 2021-08-05
CN202110894940.XA CN113564122B (zh) 2021-08-05 2021-08-05 人诱导性多能干细胞向少突胶质细胞分化的方法，试剂盒以及应用

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US18/521,956 Continuation US20240093148A1 (en) 2021-08-05 2023-11-28 Method for differentiating human induced pluripotent stem cells into oligodendrocytes, and kit and use

Publications (1)

Publication Number Publication Date
WO2023010897A1 true WO2023010897A1 (zh) 2023-02-09

Family

ID=78170402

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/088517 WO2023010897A1 (zh) 2021-08-05 2022-04-22 人诱导性多能干细胞向少突胶质细胞分化的方法，试剂盒以及应用

Country Status (3)

Country Link
US (1) US20240093148A1 (zh)
CN (1) CN113564122B (zh)
WO (1) WO2023010897A1 (zh)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113564122B (zh) * 2021-08-05 2022-04-08 呈诺再生医学科技(珠海横琴新区)有限公司 人诱导性多能干细胞向少突胶质细胞分化的方法，试剂盒以及应用
CN114262686B (zh) * 2022-03-02 2022-06-24 深圳市夏同生物医药科技有限公司 一种少突胶质细胞的制备方法及应用
CN117244064A (zh) * 2022-06-16 2023-12-19 中国科学院动物研究所 促进哺乳动物器官再生修复的物质及其应用
CN115612667B (zh) * 2022-11-02 2023-09-08 广州瑞臻再生医学科技有限公司 一种诱导多能干细胞来源的前脑少突胶质细胞的制备方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150352154A1 (en) * 2013-02-06 2015-12-10 University Of Rochester Induced pluripotent cell-derived oligodendrocyte progenitor cells for the treatment of myelin disorders
CN106456672A (zh) * 2014-05-22 2017-02-22 纽约干细胞基金会有限公司 源于多能干细胞的功能性少突胶质细胞及其制备和使用方法
CN108384755A (zh) * 2018-02-08 2018-08-10 北京呈诺医学科技有限公司 一种高效、快捷的诱导性多能干细胞向神经干细胞分化的方法
CN112469818A (zh) * 2018-04-17 2021-03-09 凯斯西储大学 人皮层球体中髓鞘少突胶质细胞的诱导
US20210246422A1 (en) * 2018-06-14 2021-08-12 Academia Sinica Method to generate induced oligodendrocyte-lineage cells and treatment using such cells
CN113564122A (zh) * 2021-08-05 2021-10-29 呈诺再生医学科技(珠海横琴新区)有限公司 人诱导性多能干细胞向少突胶质细胞分化的方法，试剂盒以及应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113564123A (zh) * 2014-10-24 2021-10-29 大日本住友制药株式会社 神经组织的制备方法
CN109251894B (zh) * 2014-11-25 2022-10-04 宾州研究基金会 人神经胶质细胞转化成用于大脑和脊髓修复的神经细胞的化学重编程
CN108624560B (zh) * 2018-06-01 2022-04-08 南京艾尔普再生医学科技有限公司 一种分化培养基及少突胶质前体细胞的制备方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150352154A1 (en) * 2013-02-06 2015-12-10 University Of Rochester Induced pluripotent cell-derived oligodendrocyte progenitor cells for the treatment of myelin disorders
CN106456672A (zh) * 2014-05-22 2017-02-22 纽约干细胞基金会有限公司 源于多能干细胞的功能性少突胶质细胞及其制备和使用方法
CN108384755A (zh) * 2018-02-08 2018-08-10 北京呈诺医学科技有限公司 一种高效、快捷的诱导性多能干细胞向神经干细胞分化的方法
CN112469818A (zh) * 2018-04-17 2021-03-09 凯斯西储大学 人皮层球体中髓鞘少突胶质细胞的诱导
US20210246422A1 (en) * 2018-06-14 2021-08-12 Academia Sinica Method to generate induced oligodendrocyte-lineage cells and treatment using such cells
CN113564122A (zh) * 2021-08-05 2021-10-29 呈诺再生医学科技(珠海横琴新区)有限公司 人诱导性多能干细胞向少突胶质细胞分化的方法，试剂盒以及应用

Also Published As

Publication number Publication date
CN113564122A (zh) 2021-10-29
CN113564122B (zh) 2022-04-08
US20240093148A1 (en) 2024-03-21

Similar Documents

Publication Publication Date Title
WO2023010897A1 (zh) 人诱导性多能干细胞向少突胶质细胞分化的方法，试剂盒以及应用
Hu et al. Differentiation of human oligodendrocytes from pluripotent stem cells
JP7116964B2 (ja) 終脳又はその前駆組織の製造方法
Xiao et al. Direct reprogramming of fibroblasts into neural stem cells by single non-neural progenitor transcription factor Ptf1a
Menendez et al. Directed differentiation of human pluripotent cells to neural crest stem cells
Liu et al. Directed differentiation of forebrain GABA interneurons from human pluripotent stem cells
Jiang et al. Characterization of Nestin-positive stem Leydig cells as a potential source for the treatment of testicular Leydig cell dysfunction
Yan et al. Efficient and rapid derivation of primitive neural stem cells and generation of brain subtype neurons from human pluripotent stem cells
Sun et al. Long-term tripotent differentiation capacity of human neural stem (NS) cells in adherent culture
Bibel et al. Generation of a defined and uniform population of CNS progenitors and neurons from mouse embryonic stem cells
AU2015336453A1 (en) Production method for nerve tissue
EP3173475B1 (en) Method for producing adenohypophysis or precursor tissue thereof
Bojnordi et al. Differentiation of spermatogonia stem cells into functional mature neurons characterized with differential gene expression
US20230233617A1 (en) Methods for differentiating stem cells into dopaminergic progenitor cells
Griffin et al. Nicotinamide alone accelerates the conversion of mouse embryonic stem cells into mature neuronal populations
Lukovic et al. Highly efficient neural conversion of human pluripotent stem cells in adherent and animal-free conditions
Chen et al. Pax 6 controls neural crest potential of limbal niche cells to support self-renewal of limbal epithelial stem cells
WO2019208788A1 (ja) 膵臓β細胞の製造方法
Machado et al. Generation of neural progenitor cells from porcine‐induced pluripotent stem cells
US20190017019A1 (en) Induction of neural progenitor cells, oligodendrocyte progenitor cells, and oligodendrocytes by stem cell differentiation using landmark transcription factors
EP4011449A1 (en) Method for producing cell aggregate including glial progenitor cells
Kuznetsova et al. Mechanisms of dedifferentiation of adult human retinal pigment epithelial cells in vitro. Morphological and molecular genetic analysis
JP6502255B2 (ja) Ｐｄｇｆを含有するｄｓ細胞用無血清培地
JP7107504B2 (ja) 内胚葉系細胞集団、及び多能性細胞から三胚葉のいずれかの細胞集団を製造する方法
Kawasaki et al. Selective neural induction from ES cells by stromal cell-derived inducing activity and its potential therapeutic application in Parkinson's disease

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22851619

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE