WO2016104574A1 - Produits de prévention et/ou de traitement contre l'ossification ectopique et méthode de criblage de ces derniers - Google Patents

Produits de prévention et/ou de traitement contre l'ossification ectopique et méthode de criblage de ces derniers Download PDF

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WO2016104574A1
WO2016104574A1 PCT/JP2015/085962 JP2015085962W WO2016104574A1 WO 2016104574 A1 WO2016104574 A1 WO 2016104574A1 JP 2015085962 W JP2015085962 W JP 2015085962W WO 2016104574 A1 WO2016104574 A1 WO 2016104574A1
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cells
inhibitor
fop
culture
imsc
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淳也 戸口田
真 池谷
佳久 松本
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国立大学法人京都大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders

Definitions

  • the present invention relates to a method for screening a substance having prophylactic and / or therapeutic activity for ectopic ossification, particularly progressive ossifying fibrodysplasia (hereinafter also referred to as “FOP”).
  • FOP progressive ossifying fibrodysplasia
  • the present invention also relates to an agent for preventing and / or treating ectopic ossification.
  • Ectopic ossification is a pathological condition in which bone formation is observed in tissues where bone formation does not naturally occur, posterior longitudinal ligament ossification, yellow ligament ossification (yellow ligament hypertrophy), progressive ossification fibrosis Symptoms are seen in erythematosis (FOP), traumatic ossifying myositis, and the like.
  • FOP is a rare genetic disease that occurs in connective tissues characterized by congenital malformations of the thumb and progressive ectopic ossification (Non-patent Document 1). Ectopic ossification in FOP begins in early childhood via the cartilage pathway and may be traumatic or sudden. FOP results in extra-articular stiffness or thorax fusion of the main joint in the mid-axis skeleton and appendage skeleton, resulting in serious disability and fatal respiratory failure.
  • ACVR1 also known as ALK2
  • a mutation (617G> A (R206H)) has been confirmed in the GS) activation domain (Non-patent Document 2).
  • R206H glycine-serine rich in ACVR1
  • Non-patent Document 2 Many researchers have experimented with overexpressing mutant ACVR1 in various cell lines, and the R206H mutation converted ACVR1 to an active form, resulting in activation of BMP signaling that does not require a ligand, It has been found to result in hypersensitivity to the ligand.
  • Non-patent Document 6 Since BMP is recognized as a protein related to bone formation and cartilage formation, it supports that the mutant ACVR1 causes ectopic bone formation of FOP.
  • induction of the mutant ACVR1 confirmed the induction of SMAD1 / 5/8.
  • some contradictions remain, such as not being seen in the C2C12 cell line (Non-Patent Documents 3, 4 and 5). Therefore, research using cells of FOP patients is necessary, but the available cells are limited and it is difficult to obtain reproducibility of symptoms due to cell aging (Non-patent Document 6).
  • Non-patent Document 7 induced pluripotent stem cells (iPS cells) from FOP patients, and further, in the FOP patient-derived iPS cells (FOP-iPSC), increased calcification and cartilage hyperplasia. It has been found that some of the pathological conditions of FOP, such as formation characteristics, are reproduced (Non-patent Document 7). However, since significant differences were observed between iPS cell clones and experiments, it was necessary to compare multiple FOP-iPSC strains and control iPSCs for detailed analysis. In addition, such variability between clones and experiments makes it difficult to elucidate the mechanism of ectopic ossification (especially FOP), and therefore, development of effective therapeutic agents has not progressed. It was.
  • FOP-iPSC iPS cells derived from patients with progressive ossifying fibrodysplasia.
  • resFOP-iPSC iPS cells
  • iPS cells derived from somatic cells of FOP patients found that cartilage tissue tends to be excessively formed as compared with resFOP-iPSC when cartilage is induced.
  • the cause of the excessive formation of the cartilage tissue was not caused by the increased proliferation of the cartilage progenitor cells but by the excessive production of the extracellular matrix from the chondrocytes. I found out.
  • gene expression profiles of various cells derived from FOP-iPSC and resFOP-iPSC are classified into several signal transduction pathways. Differences between the two were observed for the gene group.
  • a prophylactic and / or therapeutic agent for ectopic ossification comprising a plasminogen activator inhibitor 1 (PAI1) inhibitor and / or a matrix metalloproteinase 1 (MMP1) inhibitor.
  • PAI1 plasminogen activator inhibitor 1
  • MMP1 matrix metalloproteinase 1
  • PAI1 plasminogen activator inhibitor 1
  • MMP1 matrix metalloproteinase 1
  • [5] A method for preventing and / or treating ectopic ossification in a subject, comprising administering an effective amount of a PAI1 inhibitor and / or MMP1 inhibitor to the subject.
  • [6] The method according to [5], wherein the PAI1 inhibitor is tiplaxtinin.
  • [7] The method according to [5] or [6], wherein the MMP1 inhibitor is GM6001.
  • [8] The method according to any one of [5] to [7], wherein the ectopic ossification is ectopic ossification in FOP.
  • a PAI1 inhibitor and / or MMP1 inhibitor for use in the prevention and / or treatment of ectopic ossification.
  • a method for screening a substance having prophylactic and / or therapeutic activity for ectopic ossification comprising: (a) differentiating neural crest cells having a mutation in ACVR1 into mesenchymal stromal cells in the presence and absence of the test substance, (b) differentiating the mesenchymal stromal cells obtained in step (a) into chondrocytes, (c) measuring the amount of cartilage tissue in the culture obtained in step (b), and (d) Compared to the case where step (a) is carried out in the absence of the test substance, when the amount of cartilage tissue decreases when the test substance is carried out, the test substance is placed in the ectopic bone.
  • [25] The method according to any one of [17] to [20], wherein the measurement of the amount of cartilage tissue is performed by Alcian Blue staining.
  • [26] The method according to any one of [17] to [25], wherein the genetic mutation of ACVR1 is R206H.
  • the neural crest cell is a cell obtained by expanding and culturing a neural crest cell having a mutation in ACVR1.
  • Expansion of the neural crest cells is performed by culturing the neural crest cells in a culture solution containing a transforming growth factor ⁇ (TGF ⁇ ) inhibitor, epidermal growth factor (EGF), and FGF2. [27].
  • TGF ⁇ transforming growth factor ⁇
  • EGF epidermal growth factor
  • the TGF ⁇ inhibitor is SB431542.
  • the differentiation induction of the neural crest cells is performed by culturing the pluripotent stem cells in a culture solution containing a TGF ⁇ inhibitor and a glycogen synthase kinase 3 ⁇ (GSK3 ⁇ ) inhibitor. The method described.
  • the pathological condition of ectopic ossification can be expressed with good reproducibility, and thus it becomes possible to search for a prophylactic / therapeutic agent for ectopic ossification.
  • the PAI1 inhibitor and MMP1 inhibitor obtained by the screening method can suppress hyperplasia of cartilage tissue, they can be effective prophylactic / therapeutic agents for ectopic ossification.
  • FIG. 1 shows the result of producing a gene repair FOP-iPSC (resFOP-iPSC) clone.
  • FOP-iPSC FOP-iPSC
  • FIG. 1 shows the result of producing a gene repair FOP-iPSC (resFOP-iPSC) clone.
  • (b) The result of examining the repair of the mutation of ACVR1 (617 G> A) is shown. It was confirmed that the gene was repaired by determining the cDNA base sequence.
  • FOP is FOP-iPSC
  • resFOP (cl1) and resFOP (cl2) are FOP-iPSC clones that have been gene-repaired (rescue).
  • (c) shows the results of examining whether homologous recombination occurs in the 5 ′ region of ACVR1. In the 5 'region of ACVR1, a 6.8 Kb PCR product was inserted by homologous recombination.
  • (d) shows the results of examining copy number variation for the pgk-neo resistance cassette.
  • p.c. is an OSR1-GFP knock-in hiPSC clone before Cre-mediated excision of the pgk-neo cassette.
  • FIG. 2 shows the results of analysis of FOP-iPSC-derived iNCC (induced Neural Crest Cell) and iMSC (induced Mesenchymal Stromal Cell).
  • FIG. 2 shows the results of analysis of FOP-iPSC-derived iNCC (induced Neural Crest Cell) and iMSC (induced Mesenchymal Stromal Cell).
  • FIG. 2 shows the results of analysis of FOP-iPSC-derived iNCC (induced Neural Crest Cell) and iMSC (induced Mesenchymal Stromal Cell).
  • FIG. 2 shows the results of analysis of FOP-iPSC-derived iNCC (induced Neural Crest Cell) and iMSC (induced Mesenchymal Stromal Cell).
  • FIG. 2 shows the results of analysis of FOP-iPSC-derived iNCC (induced Neural Crest Cell) and iMSC (induced Mesenchymal Stromal Cell).
  • FIG. 2 shows the results of analysis of FOP-iPSC-derived iNCC (induced Neural Crest Cell) and
  • the GAG value was higher in FOP-iMSC than in resFOP-iMSC, but no significant difference was observed in the amount of DNA.
  • SD standard deviation
  • FIG. 4 shows the results of gene expression profiles of FOP-iPSC and resFOP-iPSC.
  • (a, b) shows the results of analyzing the gene expression status of FOP-iPSC and resFOP-iPSC at each stage of iPSC, iNCC and iMSC.
  • FIG. 5 shows the results of analysis of the basic activity status of SMAD1 / 5/8, SMAD2 / 3, and ERK1 / 2 pathways in FOP-iMSC and resFOP-iMSC.
  • FIG. 5 shows the results of analysis of the western blotting analysis about the phosphorylation state of SMAD1 / 5/8, SMAD2 / 3, and ERK1 / 2 in FOP-iMSC and resFOP-iMSC.
  • ID1 expression was higher in FOP-iMSC than resFOP-iMSC.
  • (e) shows the results of evaluating the activity of the TGF ⁇ -specific luciferase construct in FOP-iMSC and resFOP-iMSC. Luciferase activity was higher in FOP-iMSC than resFOP-iMSC.
  • (f) The result of having analyzed the expression state of PAI1 (target gene of TGF ⁇ ) in FOP-iMSC and resFOP-iMSC is shown. PAI1 expression was higher in FOP-iMSC than in resFOP-iMSC.
  • FIG. 6 shows the result of analyzing the regulatory pathway of PAI1 expression in iMSC.
  • FIG. 8 shows the results of analyzing the function of SMAD1 / 5/8 during MSC induction.
  • FIG. 9 shows the results of analyzing the function of SMAD2 / 3 during MSC induction.
  • a schematic diagram of the inhibitor treatment time course is shown.
  • Representative images of micromass stained with Alcian Blue after treatment with SB431542 (SMAD2 / 3-inhibitor, 10 ⁇ M) are shown.
  • ectopic ossification means a pathological condition in which bone formation is observed in a tissue where bone formation does not occur.
  • diseases associated with ectopic ossification include posterior longitudinal ligament ossification, yellow ligament ossification (yellow ligament hypertrophy), progressive ossifying fibrodysplasia (FOP), and traumatic ossification myositis Includes, but is not limited to, ectopic ossification.
  • the FOP may be accompanied by a mutation of the gene, and may be, for example, a mutation of ACVR1. Examples of mutations that occur in ACVR1 include, but are not limited to, R206H, G356D, and the like. Mutations may occur alone or multiple mutations may occur simultaneously.
  • the FOP ACVR1 mutation in the present invention may be R206H.
  • Prophylactic and / or therapeutic agent for ectopic ossification relates to a prophylactic and / or therapeutic agent for ectopic ossification, which comprises a PAI1 inhibitor and / or an MMP1 inhibitor (hereinafter referred to as “prevention / treatment of the present invention An agent).
  • a prophylactic and / or therapeutic agent for ectopic ossification which comprises a PAI1 inhibitor and / or an MMP1 inhibitor (hereinafter referred to as “prevention / treatment of the present invention An agent).
  • the term “prophylactic / therapeutic agent” means the active ingredient itself
  • the term “prophylactic / therapeutic agent” means a pharmaceutical preparation for use in prevention and / or treatment.
  • the pharmaceutical preparation may be the active ingredient alone or may be in the form of a composition containing additives other than the active ingredient.
  • PAI1 is an abbreviation for “plasminogen activator inhibitor 1”, which is tissue plasminogen activator (also referred to as “t-PA”) or urokinase-type plasminogen activator ( A serine protease inhibitor protein that specifically inhibits "u-PA”).
  • tissue plasminogen activator also referred to as “t-PA”
  • urokinase-type plasminogen activator A serine protease inhibitor protein that specifically inhibits "u-PA”
  • the ⁇ PAI1 inhibitor '' that can be used in the present invention may be any substance that inhibits the activity of PAI1 and interferes with its function, even if it specifically inhibits PAI1, It may be one that broadly inhibits PAI family members.
  • PAI1 inhibitors examples include diketopiperazine (XR330, XR334, XR1853, XR5082, etc.), 11-keto-9 (E), 12 (E) -octadecadienoic acid, tiplaxtinin, fosinopril, imidapril, captopril Enalapril, L158809, eprosartan, troglitazone, vitamin C, vitamin E, perindolpril, mifepristone (RU486), spironolactone and reactive central loop peptide, anti-PAI1 neutralizing antibody, siRNA against PAI1, etc. It is not limited to these.
  • the PAI1 inhibitor in the present invention is preferably tiplaxtinin. These substances can be produced using a synthesis method described in public literature or a normal synthesis method, or can be obtained from companies that produce and sell these substances.
  • MMP1 is an abbreviation of “matrix metalloprotease 1” and is one of the metalloproteases that are proteolytic enzymes in which a metal ion is coordinated to the active center. It is an enzyme involved in degradation.
  • the ⁇ MMP1 inhibitor '' that can be used in the present invention may be any substance that inhibits the activity of MMP1 and interferes with its function, and even a substance that specifically inhibits MMP1, It may be one that extensively inhibits MMP family members.
  • MMP1 inhibitors for example, batimastat (BB-94), marimastat (BB-2516), purinomastert (Prinomastat; AG-3340), CGS-27023A (MMI-270B), neobasstat (Neovastat AE-941), BMS 275-291, tetracyclines, matristatin, catechin, GM6001 (galaldine, iromasterat), trocade (Ro-32-3555), anti-MMP1 neutralizing antibody, siRNA against MMP1, etc.
  • the MMP1 inhibitor in the present invention is preferably GM6001.
  • These substances can be produced by referring to synthesis methods described in known literature or by using ordinary synthesis methods, and can also be obtained from the manufacture, sales, development companies, etc. of these substances. it can.
  • the PAI1 inhibitor and MMP1 inhibitor of the present invention include not only free forms but also pharmacologically acceptable salts thereof.
  • the pharmacologically acceptable salt varies depending on the type of inhibitor. For example, in the case of tiplaxtinin, an alkali metal salt (sodium salt, potassium salt, etc.), an alkaline earth metal salt (calcium salt, magnesium salt, etc.), an aluminum salt
  • Inorganic base salts such as ammonium salts, and base addition salts such as organic base salts such as trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, N, N′-dibenzylethylenediamine, or
  • Inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, phosphate, citrate, oxalate, acetate, formate, propionate, benzoate ,
  • the prophylactic / therapeutic agent of the present invention comprises a PAI1 inhibitor and / or MMP1 inhibitor as it is or mixed with a pharmacologically acceptable carrier, excipient, diluent, etc.
  • the pharmaceutical composition can be administered orally or parenterally.
  • compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), and syrups. Agents, emulsions, suspensions and the like.
  • a composition for parenteral administration for example, injections, suppositories and the like are used, and injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, and the like. Dosage forms may be included.
  • excipients eg sugar derivatives such as lactose, sucrose, sucrose, mannitol, sorbitol; starch derivatives such as corn starch, potato starch, alpha starch, dextrin; cellulose derivatives such as crystalline cellulose; Gum arabic; dextran; organic excipients such as pullulan; and silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate and magnesium metasilicate aluminate; phosphates such as calcium hydrogen phosphate; Carbonates such as calcium; inorganic excipients such as sulfates such as calcium sulfate), lubricants (eg, stearic acid metal salts such as stearic acid, calcium stearate, magnesium stearate; talc; Colloidal silica; wax like beeswax and gay wax Borax; adipic acid; sulfate such as sodium sulfate; glycol; fumedi
  • the content of the PAI1 inhibitor and the MMP1 inhibitor may be about 0.01 to 100% by weight of the whole preparation.
  • each inhibitor when a PAI1 inhibitor and an MMP1 inhibitor are used in combination, each inhibitor may be formulated alone or in combination. In the former case, each preparation can be administered to the same subject simultaneously or with a time difference.
  • the dosage of the prophylactic / therapeutic agent of the present invention can be appropriately changed in consideration of various conditions such as the type of drug, the type of administration target, body weight, age, administration route, severity of symptoms, drug acceptability and the like.
  • the amount of the active ingredient is usually about 0.1 to about 2000 mg / kg / day, preferably about 1 to 200 mg / kg / day, and this amount is once or twice to three times a day. Can be administered separately.
  • the present invention is a method for screening a test substance (hereinafter referred to as “screening of the present invention”) for searching for a prophylactic / therapeutic agent for ectopic ossification. Law).
  • a prophylactic / therapeutic agent for ectopic ossification can be screened by including the following steps; (a) differentiating neural crest cells having a mutation in ACVR1 into mesenchymal stromal cells in the presence and absence of the test substance, (b) a step of differentiating the mesenchymal stromal cells obtained in step (a) into chondrocytes, (c) measuring the amount of cartilage tissue in the culture obtained in step (b), and (d) When the amount of cartilage tissue is reduced when the step (a) is performed in the presence of the test substance compared to the case where the test substance is not present, the test substance is removed from the ectopic bone.
  • the “neural crest cell” means a cell equivalent to a cell de-epithelialized from the neural crest having the ability to migrate to various sites in the embryo. That is, the neural crest cells of the present invention include undifferentiated neural crest-derived cells in tissues derived from the neural crest (for example, bone marrow, dorsal root ganglia, heart, cornea, iris, dental pulp, and olfactory mucosa).
  • the neural crest cell may preferably be a cell positive for at least one gene of TFAP2A, SOX10, PAX3 and p75 (NGFR).
  • TFAP2A includes, as a NCBI accession number, in the case of human, NM_001032280, NM_001042425 or NM_003220, in the case of mouse, a gene having a nucleotide sequence described in NM_001122948 or NM_011547, and a protein encoded by the gene, As well as naturally occurring variants having these functions.
  • SOX10 has the NCBI accession number, NM_006941 for humans, NM_011437 for mice, NM_011437, a gene encoded by the gene, and a protein encoded by the gene, and these functions. Naturally occurring variants are included.
  • PAX3 has the nucleotide sequence described in NCBI as an accession number in humans: NM_000438, NM_001127366, NM_013942, NM_181457, NM_181458, NM_181459, NM_181460 or NM_181461, and NM_001159520 or NM_008781 in mice.
  • the gene and protein encoded by the gene, as well as naturally occurring variants having these functions are included.
  • p75 includes the gene having the nucleotide sequence described in NCNM accession number NM_002507 for humans and NM_033217 for humans, as well as proteins encoded by these genes, and these Naturally occurring variants with functions are included.
  • ACVR1 has a mutation means that ACVR1 has a mutation such as R206H (206th arginine is replaced with histidine) and / or G356D (356th glycine is replaced with aspartic acid).
  • R206H 206th arginine is replaced with histidine
  • G356D 356th glycine is replaced with aspartic acid
  • the mutation is not limited thereto. Mutations may occur alone or multiple mutations may occur simultaneously.
  • the mutation in ACVR1 may preferably be R206H.
  • ACVR1 is a receptor for BMPs also called ALK-2 (activin receptor-like kinase-2), NCBI accession number is NM_001105 or NM_001111067 for humans, and for mice, A gene having the nucleotide sequence described in NM_001110204, NM_001110205 or NM_007394 and a protein encoded by the gene.
  • ALK-2 activin receptor-like kinase-2
  • the neural crest cells used in the present invention can be proliferated by expanding and culturing neural crest cells prepared in advance by cryopreservation or the like.
  • Examples of the method for culturing neural crest cells include a method of culturing in a culture medium containing a TGF ⁇ inhibitor, EGF and FGF2.
  • the culture solution used for the expansion culture of neural crest cells can be prepared using a medium used for animal cell culture as a basal medium.
  • the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, Stem Pro34 RPMI-base medium and mixed media thereof are included.
  • ⁇ MEM medium is preferably used.
  • the medium may contain serum or may be serum-free.
  • the medium can be, for example, albumin, transferrin, Knockout Serum Replacement (KSR) (serum substitute for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acid, insulin, collagen It may contain one or more serum replacements such as precursors, trace elements, 2-mercaptoethanol (2ME), thiol glycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, It may also contain one or more substances such as growth factors, small molecule compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts.
  • KSR Knockout Serum Replacement
  • a TGF ⁇ inhibitor is a substance that inhibits signal transduction from binding of TGF ⁇ to a receptor to SMAD, a substance that inhibits binding to the ALK family of receptors, or SMAD by an ALK family.
  • the substance is not particularly limited as long as it is a substance that inhibits phosphorylation.
  • TGF ⁇ inhibitors include, for example, Lefty-1 (NCBI Accession No., mouse: NM_010094, human: NM_020997 is exemplified), SB431542, SB202190 (above, RKLindemann et al., Mol.
  • the TGF ⁇ inhibitor used for expansion of neural crest cells may preferably be SB431542.
  • the concentration of a TGF ⁇ inhibitor such as SB431542 in the culture solution is not particularly limited as long as it inhibits ALK5, but is preferably 1 to 50 ⁇ M, for example, 1 to nM, 10 to nM, 50 to nM, 100 to nM, 500 nM, 750 ⁇ M, 1 ⁇ M, 2 ⁇ M, 3 ⁇ M, 4 ⁇ M, 5 ⁇ M, 6 ⁇ M, 7 ⁇ M, 8 ⁇ M, 9 ⁇ M, 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 40 ⁇ M, Although it is 50 micro
  • the concentration of EGF in the culture medium is preferably 1 ng / ml to 100 ng / ml, for example, 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, but not limited thereto. More preferably, it is 20 ng / ml.
  • the concentration of FGF2 in the culture medium used for expansion culture of neural crest cells is preferably 1 ng / ml to 100 ng / ml, for example, 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, but not limited thereto. More preferably, it is 20 ng / ml.
  • examples of the isolation method include mechanical separation and a separation solution having protease activity and collagenase activity (for example, trypsin). And collagenase-containing solutions Accutase (TM) and Accumax (TM) (Innovative Cell Technologies, Inc.) or separation using a separation solution having only collagenase activity.
  • a ROCK inhibitor may be added to the culture solution for the purpose of suppressing cell death.
  • the ROCK inhibitor is not particularly limited as long as it can suppress the function of Rho-kinase (ROCK).
  • ROCK Rho-kinase
  • Y-27632 eg, Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000) ; Narumiya et al., Methods Enzymol. 325,273-284 (2000)
  • Fasudil / HA1077 eg, Uenata et al., Nature 389: 990-994 (1997)
  • H-1152 eg, Sasaki et al
  • Pharmacol. Ther. 93 See 225-232 (2002)
  • Wf-536 eg, Nakajima et al., Cancer Chemother Pharmacol.
  • ROCK inhibitors for example, US Patent Application Publication Nos. 2005/0209261, 2005/0192304, 2004/0014755, 2004/0002508,. 2004/0002507, 2003/0125344, 2003/0087919, and International Publications 2003/062227, 2003/059913, 2003/062225, 2002/076976 No., 2004/039796).
  • one or more ROCK inhibitors may be used.
  • a preferred ROCK inhibitor used in this step includes Y-27632.
  • the concentration of the ROCK inhibitor used in this step can be appropriately selected by those skilled in the art depending on the ROCK inhibitor to be used.For example, when Y-27632 is used as the ROCK inhibitor, 0.1 to 100 ⁇ M, preferably 1 ⁇ M to 50 ⁇ M, and more preferably 5 ⁇ M to 20 ⁇ M.
  • the expansion culture of neural crest cells of the present invention can be preferably performed by adhesion culture.
  • the culture container in order to enhance the adhesion ability of neural crest cells to the culture container, the culture container can be coated and used.
  • the coating agent include Matrigel (BD Biosciences), Synthemax (Corning), collagen, gelatin, laminin, heparan sulfate proteoglycan, entactin, or fibronectin, and fragments or combinations thereof, and preferably fibronectin.
  • the culture temperature is not limited to the following, but is about 30 to about 40 ° C., preferably about 37 ° C., and the culture is performed in an atmosphere of CO 2 -containing air.
  • the CO 2 concentration is about 2 to about 5%, preferably about 5%.
  • the neural crest cells used in the present invention may be derived from pluripotent stem cells.
  • Examples of a method for inducing neural crest cells from pluripotent stem cells include a method of culturing in a culture solution containing a TGF ⁇ inhibitor and a GSK-3 ⁇ inhibitor.
  • the neural crest cells induced by this method may be isolated and used using p75 as an indicator, or may be used as a cell population containing other cell types.
  • a method for isolating neural crest cells using p75 as an index a method well known to those skilled in the art can be used, and examples thereof include a method of labeling with an antibody of p75 and isolating using a flow cytometer.
  • the TGF ⁇ inhibitor, the culture solution, and the like used for inducing neural crest cells from pluripotent stem cells can be used under the same conditions as the above-described method for culturing neural crest cells.
  • the GSK-3 ⁇ inhibitor used for inducing neural crest cells from pluripotent stem cells is defined as a substance that inhibits the kinase activity of GSK-3 ⁇ protein (for example, the ability to phosphorylate ⁇ -catenin), Many are already known.
  • BIO also known as GSK-3 ⁇ inhibitor IX; 6-bromoindirubin 3′-oxime
  • GSK-3 ⁇ inhibitor VII which is a phenyl ⁇ bromomethyl ketone compound Acetophenone
  • L803-mts also known as GSK-3 ⁇ peptide inhibitor; Myr-N-GKEAPPAPPQSpP-NH2
  • CHIR99021 6- [2- [4- ( 2,4-Dichlorophenyl) -5- (4-methyl-1H-imidazol-2-yl) pyrimidin-2-ylamino] ethylamino] pyridine- 3-carbonitrile.
  • the concentration of a GSK-3 ⁇ inhibitor such as CHIR99021 in the culture solution is not particularly limited as long as it inhibits the kinase activity of GSK-3 ⁇ protein, but is preferably 1 nM-5 ⁇ M, for example, 1 nM, 10 nM , 50 nM, 100 nM, 500 nM, 750 nM, 1 ⁇ M, 2 ⁇ M, 3 ⁇ M, 4 ⁇ M, and 5 ⁇ M, but are not limited thereto. More preferably, it is 1 ⁇ M.
  • the method of inducing neural crest cells from pluripotent stem cells can be preferably performed by adhesion culture.
  • the adhesion culture in order to enhance the adhesion ability of neural crest cells to the culture container, the culture container can be coated and used.
  • the coating agent include Matrigel (BD Biosciences), Synthemax (Corning), collagen, gelatin, laminin, heparan sulfate proteoglycan, entactin, or fibronectin, and fragments or combinations thereof, preferably Matrigel.
  • the number of culture days for inducing neural crest cells from pluripotent stem cells is, for example, 10 days or less, for example, 1, 2, 3, 4, 5, 6, 7,
  • the culture is performed for 8 days, 9 days, and 10 days, preferably 3-9 days, particularly preferably 7 days.
  • 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 is used in the present invention. Any cell that is induced by an intermediate mesoderm cell is included. Although it does not specifically limit in a pluripotent stem cell, For example, an embryonic stem (ES) cell, the embryonic stem (ntES) cell derived from the clone embryo obtained by nuclear transfer, a sperm stem cell (GS cell), an embryonic germ cell (EG cells), induced pluripotent stem (iPS) cells, cultured fibroblasts, bone marrow stem cell-derived pluripotent cells (Muse cells), and the like.
  • ES embryonic stem
  • ntES embryonic stem
  • GS cell sperm stem cell
  • EG cells embryonic germ cell
  • iPS induced pluripotent stem
  • cultured fibroblasts bone marrow stem cell-derived pluripotent cells
  • Muse cells bone
  • 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 and other 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, as well as primary cultured cells. , Passage cells, and established cell lines are all included.
  • 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.
  • tissue stem cells such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, dental pulp stem cells
  • tissue progenitor cells such as blood cells, umbilical cord blood cells, etc.
  • lymphocytes epithelial cells, endothelial cells, muscle cells, fibroblasts (skin
  • the mammal individual from which somatic cells are collected is not particularly limited, but is preferably a human.
  • pluripotent stem cells are preferably produced from somatic cells derived from patients with progressive ossifying fibrodysplasia (FOP) because ACVR1 desirably has a mutation such as R206H or G356D.
  • FOP progressive ossifying fibrodysplasia
  • iPS cells are preferred.
  • it can also be obtained by introducing a mutation into the ACVR1 gene using a gene modification technique for a pluripotent stem cell having no mutation in the ACVR1 gene.
  • the gene modification technique various techniques known in the art can be adopted, and examples thereof include a method described in WO2013 / 042731, a method using ZFN, TALEN, CRISPR / Cas and the like. However, it is not limited to these.
  • mesenchymal stromal cells are chondrocytes, osteoblasts, bone cells, muscle cells, fat cells, It means a cell capable of differentiating into connective tissue cells such as fibroblasts, immune cells, endothelial cells and pericytes, and has the same meaning as mesenchymal stem cells unless otherwise specified.
  • the mesenchymal stromal cell in the present invention is not particularly limited, but is a cell that is positive for CD73, CD44 and CD105 and negative for CD45.
  • CD73 contains NCBI accession numbers, NM_001204813 or NM_002526 in the case of humans, NM_011851 in the case of mice, and the genes encoded by these genes, and their functions. Naturally occurring variants having are included.
  • CD44 has an NCBI accession number of NM_000610, NM_001001389, NM_001001390, NM_001001391, NM_001001392, NM_001202555, NM_001202556 or NM_001202557 for humans, NM_001039150, NM_001039151, NM_001177785, NM_001177786, As well as the proteins encoded by the genes and naturally occurring variants having these functions.
  • CD105 is encoded by NCBI as an accession number of a gene having a nucleotide sequence described in NM_000118, NM_001114753 or NM_001278138 in the case of human, NM_001146348, NM_001146350 or NM_007932 in the case of a mouse, and the gene. Proteins as well as naturally occurring variants having these functions are included.
  • CD45 is encoded by NCBI as an accession number of a gene having a nucleotide sequence described in NM_001267798, NM_002838 or NM_080921 in the case of humans, NM_001111316, NM_001268286 or NM_011210 in the case of humans, and the gene. Proteins as well as naturally occurring variants having these functions are included.
  • the step of inducing mesenchymal stromal cells from neural crest cells may be performed by suspension culture, or may be performed by adhesion culture using a coated culture dish. Preferably, it is cultured by adhesion culture.
  • adhesion culture a coated culture vessel may be used, or culture may be performed on feeder cells.
  • a medium for inducing mesenchymal stromal cells can be prepared using a medium used for culturing animal cells as a basal medium.
  • the basal medium include IMDM medium, MediumMedi199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, StemPro34 medium , RPMI-base medium and mixed media thereof.
  • ⁇ MEM medium is preferably used.
  • the medium may contain serum or may be serum-free.
  • the medium can be, for example, albumin, transferrin, Knockout Serum Replacement (KSR) (serum substitute for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acid, insulin, collagen It may contain one or more serum replacements such as precursors, trace elements, 2-mercaptoethanol (2ME), thiol glycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, It may also contain one or more substances such as growth factors, small molecule compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts.
  • a preferred basal medium is ⁇ MEM medium containing serum.
  • the concentration of FGF2 in the culture medium used for induction of mesenchymal stromal cells from neural crest cells is preferably 1 ng / ml to 20 ng / ml, for example, 1 ng / ml, 2 ng / ml, 3 ng / ml ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml It is not limited to. More preferably, it is 5 ng / ml.
  • the number of days of culture for induction of mesenchymal stromal cells from neural crest cells does not affect the induction of mesenchymal stromal cells by culturing for a long time, so an upper limit is not particularly required. 2 days or more, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days and 14 days, preferably 7 days or longer, particularly preferably 13 days.
  • Induction of mesenchymal stromal cells from neural crest cells can be preferably performed by adhesion culture.
  • the adhesion culture in order to enhance the adhesion ability of neural crest cells to the culture container, the culture container can be coated and used.
  • the coating agent include Matrigel (BD Biosciences), Synthemax (Corning), collagen, gelatin, laminin, heparan sulfate proteoglycan, entactin, or fibronectin, and fragments or combinations thereof, and preferably fibronectin.
  • the cells may be separated with a known separation solution such as trypsin-EDTA and re-seeded under the same conditions, and the passage may be performed.
  • the agent may be changed.
  • the first culture can be performed in a culture vessel coated with fibronectin, and the subsequent culture can be performed by adhesion culture without using the coating agent.
  • the number of days of culture on fibronectin is, for example, 5 days or less, for example, 1 day, 2 days, 3 days, 4 days, 5 days of culture, preferably 1-3 days, particularly preferably 2 days.
  • the culture temperature is not limited to the following, but is about 30 to about 40 ° C., preferably about 37 ° C., and the culture is performed in an atmosphere of CO 2 -containing air.
  • the CO 2 concentration is about 2 to about 5%, preferably about 5%.
  • chondrocytes are cells that produce extracellular matrix constituting cartilage or cartilage tissue such as collagen and glycosaminoglycan (GAG), or It means a progenitor cell that becomes such a cell.
  • the chondrocytes may form a population only by the chondrocytes, or may be in the state of a culture (particle) composed of chondrocytes and an extracellular matrix produced from the chondrocytes (cartilage-like tissue). May be.
  • Such chondrocytes may be cells that express a chondrocyte marker, and examples of chondrocyte markers include type II collagen (COL2A1), SOX9 or AGGRECAN (ACAN).
  • COL2A1 includes NCBI accession numbers, NM_001844 or NM_033150 for humans, NM_001113515 or NM_031163 for mice, and proteins encoded by these genes, and these Naturally occurring variants having the following functions are included.
  • SOX9 has a gene having a nucleotide sequence described as NCBI accession number NM_000346 for humans and NM_011448 for mice, a protein encoded by the gene, and these functions. Naturally occurring variants are included.
  • AGGRECAN includes NCBI accession numbers as follows: in the case of humans, NM_001135 or NM_013227, in the case of mice, the gene having the nucleotide sequence described in NM_007424, the protein encoded by the gene, and their functions Naturally occurring variants having are included.
  • the chondrocytes in the present invention can be stained with Alcian Blue.
  • a two-dimensional (2D) micromass culture method or a three-dimensional (3D) pellet culture method can be used as a method for inducing chondrocytes from mesenchymal stromal cells.
  • mesenchymal stromal cells can be separated by any method and cultured by adhesion culture.
  • a separation method a mechanical method or an enzymatic method can be used.
  • the separation can be preferably performed by TrypLE Select (Invitrogen).
  • the adhesion culture in the 2D micromass culture method can be performed by culturing using a culture vessel coated with an extracellular matrix.
  • the coating treatment can be performed by placing a solution containing an extracellular matrix in a culture container and then removing the solution as appropriate.
  • coating with fibronectin is preferable.
  • the culture solution used in the 2D micromass culture method can be prepared by adding PDGF-BB or a functional equivalent thereof to a basal medium used for animal cell culture. TGF ⁇ 3, BMP4, or a functional equivalent thereof can be further added to the culture solution.
  • the factors added to these basal media may be added simultaneously, or may be added separately at any stage of the culture process.
  • Examples of functional equivalents of PDGF-BB include, but are not limited to, PDGF-AA, PDGF-AB, PDGF-CC, and PDGF-DD.
  • Examples of functional equivalents of TGF ⁇ 3 include, but are not limited to, TGF ⁇ 1, TGF ⁇ 2, and the like.
  • Examples of functional equivalents of BMP4 include, but are not limited to, BMP2, BMP6, BMP7 and the like.
  • Examples of the basic medium used in the 2D micromass culture method include IMDM medium, Medium ⁇ 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 Examples thereof include a medium, Fischer's medium, and a mixed medium thereof.
  • the medium may contain serum (eg, FCS) or may be serum-free.
  • albumin transferrin, KnockOut Serum Replacement (KSR) (serum substitute for FBS during ES cell culture) (Invitrogen), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, It may contain one or more serum substitutes such as sodium selenate, collagen precursor, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non It may also contain one or more substances such as essential amino acids (NEAA), vitamins, growth factors, low molecular compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts and the like.
  • the basal medium is a serum-free chondrogenic medium in which DMEM medium and Ham's F12 medium are mixed at a ratio of 1: 1.
  • the concentration of PDGF-BB added to the basal medium is, for example, in the range of 1-100 ng / ml, preferably in the range of 20-60 ng / ml, for example, 1 ng / ml, 10 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 35 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 Although it is ng / ml, it is not limited to these. Preferably, it is 40 ng / ml.
  • the concentration of TGF ⁇ 3 added to the basal medium is, for example, in the range of 1-100 ng / ml, preferably in the range of 5-20 ng / ml, for example, 1 ng / ml, 2 ng.
  • ng / ml 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, 11 ng / ml, 12 ng / ml, 13 ng / ml, 14 ng / ml, 15 ng / ml, 16 ng / ml, 17 ng / ml, 18 ng / ml, 19 ng / ml, 20 ng / ml, 25 ng / ml, 50 ng / ml, 75 ng / ml, and 100 ng / ml, but not limited thereto.
  • it is 10 ng / ml.
  • the concentration of BMP4 added to the basal medium is, for example, in the range of 1-100 ng / ml, preferably in the range of 5-20 ng / ml, for example, 1 ng / ml, 2 ng.
  • ng / ml 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, 11 ng / ml, 12 ng / ml, 13 ng / ml, 14 ng / ml, 15 ng / ml, 16 ng / ml, 17 ng / ml, 18 ng / ml, 19 ng / ml, 20 ng / ml, 25 ng / ml, 50 ng / ml, 75 ng / ml, and 100 ng / ml, but not limited thereto.
  • it is 10 ng / ml.
  • the culture temperature is not particularly limited, but is about 30 to about 40 ° C., preferably about 37 ° C., and the culture is performed in an atmosphere of CO 2 -containing air.
  • the CO 2 concentration is about 2 to about 5%, preferably about 5%.
  • the condition in which the O 2 content in the air is lower than the normal 20% may be used, and examples thereof include 15%, 10%, or 5%.
  • the culture time in this step is, for example, 20 days or less, preferably 10 days.
  • the addition of PDGF-BB, TGF ⁇ 3 and BMP4 to the basal medium may be performed simultaneously or separately at any stage of the culture process. Preferably, they are added in any combination in any order depending on the stage of the culturing process.
  • the addition of PDGF-BB, TGF ⁇ 3, and BMP4 to the basal medium can be added directly to the medium being cultured, or can be added when the medium is changed.
  • the addition of PDGF-BB, TGF ⁇ 3 and BMP4 to the basal medium in this step can be performed, for example, in the following order and combination.
  • the culture period in step (1) is, for example, 10 days or less, preferably 3 days.
  • the culture period in step (2) is, for example, 8 days or less, preferably 7 days.
  • the culture in the step (3) may be omitted, and the period for performing the step is, for example, 8 days or less, preferably 4 days.
  • PDGF-BB, TGF ⁇ 3 and BMP4 can be further performed in combination with other differentiation inducing factors.
  • other differentiation-inducing factors include, but are not limited to, Wnt3A, Activin, FGF2, Follistatin, GDF5, and NT4.
  • (Ii) Three-dimensional (3D) pellet culture method prior to 3D pellet culture, a step of subculturing mesenchymal stromal cells in a medium supplemented with FGF2 and TGF ⁇ 3 can be included.
  • the passage period is not particularly limited, but is a period of 5 days or less, preferably 3 days.
  • the concentration of FGF2 in the medium is, for example, in the range of 0.1-50 ng / ml, preferably in the range of 0.5-20 ng / ml, such as 0.5 ng / ml, 0.6 ng / ml, 0.7 ng / ml, 0.8 ng / ml, 0.9 ng / ml, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, Examples include, but are not limited to, 9 ng / ml, 10 ng / ml, 12 ng / ml, 14 ng / ml, 16 ng / ml, 18 ng / ml, and 20 ng / ml.
  • the concentration of TGF ⁇ 3 in the medium is, for example, in the range of 1-100 ng / ml, preferably in the range of 5-20 ng / ml, such as 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, 11 ng / ml, 12 ng / ml, 13 ng / ml, 14 ng / ml, 15 ng / ml, 16 ng / ml, 17 ng / ml, 18 ng / ml, 19 ng / ml, 20 ng / ml, 25 ng / ml.
  • the subcultured cells may be further centrifuged to form a pellet.
  • the number of cells used in the centrifuge is not particularly limited. For example, 2.5 ⁇ 10 5 cells can be used.
  • the culture medium used in the 3D pellet culture method can be performed using the same medium as in the 2D micromass culture method.
  • the culture temperature is not particularly limited, but is about 30 to about 40 ° C., preferably about 37 ° C., and the culture is performed in an atmosphere of CO 2 -containing air.
  • the CO 2 concentration is about 2 to about 5%, preferably about 5%.
  • the culture time in this step is, for example, 40 days or less, preferably 28 days.
  • the test substance with a reduced amount of cartilage tissue is prevented from ectopic ossification compared to the case where the test substance is brought into contact with the cells described above. ⁇ Select as a therapeutic agent.
  • the measurement of the amount of cartilage tissue in the screening method of the present invention is to measure the size of the chondrocyte tissue induced per unit cell of mesenchymal stromal cells, and to measure the expression level of the chondrocyte marker gene Or by measuring the amount of chondrocyte extracellular matrix.
  • the contact between the test substance and the cells may be performed at least during induction from neural crest cells to mesenchymal stromal cells, and induced from mesenchymal stromal cells to chondrocytes. It does not prevent the test substance from coming into contact with the cells during the process.
  • the measurement of the tissue size of chondrocytes in the present invention can be performed by measuring the area or diameter stained with a substance that specifically stains chondrocytes.
  • a substance that specifically stains chondrocytes include, but are not limited to, Alcian Blue, Safranin O, and analogs thereof.
  • chondrocyte marker gene in the present invention examples include, but are not limited to, SOX9, ACAN, COL2A1, and the like.
  • the chondrocyte marker gene is measured as mRNA expression level or protein level, and can be performed by methods well known to those skilled in the art. For example, Northern blotting, RT-PCR, Western blotting, flow site Examples include a method such as measurement.
  • Measurement of the amount of extracellular matrix in the present invention can be performed using methods well known to those skilled in the art.
  • the amount of extracellular matrix can be measured using Blyscan ⁇ Glycosaminoglycan Assay (Biocolor), which targets glycosaminoglycan (GAG), but is not limited thereto.
  • any test substance can be used, and any known compound and novel compound may be used, for example, cell extracts, cell culture supernatants, microbial fermentation products, marine organism-derived extracts.
  • Product plant extract, purified protein or crude protein, peptide, non-peptide compound, synthetic low molecular weight compound, natural compound and the like.
  • the test substance is also (1) biological library method, (2) synthetic library method using deconvolution, (3) “one-beadoneone-compound” live Can be obtained using any of a number of approaches in combinatorial library methods known in the art, including rally methods, and (4) synthetic library methods using affinity chromatography sorting.
  • Biological library methods using affinity chromatography sorting are limited to peptide libraries, but the other four approaches are applicable to peptide, non-peptide oligomer, or small molecule compound libraries of compounds (Lam (1997) Anticancer Drug Des. 12: 145-67).
  • Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422-6; Zuckermann et al. (1994) J. Med. Chem. 37: 2678-85; Cho et al.
  • Example 1 Establishment of resFOP-iPSC clone by homologous recombination using BAC
  • homologous recombination technology using BAC was used.
  • BAC recombination method Ikeya M., Int. J. Dev. Biol. 49: 807-823, 2005
  • BAC recombination method Ikeya M., Int. J. Dev. Biol. 49: 807-823, 2005
  • human BAC clone CTD-20251l1 was purchased from life technologies (Carlsbad, CA, USA).
  • a targeting vector was then constructed by inserting the floxed pgk-neo cassette into the 6th intron and shortening the 5 ′ region to 5 KB.
  • the resulting targeting vector is a construct comprising 5 KB of 5′-short arm and 120 Kb of 3′-long arm as shown in FIG. 1a.
  • targeting vectors were introduced by electroporation using a method modified from the method of Mae et al. (Mae S, et al., Nat Commun. 4: 1367, ⁇ 2013), and two types of resFOP-iPSC clones were established.
  • ACVR1 targeting vector was linearized with FspA1 restriction enzyme and sterilized by ethanol precipitation.
  • FOP-iPSC vFOP4-1 strain; Matsumoto Y., et al. Orphanet J Rare Dis. 8 (1): 190, 2013
  • FOP-iPSC A total of 30 mg of linearized DNA was added to the resuspended FOP-iPSC, and cells were subjected to electrical stimulation of single-250 V, 500-mF pulse (Gene-pulser-CE, Bio-Rad) at room temperature.
  • FOP-iPSC was seeded on a SNL feeder layer treated with mitomycin-C in a medium supplemented with ROCK inhibitor (Y-27632, 10 ⁇ M).
  • ROCK inhibitor Y-27632, 10 ⁇ M
  • G418 75 ⁇ g / ml
  • Genomic DNA was extracted from the obtained colonies, and the base sequence of the genomic DNA was determined, whereby the gene repair clone was subjected to primary screening.
  • resFOP-iPSC cl1
  • resFOP-iPSC cl2
  • FOP-iPSC measure pluripotency marker gene expression, teratoma formation ability, karyotype and morphology (Fig. 1e), and there is no difference in pluripotency It was confirmed. Moreover, it was confirmed that there is no difference in resFOP-iPSC and FOP-iPSC in the cartilage region in teratomas.
  • Example 2 Cell culture iPSCs were maintained in primate ES cell medium (ReproCELL, Tokyo, Japan) supplemented with 4 ng / ml recombinant human basic fibroblast growth factor (FGF2) (WAKO).
  • primate ES cell medium ReproCELL, Tokyo, Japan
  • FGF2 human basic fibroblast growth factor
  • NCC neural crest cells
  • CDM is 1x chemically defined lipid concentrate (GIBCO), 15 ⁇ g / ml apo-transferrin (Sigma), 450 ⁇ M monothioglycerol (Sigma), 5 mg / ml purified BSA (99% purified by crystallization; Sigma), 7 ⁇ g / ml Insulin (WAKO) and penicillin / streptomycin (Invitrogen) were added to Iscove's modified Dulbecco's medium / Ham's F-12 1: 1.
  • differentiation induction from iNCC to mesenchymal stromal cells was performed by the following method. Extract p75-positive cells by FACS using the anti-p75 antibody (BD, Cat No. 560326) from the induced cells described above, inoculate on a 12-well plate coated with Fibronectin at 2 ⁇ 10 5 / well, and 10 ⁇ M After culturing in CDM supplemented with Y-27632 and 10 ⁇ M SB431542 for 1 day, the medium was replaced with CDM supplemented with 10 ⁇ M SB431542, 20 ng / ml FGF2 and 20 ng / ml epidermal growth factor (EGF) (R & D) Maintenance culture was performed for 9 days.
  • CDM supplemented with Y-27632 and 10 ⁇ M SB431542 for 1 day
  • the medium was replaced with CDM supplemented with 10 ⁇ M SB431542, 20 ng / ml FGF2 and 20 ng / ml epidermal growth factor
  • MSC medium minimum essential medium alpha modification ( ⁇ MEM; Invitrogen Co.) supplemented with 5 ng / ml FGF2, 10% fetal bovine serum (FBS) (Nichirei Inc.) and 0.5% penicillin and streptomycin (Invitrogen)
  • FBS fetal bovine serum
  • Invitrogen penicillin and streptomycin
  • Example 3 Analysis of iNCC / iMSC differentiation ability of FOP-iPSC It was previously reported that overexpression of ACVR1 (R206H) promotes cartilage induction in FOP-iPSC (Matsumoto Y., et al. Orphanet J Rare Dis. 8 (1): 190 (2013)). Therefore, iNCC (induced Neural Crest Cell) and iMSC (induced Mesenchmal Stromal Cell) were induced from FOP-iPSC and resFOP-iPSC, respectively, and their chondrocyte differentiation characteristics were compared. On the 7th day from the start of iNCC induction, cells were sorted with anti-p75 antibody using FACS, and the morphology of iNCC was observed.
  • ACVR1 induced Neural Crest Cell
  • iMSC induced Mesenchmal Stromal Cell
  • PCR was performed with ExTaq (Takara, Shiga, Japan). Quantitative PCR was performed using Thunderbird SYBR qPCR Mix (TOYOBO, Osaka, Japan), and analysis was performed using StepOne real-time PCR system (Applied Biosystems, Forester City, CA).
  • NCC markers TFAP2A, SOX10 and PAX3
  • iNCC derived from FOP-iPSC and resFOP-iPSC (cl1) and (cl2)) for all genes of p75 (NGFR) at similar levels. It was expressed specifically (Fig. 2b).
  • Example 4 Analysis of chondrocyte differentiation ability of FOP-iMSC
  • FOP-iMSC and resFOP-iMSC were differentiated into chondrocytes using a 2D micromass culture system and analyzed.
  • the 2D micromass culture system was performed as described by Umeda et al. (Umeda, K. et al. Scientific Reports 2 (2012)).
  • Induced MSC (1.5 x10 5 cells) in 5 ⁇ l of cartilage medium (40 ng / ml PDGF-BB (R & D) and 1% FBS (Nichirei) in serum-free chondrogenic medium (DMEM: F12 (1: 1) (Invitrogen), 1% (v / v) ITS + mix (BD), 0.17 mM AA2P, 0.35 mM Proline (Sigma), 0.1 ⁇ M dexamethasone (Sigma), 0.15% (v / v) glucose (Sigma), 1 mM Na -pyruvate (Invitrogen), 2 mM GlutaMax, 0.05 mM MTG) and spotted on a fibronectin-coated 24-well plate (BD).
  • cartilage medium 40 ng / ml PDGF-BB (R & D) and 1% FBS (Nichirei) in serum-free chondrogenic medium (DMEM: F12 (1: 1) (Invitrogen
  • cartilage medium was added to make 1 ml. From day 3 to day 10, 10 ng / ml TGF ⁇ 3 (R & D system) was added to the medium and cultured. Micromass culture was performed at 5% CO 2 and 37 ° C. for 10 days. Cartilage induction was evaluated by Alcian Blue staining, glycosaminoglycan (GAG) amount, and DNA amount.
  • GAG glycosaminoglycan
  • Alcian Blue staining was performed by fixing induced cells with 10% formalin (Sigma) for 30 minutes, washing with PBS, and then using Alcian Blue solution (3% glacial acetic acid and 1% HCl, pH 1 1% Alcian Blue (MUTO PURE CHEMICAL CO., LTD)) overnight and destained with acetic acid solution.
  • the amount of GAG was measured by quantifying the amount of GAG in the pellet using BLYSCAN Dye and Dissociation reagents (BIOCOLOR, Harbor, UK). On the other hand, the amount of DNA was measured using PicoGreen dsDNA Quantitation kit (Invitrogen).
  • each chondrocyte differentiation marker of FOP-iMSC and resFOP-iMSC induced to differentiate into chondrocytes for 10 days was analyzed by RT-PCR.
  • all chondrocyte differentiation markers SOX9, COL2A1 and ACAN
  • ACVR1 mutations contribute not to cell proliferation but to promotion of differentiation into chondrocytes and maturation of chondrocytes.
  • Example 5 Analysis on gene expression profile of FOP-iPSC
  • differentiation differentiation into chondrocytes was shown between FOP-iMSC and resFOP-iMSC.
  • Microarray analysis was performed to compare gene expression profiles at each stage. Microarray analysis was performed as follows. Total RNA was prepared using RNeasy Mini Kit (Qiagen) and then cDNA was synthesized using GeneChip WT (Whole Transcript) Sense Target Labeling and Control Reagents kit as described by the manufacturer (Affymetrix). . After that, hybridization, washing and scanning into GeneChip Human Gene 1.0 ST expression arrays were performed according to the manufacturer's (Affymetrix) protocol.
  • FIG. 4e MMP1 and PAI1 are shown in the figure as genes that rise in FOP-iMSC.
  • 191 genes whose expression was increased 2-fold or more in FOP-iMSC and 110 genes whose expression was decreased 2-fold or more were confirmed, and the top 20 of these genes are shown in FIG. 4f. From the above, it was confirmed that FOP cells and resFOP cells were almost identical at the stage of iPS cells, NCC and MSC, but several gene expressions were found to be different at the stage of MSC.
  • Example 6 Analysis of basic activation state of SMAD1 / 5/8, SMAD2 / 3 and ERK1 / 2 pathway in iMSC BMP signal is SMAD binding element (SBE) of SMAD1 / 5/8 gene activated by BMP It is transmitted by the canonical pathway by binding to the transcription factor and the non-canonical pathway by binding of the transcription factor to the AP-1 binding site. Therefore, in order to confirm whether the expression of the gene in FOP-iMSC is caused by BMP signaling, we examined whether there are SBE and AP-1 binding site in the regulatory region of the gene whose expression is increased. It was confirmed that there was one or more SBE and AP-1 binding sites.
  • SBE SMAD binding element
  • reporter assay using BMP-specific luciferase reporter construct (BRE-luciferase), and expression of ID1, which is a downstream gene of BMP State analysis was performed.
  • BMP-specific luciferase reporter construct (BRE-luciferase)
  • ID1 which is a downstream gene of BMP State analysis was performed.
  • FOP-iMSC and resFOP-iMSC were dissolved, and analyzed according to dual ⁇ luciferase reporter assay system (Promega) according to the manufacturer's instructions.
  • dual ⁇ luciferase reporter assay system Promega
  • TGF ⁇ signal a reporter assay using a TGF ⁇ -specific luciferase reporter construct (CAGA-lusiferase) and an analysis of the expression state of PAI1, which is a downstream gene of TGF ⁇ , were performed. As a result, it was observed that both luciferase activity and PAI1 expression were higher in FOP-iMSC than in resFOP-iMSC (FIGS. 5e and f). On the other hand, luciferase assay was similarly performed for AP-1, but no difference was found between the two.
  • CAGA-lusiferase TGF ⁇ -specific luciferase reporter construct
  • Example 7 Analysis of PAI1 expression control pathway in iMSC
  • activation of SMAD1 / 5/8, SMAD2 / 3, and ERK1 / 2 pathway was observed in FOP-iMSC. It was investigated whether the expression was controlled.
  • Cells differentiated into iMSC by the same method as in Example 3 FOP-iMSC and resFOP-iMSC were treated with DMH1 (SMAD1 / 5/8 inhibitor, 2 ⁇ M), SB431542 (SMAD2 / 3 inhibitor, 10 ⁇ M) and U0126 ( MEK1 / 2 inhibitor, 1 ⁇ M), and 24 hours later, the expression of PAI1 was analyzed by RT-PCR.
  • PAI1 expression decreased when treated with DMH1 or SB431542 (FIG. 6a).
  • BMP4 10 ng / ml
  • BMP7 100 ng / ml
  • TGF ⁇ 3 10 ng / ml
  • BMP4, BMP7, or TGF ⁇ 3 increased both FOP-iMSC and resFOP-iMSC (FIG. 6b).
  • Example 8 Analysis of functions of PAI1, MMP1, SMAD1 / 5/8 and SMAD2 / 3 in chondrocyte differentiation Since expression of PAI1 and MMP1 was higher in FOP-iMSC than resFOP-iMSC (FIG. 4e), PAI1 And to examine whether MMP1 plays an important role in chondrocyte differentiation, during induction of NCC to MSC (phase I), during induction of MSC to chondrocytes (phase II), and from NCC to chondrocytes At each stage during induction of (phase I + II) (FIGS.
  • PAI1, MMP1, SMAD1 / 5/8 and SMAD2 / 3 10 ⁇ M Tiplaxtinin (PAI1 inhibitor) ( Axon Medchem), 10 nM GM6001 (MMP1 inhibitor) (Calbiochem), 2 ⁇ M DMH1 (SMAD1 / 5/8 inhibitor) and 10 ⁇ M SB431542 (SMAD2 / 3 inhibitor)) are added to the chondrocyte size and GAG The value was examined. Differentiation induction into NCC was performed in Example 3, and differentiation induction into MSC and chondrocytes was performed in the same manner as in Examples 3 and 4.
  • Ectopic ossification in FOP patients is caused by endochondral ossification, where cartilage is first formed and then replaced by bone, so drugs that have the effect of inhibiting cartilage formation can be used to treat ectopic ossification. It is considered useful. Furthermore, since it is thought that chondrogenic ability is determined in the process of induction of MSC in the ectopic ossification of FOP, PAI1 inhibitor and MMP1 inhibitor act at the induction of MSC and suppress MSC cartilage differentiation. It is considered effective for the treatment of ectopic ossification of FOP.
  • the present invention provides a new screening method for a prophylactic / therapeutic agent for ectopic ossification.
  • a prophylactic / therapeutic agent for ectopic ossification By using an appropriate differentiation induction system, more effective screening can be performed.
  • PAI1 inhibitors and MMP1 inhibitors newly found by the screening system are extremely useful for ectopic ossification, especially for the prevention and treatment of FOP.

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Abstract

L'invention concerne des produits de prévention et/ou de traitement contre l'ossification ectopique, la préparation pharmaceutique contenant en tant que principe actif (i) un inhibiteur du PAI1 et/ou (ii) un inhibiteur de la MMP1. L'invention concerne également une méthode de criblage de produits de prévention et/ou de traitement contre l'ossification ectopique, la méthode comprenant (a) une étape de différenciation de cellules des crêtes neurales possédant une mutation de ACVR1 en cellules stromales mésenchymateuses, éventuellement en présence d'un des composés testés, (b) une étape de différenciation des cellules stromales mésenchymateuses obtenues à l'étape (a) en chondrocytes, (c) une étape de mesure de la masse de tissu cartilagineux au sein de la culture obtenue en (b), et (d) une étape de sélection du composé testé comme étant un produit de prévention et/ou de traitement contre l'ossification ectopique s'il apparaît que la masse de tissu cartilagineux diminue lorsque le composé testé est présent à l'étape (a) par rapport au moment où le composé testé n'est pas présent.
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JP2019531053A (ja) * 2016-07-26 2019-10-31 テテック ティシュー エンジニアリング テクノロジーズ アクチェンゲゼルシャフト 細胞培養の組成または純度を定量し軟骨細胞または滑膜細胞の同一性を生体外で定量するためのマーカーと方法
JP7001674B2 (ja) 2016-07-26 2022-02-04 テテック ティシュー エンジニアリング テクノロジーズ アクチェンゲゼルシャフト 細胞培養の組成または純度を定量し軟骨細胞または滑膜細胞の同一性を生体外で定量するためのマーカーと方法
WO2018199142A1 (fr) * 2017-04-27 2018-11-01 国立大学法人京都大学 Procédé de production de cellules de crête neurale et de neurones sympathiques
JP7094567B2 (ja) 2017-04-27 2022-07-04 国立大学法人京都大学 神経堤細胞および交感神経細胞の製造方法
JPWO2018199142A1 (ja) * 2017-04-27 2020-03-12 国立大学法人京都大学 神経堤細胞および交感神経細胞の製造方法
JP2021500008A (ja) * 2017-09-07 2021-01-07 メモリアル スローン ケタリング キャンサー センター 幹細胞由来外胚葉系統前駆体を分化する方法
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JPWO2019107485A1 (ja) * 2017-11-30 2020-11-26 国立大学法人京都大学 細胞の培養方法
JP7330466B2 (ja) 2017-11-30 2023-08-22 国立大学法人京都大学 細胞の培養方法
WO2019107485A1 (fr) * 2017-11-30 2019-06-06 国立大学法人京都大学 Méthode de culture de cellules
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CN112585262A (zh) * 2018-08-22 2021-03-30 国立大学法人京都大学 肠神经前体细胞的制造方法
JPWO2020040166A1 (ja) * 2018-08-22 2021-08-26 国立大学法人京都大学 腸管神経前駆細胞の製造方法
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
JP7341433B2 (ja) 2018-08-22 2023-09-11 国立大学法人京都大学 腸管神経前駆細胞の製造方法
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