WO2018199142A1 - Procédé de production de cellules de crête neurale et de neurones sympathiques - Google Patents

Procédé de production de cellules de crête neurale et de neurones sympathiques Download PDF

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WO2018199142A1
WO2018199142A1 PCT/JP2018/016758 JP2018016758W WO2018199142A1 WO 2018199142 A1 WO2018199142 A1 WO 2018199142A1 JP 2018016758 W JP2018016758 W JP 2018016758W WO 2018199142 A1 WO2018199142 A1 WO 2018199142A1
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cells
sympathetic
cell
producing
neural crest
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中畑　龍俊
潤 齋藤
浩輔 桐野
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国立大学法人京都大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal 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
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies

Definitions

  • the present invention relates to a method for producing neural crest cells from pluripotent stem cells, and a method for producing sympathetic progenitor cells and further sympathetic neurons from neural crest cells.
  • Sympathetic nerves are peripheral autonomic nerves that control involuntary functions such as circulation, breathing, digestion, sweating / temperature regulation, endocrine function, reproductive function, and metabolism.
  • sympathetic neurons are known to be derived from trunk neural crest cells, and various factors involved in differentiation induction have been identified in animal models. Although there have been some reports on methods for inducing sympathetic neurons from pluripotent stem cells, differentiation efficiency is not sufficient (Non-Patent Documents 1 to 4).
  • an object of the present invention is to efficiently produce sympathetic neurons from pluripotent stem cells.
  • the inventors first developed an efficient protocol for obtaining functional sympathetic neurons via neural crest cells (NCC) by examining each differentiation stage in detail using PHOX2B-eGFP reporter-expressing pluripotent stem cells Established.
  • NCC neural crest cells
  • the present inventors have found that sympathetic neurons can be obtained from pluripotent stem cells with high purity by this protocol, and the present invention has been completed.
  • a method for producing neural crest cells comprising culturing pluripotent stem cells in a culture medium containing FGF (Fibroblast growth factor) 2, retinoic acid and BMP (Bone morphogenetic protein) 4.
  • FGF Fibroblast growth factor
  • BMP Breast morphogenetic protein
  • the concentration of FGF2 is 1 ng / ml to 100 ng / ml
  • the concentration of retinoic acid is 10 nM to 10 ⁇ M
  • the concentration of BMP4 is 5 ng / ml to 150 ng / ml.
  • Pluripotent stem cells are cultured in a culture solution containing GSK (Glycogen synthase kinase) 3 ⁇ inhibitor and TGF (Transforming growth factor) ⁇ inhibitor, and then in a culture solution containing FGF2, retinoic acid and BMP4.
  • [5] The method for producing neural crest cells according to any one of [1] to [4], comprising a selection step using CD49d.
  • a method for producing sympathetic progenitor cells comprising culturing neural crest cells in a culture solution containing cytokine and BMP4 to induce sympathetic progenitor cells.
  • the cytokines are EGF (Epidermal Growth Factor) and FGF2.
  • the concentration of BMP4 is 5 ng / ml to 150 ng / ml
  • the concentration of EGF is 1 ng / ml to 100 ng / ml
  • the concentration of FGF2 is 1 ng / ml to 100 ng / ml.
  • a neural crest cell is produced by the method according to any one of [1] to [6], and the obtained neural crest cell is used to sympathize with the method according to any one of [7] to [10].
  • a method for producing sympathetic neural progenitor cells which produces neural progenitor cells.
  • a step of producing sympathetic progenitor cells by the method according to any one of [7] to [12], and sympathy by culturing the obtained sympathetic progenitor cells in a culture solution containing neurotrophic factor A method for producing a sympathetic nerve cell, comprising a step of maturing a neural progenitor cell into a sympathetic nerve cell.
  • NGF Nemasarcoma growth factor
  • BDNF Brain-derived neurotrophic factor
  • GDNF Glial cell line-derived neurotrophic factor
  • a sympathetic nerve cell obtained by the method according to [13] or [14].
  • a composition for treating sympathetic neuropathy comprising sympathetic nerve cells obtained by the method according to [13] or [14].
  • human sympathetic nerve cells which have conventionally been difficult to collect, can be produced from human pluripotent stem cells and used for many studies.
  • a wide variety of neural cells have been induced to differentiate from human pluripotent stem cells and used for regenerative medicine, creation of disease models, drug discovery screening, neurotoxicity evaluation, and the like.
  • sympathetic nerve cells induced to differentiate in the present invention are considered to be applicable to regenerative medicine, creation of disease models, drug discovery, and the like.
  • FIG. 1 The figure which shows the analysis result about the differentiation conditions, cell profile, etc. of PHOX2B :: eGFP introduction
  • A Diagram of culture conditions for adjusting determination of hPSC anteroposterior and dorsoventral axes.
  • B Heat map image showing the percentage of eGFP + cells on day 10 of differentiation of KhES1 PHOX2B :: eGFP strain differentiated under various conditions.
  • C Representative FCM plots of KhES1 PHOX2B :: eGFP strain-derived aggregates on day 10 under conditions (i)-(iv) of b.
  • FIG. 1 The figure which shows the analysis result about the differentiation conditions, marker expression profile, etc. of PHOX2B :: eGFP introduction
  • A Diagram of culture conditions for sympathetic nervous system NCC differentiation.
  • (B) Representative image of neurosphere on the third day after sorting under EF and EFB conditions (scale bar 100 ⁇ m).
  • (C) Quantification by FCM analysis of eGFP + cells in neurospheres 7 days after sorting under EF and EFB conditions (mean ⁇ SEM, n 3, P ⁇ 0.01).
  • (D) Changes in cell number, percentage of eGFP + cells and percentage of SOX10 + cells in 28 days of culture under EFB conditions after sorting (mean ⁇ SEM, n 3). The cell number is described as the rate of change relative to the number on day 0.
  • (D) Immunocytochemical analysis for eGFP, TH, PRPH and DBH on day 32 of differentiation (scale bar 50 ⁇ m).
  • (E) Quantification of PRPH + cells, TH + cells and DBH + cells in d (mean ⁇ SEM, n 3).
  • (F) Immunocytochemical analysis for PHOX2B and SOX10 on days 10 and 17 and for TH, DBH and PRPH on day 32 (scale bar 50 ⁇ m).
  • the white line box in the image on the 10th day indicates the enlarged area on the left side of the same panel.
  • assembly of PHOX2B :: eGFP knock-in hPSC clone.
  • FIG. 1 Schematic diagram of PHOX2B gene targeting using TALEN-mediated genome editing.
  • B Genomic PCR showing targeted integration at the 3′UTR region of PHOX2B.
  • C Quantification of eGFP + PHOX2B + cells in cells (PHOX2B / eGFP) Quantitative and PHOX2B + eGFP + cells in cells (eGFP / PHOX2B).
  • DSB double strand break
  • CHIR CHIR99021
  • RA retinoic acid.
  • A RT-PCR analysis for SOX2, BRACKYURY, TBX6, HOXB1, HOXB2, HOXB4 and HOXB6 in day 3 aggregates using various concentrations of CHIR99021.
  • A Representative FCM plot of aggregates from day 10 KhES1 PHOX2B :: eGFP strain under CHIR 2.0 ⁇ M BMP RA 0 nM and CHIR 2.0 ⁇ M BMP RA 100 nM conditions.
  • FIG. 1 The figure which shows the analysis result of the cell in the aggregate of CHIR 2.0micromol BMP4RA 100nM conditions.
  • A Time course FCM analysis of eGFP expression and CD49 expression.
  • C FCM analysis of eGFP, CD49d and TUBBIII in day 10 aggregates. Intracellular eGFP and TUBBIII were stained after extracellular staining, fixation, and permeabilization of CD49d.
  • FIG. 1 The figure which shows the result about the induction
  • A FCM analysis (gray) of eGFP expression in neurosphere cells 7 days after sorting with or without BMP4. A parent clone without the PHOX2B :: eGFP reporter was used as a negative control (white).
  • B Immunocytochemical analysis for SOX10 on day 7, 14 and 28 after selection.
  • C FCM analysis of TUBBIII expression in neurosphere cells at day 7, 14, 21, and 28 after selection (grey). An isotype control was used to quantify the negative population (white).
  • (D) Quantification of TUBBIII + cells in FCM analysis of c (mean ⁇ SEM, n 3).
  • E Immunocytochemical analysis for eGFP and SMA of cells 14 days after selection that are adherently cultured using BMP4.
  • F Neurosphere cell morphology with or without NF treatment.
  • A Diagram of culture conditions for neuronal cell induction in neurosphere culture.
  • D Diagram of neurosphere cell storage and culture after thawing.
  • the present invention provides a method for producing sympathetic neurons from pluripotent stem cells.
  • the production method includes (1) a step of inducing neural crest cells from pluripotent stem cells, (2) Inducing sympathetic progenitor cells from neural crest cells, and (3) Inducing sympathetic neurons from sympathetic progenitor cells. This will be described below.
  • Pluripotent stem cell A pluripotent stem cell is a stem cell having pluripotency that can be differentiated into many cells existing in a living body and also having proliferative ability, and at least hematopoiesis used in the present invention. Any cell that is derived from a progenitor cell is included. Pluripotent stem cells are preferably derived from mammals, and more preferably derived from humans.
  • pluripotent stem cells include, but are not limited to, embryonic stem (ES) cells, cloned embryo-derived embryonic stem (ntES) cells obtained by nuclear transfer, sperm stem cells (“GS cells”), embryonic Examples include germ cells (“EG cells”), induced pluripotent stem (iPS) cells, cultured fibroblasts, umbilical cord blood-derived pluripotent stem cells, bone marrow stem cell-derived pluripotent cells (Muse cells), and the like.
  • ES embryonic stem
  • ntES cloned embryo-derived embryonic stem
  • GS cells sperm stem cells
  • EG cells germ cells
  • iPS induced pluripotent stem
  • a preferred pluripotent stem cell is an iPS cell, more preferably a human iPS cell, from the viewpoint that it can be obtained without destroying an embryo, an egg or the like in the production process.
  • IPS cell production methods are known in the art and can be produced by introducing reprogramming factors into any somatic cells.
  • the reprogramming factor is, for example, Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15 -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3 or Glis1, etc. genes or gene products are exemplified, and these reprogramming factors may be used alone or in combination. Also good.
  • Somatic cells include, but are not limited to, fetal (pup) somatic cells, neonatal (pup) somatic cells, and mature healthy or diseased somatic cells. , Passage cells, and established cell lines.
  • somatic cells are, for example, (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, dental pulp stem cells, (2) tissue progenitor cells, (3) blood cells (peripheral) Blood cells, umbilical cord blood cells, etc.), lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (pancreatic exocrine cells) Etc.), differentiated cells such as brain cells, lung cells, kidney cells and fat cells.
  • the mammalian individual from which somatic cells are collected is not particularly limited, but is preferably a human
  • Neural crest is a process of inducing neural crest cells from pluripotent stem cells. It is a transient tissue that appears at the boundary between neuroectodermal and epidermal germ layers during neural tube formation in vertebrate development. A group of cells that migrates after deepithelialization is called neural crest cells (J Cell Biochem 107, 1046-52 (2009)). The neural crest-derived cells are defined by the presence of neural crest markers such as SOX10 and FOXD3, for example.
  • the step of inducing neural crest cells from pluripotent stem cells preferably includes the following steps. (I) culturing pluripotent stem cells in a culture medium containing a TGF ⁇ inhibitor and a GSK3 ⁇ inhibitor; (Ii) A step of culturing the obtained cells in a medium containing FGF2, retinoic acid and BMP4.
  • the culture medium used for the culture of pluripotent stem cells for the production of neural crest cells is not particularly limited, but the medium used for animal cell culture is transferred to the basal medium as a TGF ⁇ inhibitor, GSK3 ⁇ inhibitor or FGF2, retinoic acid and It can be prepared by adding BMP4 or the like.
  • Basal media include, for example, Iscove's'Modified Dulbecco's Medium (IMDM), Medium 199, Eagle's Minimum Essential Medium (EMEM), ⁇ MEM, Dulbecco's modified Eagle's Medium (DMEM), Ham's F1 ', , Essential ⁇ ⁇ 6 medium, Neurobasal ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ Medium (Life Technologies) and mixed media thereof.
  • Serum may be contained in the medium, or serum-free may be used.
  • the basal medium can be, for example, albumin, insulin, transferrin, selenium, fatty acid, trace elements, 2-mercaptoethanol, thiolglycerol, ROCK inhibitor, Purmorphamine, lipids, amino acids, L-glutamine, non-essential amino acids, It may also contain one or more substances such as vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, heparin and the like.
  • the TGF ⁇ inhibitor used in step (i) is a small molecule inhibitor that interferes with TGF ⁇ family signaling, such as SB431542, SB202190 (RKLindemanndeet al.,. Mol. Cancer 2:20 (2003)), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories) and the like, for example, when the TGF ⁇ inhibitor is SB431542, the concentration in the medium is 0.5 ⁇ M to 100 ⁇ M, more preferably 5 ⁇ M to 30 ⁇ M.
  • the GSK3 ⁇ inhibitor used in step (i) is defined as a substance that inhibits the kinase activity of GSK (glycogen synthase kinase) -3 ⁇ protein (for example, the ability to phosphorylate ⁇ -catenin). Already known.
  • indirubin derivatives such as BIO (also known as GSK-3 ⁇ inhibitor IX; 6-bromoindirubin-3′-oxime), SB216763 (3- (2,4-dichlorophenyl) -4- (1 Maleimide derivatives such as -methyl-1H-indol-3-yl) -1H-pyrrole-2,5-dione), ⁇ -bromomethyl ketone compounds such as GSK-3 ⁇ inhibitor VII ⁇ ⁇ (4-dibromo-acetophenone), CHIR99021 ( 6-[(2- ⁇ [4- (2,4-dichlorophenyl) -5- (4-methylimidazol-2-yl) pyrimidin-2-yl] amino ⁇ ethyl) amino] pyridine-3-carbonitrile) ( WO1999 / 65897; CAS number 252917-06-9), cell membrane permeable phosphorylated peptides such as L803-mts, and derivatives thereof.
  • BIO also known as GSK
  • the concentration of retinoic acid in the medium used in step (ii) is usually 10 nM to 10 ⁇ M, preferably 50 nM to 5 ⁇ M.
  • the concentration of BMP4 (Bone morphogenetic protein 4) in the medium used in step (ii) is usually 5 ng / ml to 150 ng / ml, preferably 10 ng / ml to 100 ng / ml, More preferably, it is 20 ng / ml to 80 ng / ml.
  • the concentration of FGF2 (fibroblast growth factor 2: aka bFGF) in the medium used in step (ii) is, for example, 1 ng / ml to 100 ng / ml, preferably 5 ng / ml to 50 ng / ml, more preferably 10 ng / ml. ⁇ 30 ng / ml.
  • the culture method of pluripotent stem cells may be adhesion culture or suspension culture, but suspension culture is preferred.
  • pluripotent stem cells can be subjected to suspension culture after separating colonies cultured until they are 80% confluent with respect to the used dish and dissociating them into single cells.
  • methods for separating pluripotent stem cells include, for example, a method for separating mechanically, a separation solution having protease activity and collagenase activity (eg, Accutase TM and Accumax TM), or a separation solution having only collagenase activity. The separation method using is mentioned.
  • Suspension culture means culturing cells in a non-adherent state in a culture vessel.
  • artificial treatment for example, coating treatment with an extracellular matrix or the like to improve adhesion to cells.
  • Culture vessels that have not been treated, or treatment that artificially suppresses adhesion for example, coating treatment with polyhydroxyethylmethacrylic acid (poly-HEMA) or nonionic surfactant polyol (Pluronic F-127, etc.)
  • poly-HEMA polyhydroxyethylmethacrylic acid
  • Pluronic F-127 nonionic surfactant polyol
  • the temperature conditions for culturing for producing neural crest cells are not particularly limited, but for example, about 37 ° C. to about 42 ° C., about 37 ° C. to about 39 ° C. are preferable.
  • the culture period is not particularly limited as long as neural crest cells can be obtained.
  • the culture period in step (i) is 1 to 4 days, and the culture period in step (ii) is, for example, 3 to 10 Day.
  • the obtained neural crest cells are preferably purified and used in the next step.
  • purification for example, CD49d (integrin ⁇ 4) can be used, and the purification method can be a method well known to those skilled in the art, for example, by flow cytometry using an anti-CD49d antibody.
  • purification etc. is mentioned.
  • sympathetic progenitor cells mean cells that can be differentiated into sympathetic nerves by adding neurotrophic factor and culturing, for example, PHOX2B (Nature 399, 366-370 (1999)) and CD49d can be recognized as positive.
  • Sympathetic neural progenitor cells can be obtained by culturing neural crest cells in a culture medium containing cytokines and BMP4.
  • the culture medium used for the production of sympathetic progenitor cells in the present invention is not particularly limited, but can be prepared by using a medium used for animal cell culture as a basal medium and adding cytokine and BMP4 thereto.
  • a basal medium the same medium as described above can be used.
  • the concentration of BMP4 is, for example, 5 ng / ml to 150 ng / ml, preferably 10 ng / ml to 100 ng / ml, more preferably 20 ng / ml to 80 ng / ml.
  • EGF Epidermal Growth Factor
  • FGF2 FGF2
  • concentration of EGF is, for example, 1 ng / ml to 100 ng / ml, preferably 5 ng / ml to 50 ng / ml, more preferably 10 ng / ml to 30 ng / ml.
  • concentration of FGF2 is, for example, 1 ng / ml to 100 ng / ml, preferably 5 ng / ml to 50 ng / ml, more preferably 10 ng / ml to 30 ng / ml.
  • pluripotent stem cells are cultured by forming embryoid bodies (EBs), After obtaining the dyke cells, it is preferable to dissociate the obtained EBs and culture them in a culture solution to which cytokines and BMP4 have been added to form neurospheres and culture.
  • EBs embryoid bodies
  • the culture temperature conditions for culturing neural crest cells to produce sympathetic progenitor cells are not particularly limited, but for example, about 37 ° C. to about 42 ° C., preferably about 37 ° C. to about 39 ° C. are preferable.
  • the culture period is not particularly limited as long as sympathetic progenitor cells can be obtained.For example, 10 days or more, 12 days or more, 14 days or more, 16 days or more, 18 days or more, 20 days or more, or 24 More than 30 days, less than 30 days, or less than 48 days.
  • the sympathetic neurons are identified as, for example, positive cells of tyrosine hydroxylase (TH) and / or dopamine ⁇ -hydroxylase (DBH), preferably more positive for peripherin (PRPH) It is.
  • TH tyrosine hydroxylase
  • DH dopamine ⁇ -hydroxylase
  • PRPH peripherin
  • the sympathetic nerve cell can be produced by a method including a step of culturing the sympathetic neural progenitor cell in a culture solution to which a neurotrophic factor is added.
  • neurotrophic factors are ligands to membrane receptors that play an important role in motor neuron survival and function maintenance, such as Nerve Growth Factor (NGF), Brain-derived Neurotrophic Factor (BDNF), Neurotrophin. 3 (NT-3), Neurotrophin 4/5 (NT-4 / 5), Neurotrophin 6 (NT-6), Glia cell line-derived Neurotrophic Factor (GDNF), Ciliary Neurotrophic Factor (CNTF) and LIF .
  • Preferred neurotrophic factors are those selected from the group consisting of NGF, BDNF and GDNF.
  • the concentration of the neurotrophic factor to be added may be appropriately selected by those skilled in the art depending on its efficacy, and is, for example, 1 ng / ml to 100 ng / ml, and preferably 5 ng / ml to 50 ng / ml.
  • the culture solution used for the production of sympathetic nerve cells is not particularly limited, but can be prepared by adding a neurotrophic factor to a basal medium as a medium used for culturing animal cells.
  • a basal medium a medium as described above can be used.
  • the temperature conditions for culturing sympathetic progenitor cells for producing neurotrophic factors are not particularly limited, but for example, about 37 ° C. to about 42 ° C., preferably about 37 ° C. to about 39 ° C. are preferable.
  • the culture period is not particularly limited as long as sympathetic neurons can be obtained.For example, for example, 10 days or more, 12 days or more, 14 days or more, 16 days or more, 18 days or more, 20 days or more, or 24 days or more, 30 days or less, or 48 days or less.
  • the sympathetic nerve cells obtained by the method of the present invention can also be used for screening for compounds for treating sympathetic neuropathy (for example, pharmaceutical compounds, solvents, small molecules, peptides, or polynucleotides).
  • a candidate pharmaceutical compound can be evaluated by adding a sympathetic nerve cell and changing the morphology or function of the cell.
  • a functional change it can be evaluated by measuring the amount of norepinephrine produced from the cell.
  • the artificial neural progenitor cell from which the sympathetic nerve cell is derived is preferably a cell exhibiting a phenotype similar to that of the sympathetic neuropathy to be treated, and particularly preferably prepared from a somatic cell affected by the sympathetic neuropathy. It is an induced pluripotent stem cell.
  • the sympathetic nerve cell obtained by the method of the present invention can be effectively used in the field of regenerative medicine for normalizing damaged sympathetic nervous system tissue. Therefore, this cell can be a therapeutic cell for diseases associated with disorders of all sympathetic nervous system cells (such as sympathetic nerve damage and autonomic dysfunction).
  • KhES1 and KhES3 hESC lines were provided by Dr. Hirofumi Suemori (Research Institute for Regenerative Medicine, Kyoto University).
  • Human iPS cell lines 409B2 and 604A1 were provided by Dr. Shinya Yamanaka (iPS Cell Research Institute, Kyoto University). These cell lines were maintained on Growth Factor Reduced Matrigel matrix (Corning) coated cell culture plates containing mTeSR1 medium (STEMCELL Technologies).
  • Plasmid construction TALEN Nucleotide Targeter 2.0 (https://tale-nt.cac.cornell.edu/) is used to construct a transcriptional activator-like effector nuclease (TALEN) plasmid.
  • a group (RVD) was designed.
  • the TALEN encoding plasmid was assembled using Golden Gate TALEN and TAL Effector Kit 2.0 and its protocol for assembly of the TALEN encoding plasmid (Addgene).
  • a mammalian expression vector containing the modified FokI was received from Dr. Takashi Yamamoto of Hiroshima University.
  • a 1 kbp PCR amplified homology arm was cloned 3 ′ to the loxP-neo-loxP cassette vector for the construction of the targeting vector.
  • a 1 kbp 5 ′ homology arm PCR amplified product
  • a T2A peptide sequence annealed oligonucleotide pair
  • an eGFP open reading frame first ORF without 1ATG; PCR amplification product
  • transfection and generation of stable strains For genome editing by TALEN transfection was performed using the superelectroporator NEPA21 (Neppagene) according to the manufacturer's instructions. Specifically, the cells were dissociated into individual cells using StemPro Accutase Cell Dissociation Reagent (Gibco). Next, one million cells in one cuvette were transfected with 2 ⁇ g of each TANEN plasmid and 6 ⁇ g of targeting vector plasmid, and growth factor containing mTeSR1 medium supplemented with 50 ⁇ M Y27632 (Merck Millipore). Reduced Matrigel matrix-coated 6 cm cell culture dishes (BD Falcon) were immediately replated.
  • the living cells were dissociated, and mitosis containing primate ES cell culture medium (Reprocell Co., Ltd.) supplemented with 5 ng / mL FGF2 (Wako Pure Chemical Industries, Ltd.) and 10 ⁇ M Y27632. Those surviving cells were passaged onto inactivated SNL feeder cells. The next day, Y27632 was removed. Individual colonies were isolated 14 days after passage and grown for subsequent experiments.
  • Genomic PCR Genomic DNA was extracted using QIAamp DNA Blood Mini Kit (QIAGEN) according to the manufacturer's instructions. Genomic PCR was performed to detect integration into the genome at the target site using PrimSTAR GXL DNA polymerase.
  • hPSCs Differentiation of hPSCs Dissociated hPSCs that had been maintained using the StemProactase cell dissociation reagent for NMP-like cell induction into individual cells, and 96-well ultra-low attachment multi-wall plates (Corning) were used.
  • the cells were immediately reaggregated in 100 ⁇ L Essential 6 medium (Gibco) supplemented with 10 ⁇ M SB431542 (Sigma Aldrich), various concentrations of CHIR99021 (Merck Millipore) and 10 ⁇ M Y27632. 10,000 cells / well).
  • the medium was changed every other day until day 10. Aggregates from day 3 treated with 1.5 ⁇ M CHIR99021 for cranial motor neuron differentiation up to day 10 in Essential 6 medium supplemented with 20 ng / mL FGF2, 1 ⁇ M RA and 1 ⁇ M Purmorphamine. Cultured. The medium was changed every other day. On day 10, the cells were dissociated into individual cells using the StemProactase cell dissociation reagent and eGFP + CD49d ⁇ cells were selected by fluorescence activated cell sorting (FACS, see below).
  • FACS fluorescence activated cell sorting
  • Glutamax I Glutamax I (Gibco), N2 and B27 supplements, 20 ng / mL FGF2, 20 ng / mL EGF (R & D) in an ultra-low attachment dish (10 cm) or multi-wall plate (6 wells) (Corning) Systems), sorted cells in Neurobasal medium supplemented with 50 ng / mL BMP4 and 2 ⁇ g / mL heparin (Sigma Aldrich) were cultured at a density of 500,000 cells / mL.
  • the medium is changed every 3-4 days, and 0.05% trypsin and 10 ⁇ g / mL DNaseI (STEMCELL Technologies) are used, followed by gentle pipetting to dissociate the cells and remove the cell mass every 7 days. Passage treatment.
  • Neurobasal medium supplemented with 1 ⁇ Glutamax I (Gibco), N2 and B27 supplements, neurotrophic factor (NF) NGF (R & D Systems), BDNF and GDNF (10 ng / ml each) for neuronal maturation
  • the cell mass was transferred onto an ultra-low attachment dish or multi-wall plate containing.
  • the medium was changed every 3-4 days.
  • Flow cytometry analysis and FACS PE-conjugated mouse anti-CD49d antibody (BioLegend), Alexa Fluor488 rat anti-GFP antibody (BioLegend) and Alexa Fluor647 mouse anti-class III beta tubulin (TUBBIII) antibody (BD Biosciences) were used according to the manufacturer's protocol. Flow cytometry was performed using a MACSQuant Analyzer 10 (Miltenyi Biotech). FACS was performed by BD FACSAria II (BD Bioscience). Isotype controls were used as a control population in all experiments.
  • RNA isolation and RT-PCR Total RNA extraction from the cells was performed using RNeasy Mini kit (QIAGEN). Total RNA (1 ⁇ g) was used for reverse transcription using PrimeScript RT Master Mix (TaKaRa). RT-PCR was performed using Ex Taq Hot Start version (TaKaRa) or PrimeStar GXL DNA polymerase (TaKaRa).
  • Immunocytochemistry and microscopy Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature and permeabilized in 0.2% Triton X-100 for 10 minutes at room temperature. The cells are then incubated with Block Ace (DS Pharma Biomedical) to prevent any non-specific binding and then incubated with the primary antibody for 12 hours at 4 ° C. or 2 hours at room temperature. I left it alone. Isothermal with a secondary antibody using an appropriate species-specific antiserum bound to either FITC, Alexa647, Cy-3 (Jackson ImmunoResearch; 1/200) or Alexa555 (Invitrogen; 1/1000) The standing was carried out for 1 hour.
  • Block Ace DS Pharma Biomedical
  • Norepinephrine quantification Norepinephrine concentration in the culture supernatant was measured as reported in Cell Stem Cell 19, 95-106 (2016).
  • the cultured SN was cultured for 15 minutes using HBSS (Gibco).
  • the medium was collected as a control.
  • the cells were cultured for an additional 15 minutes in HBSS containing 50 mM KCl, after which the medium was collected. After media collection, the media was centrifuged at 300 g for 5 minutes to eliminate cells or cell debris.
  • 1 mM EDTA (Gibco) and 4 mM sodium metabisulfite (Nacalai Tesque) were added to the samples and the samples were stored at ⁇ 80 ° C. until analysis.
  • norepinephrine levels in the samples were quantified using an epinephrine / norepinephrine ELISA kit (Abnova) according to the manufacturer's instructions.
  • the norepinephrine release level was determined by subtracting the calculated epinephrine level in the control sample from the epinephrine level in the sample treated with 50 mM KCl.
  • PHOX2B is an essential transcription factor for the development of autonomic nervous system neural crest derivatives such as sympathetic ganglia, parasympathetic ganglia and intestinal ganglia in mice.
  • PHOX2B expression may be useful in optimizing differentiation protocols.
  • PHOX2B eGFP targeting the 3′UTR region of the PHOX2B locus from two types of hPSC clones, namely human embryonic stem cells (hESC, cell line: KhES1) and human induced pluripotent stem cells (hiPSC, cell line: 409B2)
  • hESC human embryonic stem cells
  • hiPSC human induced pluripotent stem cells
  • SN is derived from trunk NCC, and trunk NCC is derived from NMP.
  • NMP is amphoteric for the posterior neural plate and paraxial mesoderm during embryonic body axis development.
  • WNT-mediated retroversion of hPSC is important for NMP induction.
  • the effect of WNT activator CHIR99021 during the first 3 days of agglutination culture was first evaluated. Treatment with 1.5 ⁇ M or more of CHIR was effective in increasing the expression of the Hox gene. This indicates that cells under these conditions began to retrograde during 3 days of initial differentiation (FIG. 6a).
  • the NMP markers BRACURY and SOX2 were also expressed under these conditions, confirming that the CHIR-treated aggregate on the third day had the characteristics of NMP. Since expression of the mesoderm-specific transcription factor TBX6 was increased, treatment with a higher dose of CHIR (5 ⁇ M) is likely to lead hPSCs to developmental fate to the mesoderm.
  • NPCs neural progenitor cells
  • NCC neural progenitor cells
  • SHH sonic hedgehog
  • BMP4 and SHH agonist purmorphamine are 1.5 ⁇ M.
  • retinoic acid is effective in inducing NPC via NMP, and neuronal induction of primary neural crest stem cells from extraneural tubes or embryonic autonomic ganglia has been performed in the presence of RA.
  • RA was used in this situation.
  • PHOX2B eGFP + cells were detected with a purity of over 40% (FIGS. 1b and 6b).
  • CHIR 1.5 ⁇ M Pur + RA 1000nM CHIR 2.0 ⁇ M Pur + RA 1000nM
  • the 3.0 ⁇ M CHIR treatment detected eGFP + cells at a relatively low frequency (0-10%) compared to 1.5 ⁇ M or 2.0 ⁇ M CHIR treatment (data not shown).
  • PHOX2B is expressed not only in autonomic crest derivatives but also in the central nervous system (CNS) neurons and their neural progenitors in the hindbrain. Since CD49d (integrin ⁇ 4) is expressed in migratory NCC and their derivatives, CD49d was used to distinguish NCC from other lineages such as NPC in the CNS. More CD49d + cells were detected under BMP treatment conditions (conditions (iii) and (iv)) than under Pur treatment conditions (conditions (i) and (ii)) (FIG. 1c).
  • CHIR treatment at 1.5 ⁇ M assigns cells to the hindbrain and cervical spinal cord region (HOXB4 + HOXB8 + HOXC9 ⁇ ), while 2.0 ⁇ M CHIR treatment causes cells to move from the cervical to thoracic spinal cord region (HOXB4 ⁇ HOXB8 + HOXC9 + ) (FIG. 1d).
  • eGFP + cells Under Pur conditions, the majority of eGFP + cells did not express CD49d (FIGS. 1c and 6c). Since SHH signals ventralize neuroepithelial cells, it was hypothesized that, under conditions (i, ii), eGFP + cells were progenitors of cranial motor neurons in the ventral posterior brain. In fact, these CD49d ⁇ eGFP + cells differentiated into neurons expressing choline acetyltransferase (ChAT), a motor neuron marker (FIG. 6d, e).
  • ChAT choline acetyltransferase
  • CD49d ⁇ eGFP + cells are in the late stage of neuronal differentiation lineage determination.
  • the majority of CD49d + eGFP + cells were double positive for SOX10 and PHOX2B (FIGS. 2c, d and 8e). This suggests that these cells correspond to double positive progenitor cells of SOX10 and PHOX2B that are localized in mouse embryonic sympathetic ganglia. Since the majority of CD49d + eGFP + cells express HOXB7 (FIGS. 2e, f and 8f), these cells were defined on the anterior-posterior axis at the trunk level.
  • CD49d + eGFP + cell culture conditions were optimized for SN induction. SN maintained PHOX2B expression throughout differentiation, whereas sympathetic NCC-derived non-neuronal cells lost PHOX2B expression, so eGFP expression was followed again. Because the neurosphere culture method can selectively propagate sympathetic NCC in embryonic mouse sympathetic ganglia in vitro, CD49d + eGFP + sympathetic NCC enrichment selected using EGF and FGF2 Cells were cultured in suspension to form aggregates (FIG. 3a). However, about half of those cells lost eGFP expression during the first 7 days of culture after sorting ( Figure 3b, c).
  • BMP4 is required to determine the initial neuronal differentiation lineage of the sympathetic nervous system NCC. Therefore, BMP4 was added and the expression of eGFP was maintained in the majority of cells (> 90%) by the BMP4. Furthermore, when the cells were cultured longer (up to 28 days after sorting), this condition expanded the cell number by more than 10-fold without losing eGFP expression (FIG. 3d). During the prolonged agglutination culture, the expression of SOX10 declined rapidly, with few cells expressing SOX10 after 14 days (FIGS. 3d and 9b).
  • SN was induced from the neurosphere culture, and the purity of those SNs was evaluated.
  • the seeded BMP4 treated aggregates did not show neuronal morphology (Fig. 9f), probably due to their immaturity.
  • Sympathetic neurons except for EGF, FGF2 and BMP4, which are all reported to be involved in the determination of the initial neuronal differentiation lineage of SN due to the maturation of neural progenitor cells, but are not sufficient for the induction of mature neurons NGF, BDNF, and GDNF, which are neurotrophic factors (NF) that promote development, were added (FIG. 3e). After 14 days of culture using this modified protocol, the cells exhibited axonal morphology (FIG. 9f).
  • TH tyrosine hydroxylase
  • DBH dopamine ⁇ -hydroxylase
  • HPSC was successfully differentiated into SN in stages by improving the culture conditions.
  • such differentiation was only possible when using the PHOX2B :: eGFP reporter hPSC line, since a cell sorting step is required for the purification of PHOX2B expressing NCC. Since our ultimate goal is to develop a robust and universal differentiation method that can be applied to various hPSC strains, we next tried to apply our system to hPSC strains without the PHOX2B reporter It was.
  • eGFP + cells were purified with high frequency (day 17; 75% -85%) after transfer to neurosphere culture, and high frequency (day 31; 75%) after neuronal maturation steps. Time course analysis showed that it was maintained at ( ⁇ 90%) (FIGS. 4b, c). Immunostaining revealed PHOX2B + TH + DBH + PRPH + peripheral noradrenergic neurons in 75% -80% of those cells (FIGS. 4d, e). This indicates that the protocol can selectively propagate sympathetic NCC and their derivatives without cell sorting.

Abstract

Des cellules souches pluripotentes sont mises en culture dans une solution de culture contenant du FGF2, de l'acide rétinoïque et du BMP4 afin de produire des cellules de crête neurale. Les cellules de crête neurale sont mises en culture dans une solution de culture contenant une cytokine et du BMP4 afin de produire des précurseurs de neurones sympathiques. En outre, les précurseurs de neurones sympathiques sont mis en culture dans une solution de culture contenant un facteur neurotrophique afin de produire des neurones sympathiques.
PCT/JP2018/016758 2017-04-27 2018-04-25 Procédé de production de cellules de crête neurale et de neurones sympathiques WO2018199142A1 (fr)

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WO2020040166A1 (fr) * 2018-08-22 2020-02-27 国立大学法人京都大学 Procédé de production d'une cellule précurseur de nerf du tractus intestinal
WO2021045217A1 (fr) * 2019-09-06 2021-03-11 学校法人慶應義塾 Procédé de production d'agrégat cellulaire comprenant des cellules progénitrices gliales
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