JP6673586B2 - Agent for preventing and treating ectopic ossification and screening method thereof - Google Patents

Description

  The present invention relates to a method for screening a substance having a prophylactic and / or therapeutic activity for ectopic ossification, especially progressive ossifying fibrodysplasia (hereinafter also referred to as “FOP”). The present invention also relates to an agent for preventing and / or treating ectopic ossification.

  Ectopic ossification is a condition in which osteogenesis is observed in tissues where bone formation does not originally occur. Posterior longitudinal ligament ossification, yellow ligament ossification (yellow ligament hyperplasia), progressive ossifying fibrosis The symptoms are seen in plaque (FOP), traumatic ossifying myositis and the like.

  FOP is a rare genetic disease that occurs in connective tissue characterized by congenital malformation 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 sporadic. FOP causes extraarticular stiffness or thorax fusion of the main joints in the medial and appendicular skeletons, resulting in severe disability and fatal respiratory failure.

  The gene responsible for FOP is thought to be ACVR1 (also known as ALK2), which encodes one of the type I receptors for bone morphogenetic protein (BMP), and the glycine-serine-rich ( A mutation (617G> A (R206H)) has been confirmed in the GS) activation domain (Non-Patent Document 2). Experiments overexpressing the mutant ACVR1 in various cell lines have been performed by many researchers, and the R206H mutation converts ACVR1 to an active form, thereby activating BMP signaling that does not require a ligand, It has been found that it results in hypersensitivity to the ligand. BMP is recognized as a protein involved in bone formation and cartilage formation, thus supporting that mutant ACVR1 is responsible for the ectopic bone formation of FOP. However, despite these important findings using the mutant ACVR1 overexpression system, for example, induction of SMAD1 / 5/8 was confirmed by introduction of the mutant ACVR1 (R206H) in COS-7 and MC3T3-E1 cell lines. However, some inconsistencies remain, such as the fact that such a phenomenon has not been observed in the C2C12 cell line (Non-Patent Documents 3, 4, and 5). Therefore, research using cells of FOP patients is required, but available cells are limited, and it is difficult to obtain reproducibility of symptoms due to cell aging and the like (Non-Patent Document 6).

  Recently, the present inventors have succeeded in producing an induced pluripotent stem cell (iPS cell) from a FOP patient, and furthermore, in the FOP patient-derived iPS cell (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, there are considerable differences between iPS cell clones and between experiments, so it was necessary to compare multiple FOP-iPSC strains and control iPSCs for detailed analysis. In addition, such variability between clones and between experiments makes it difficult to elucidate the mechanism of ectopic ossification (particularly, FOP), and therefore, the development of effective therapeutic agents has not progressed. Was.

Kaplan FS, et al., Dis Model Mech. 5, 756-762 (2012) Shore EM, et al., Nat Genet. 38, 525-527 (2006) Fukuda T, et al., J Biol Chem. 284, 7149-56 (2009) Shen Q, et al., J Clin Invest. 119, 3462-3472 (2009) van Dinther M et al., J Bone Miner Res. 25, 1208-1215 (2010) Billings PC et al., J Bone Miner Res. 23, 305-313 (2008) Matsumoto Y., et al. Orphanet J Rare Dis. 8 (1): 190 (2013)

  An object of the present invention is to provide a highly accurate screening method for a substance having a preventive and / or therapeutic activity for ectopic ossification, especially FOP (hereinafter, also referred to as “prophylactic / therapeutic agent”). Another object of the present invention is to provide a preventive and / or therapeutic agent for ectopic ossification, which contains a substance obtained by the screening method.

  The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, by applying gene repair technology to iPS cells (FOP-iPSC) derived from patients with progressive ossifying fibrodysplasia, The iPS cells (resFOP-iPSC) with the same background and only the mutation repaired were successfully produced. Subsequently, when FOP-iPSCs were induced to differentiate into chondrocytes via neural crest cells and mesenchymal stromal cells, they succeeded in reproducing the pathological condition of FOP-iPSCs that promotes chondrocyte differentiation, which is unique to FOP-iPSCs. . That is, it was found that iPS cells derived from somatic cells of FOP patients tended to form cartilage tissue excessively when cartilage was induced, as compared to resFOP-iPSC. In addition, when the cause of the hyperplasia of cartilage tissue was examined, it was not due to the increased proliferation of cartilage progenitor cells, but the excessive production of extracellular matrix from chondrocytes was the cause of cartilage tissue hyperplasia. Was found. Furthermore, in order to find out the mechanism of ectopic ossification represented by FOP, when comparing gene expression profiles of various cells derived from FOP-iPSC and resFOP-iPSC, they are classified into several signal transduction pathways Differences were observed between the two in the gene group. When PAI1 inhibitor and MMP1 inhibitor among these signal inhibitors are applied to neural crest cells derived from FOP-iPSC, cartilage tissue hyperplasia is suppressed, so the inhibitor is ectopic ossification, especially The present inventors have found that FOP is useful as a prophylactic / therapeutic agent for ectopic ossification, and completed the present invention.

That is, the present invention provides the following items.
[1] A preventive and / or therapeutic agent for ectopic ossification, comprising a plasminogen activator inhibitor 1 (PAI1) inhibitor and / or a matrix metalloproteinase 1 (MMP1) inhibitor.
[2] The agent according to [1], wherein the PAI1 inhibitor is tiplaxtinin.
[3] The agent according to [1] or [2], wherein the MMP1 inhibitor is gallardine (GM6001).
[4] The agent according to any one of [1] to [3], wherein the ectopic ossification is ectopic ossification in progressive ossifying fibrodysplasia (FOP).
[5] A method for preventing and / or treating ectopic ossification in a subject, comprising administering to the subject an effective amount of a PAI1 inhibitor and / or an MMP1 inhibitor.
[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.
[9] A PAI1 inhibitor and / or an MMP1 inhibitor for use in preventing and / or treating ectopic ossification.
[10] The inhibitor according to [9], wherein the PAI1 inhibitor is tiplaxtinin.
[11] The inhibitor according to [9] or [10], wherein the MMP1 inhibitor is GM6001.
[12] The inhibitor according to any one of [9] to [11], wherein the ectopic ossification is ectopic ossification in FOP.
[13] Use of a PAI1 inhibitor and / or an MMP1 inhibitor for the manufacture of a prophylactic and / or therapeutic agent for ectopic ossification.
[14] The use of [13], wherein the PAI1 inhibitor is tiplaxtinin.
[15] The use of [13] or [14], wherein the MMP1 inhibitor is GM6001.
[16] The use according to any one of [13] to [15], wherein the ectopic ossification is ectopic ossification in FOP.
[17] A method for screening a substance having a prophylactic and / or therapeutic activity for ectopic ossification, comprising:
(A) a step of differentiating neural crest cells having a mutation in ACVR1 in the presence and absence of a test substance into mesenchymal stromal cells,
(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 as compared with the case where the step (a) is performed in the absence of the test substance, the test substance is transferred to the ectopic bone. Selecting as a substance having a prophylactic and / or therapeutic activity of keratinization,
Including, methods.
[18] The method according to [17], wherein the step (b) is performed in the presence and absence of a test substance.
[19] The method according to [17] or [18], wherein the step (a) is performed by culturing neural crest cells in a culture solution containing basic fibroblast growth factor (FGF2).
[20] The above step (b) is performed by culturing mesenchymal stromal cells in a culture solution containing platelet-derived growth factor B chain homodimer (PDGF-BB), [17] to [19]. The method according to any one of claims 1 to 4.
[21] The method according to any one of [17] to [20], wherein the measurement of the amount of cartilage tissue is performed by measuring the amount of extracellular matrix.
[22] The method according to [21], wherein the extracellular matrix is glycosaminoglycan (GAG).
[23] The method according to any one of [17] to [20], wherein the measurement of the amount of cartilage tissue is performed by measuring the expression level of a chondrocyte marker gene.
[24] The method according to [23], wherein the chondrocyte marker gene is at least one gene selected from the group consisting of SOX9, ACAN and COL2A1.
[25] The method according to any one of [17] to [20], wherein the measurement of the cartilage tissue amount 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.
[27] The method according to any one of [17] to [26], wherein the neural crest cell is a cell obtained by expanding a neural crest cell having a mutation in ACVR1.
[28] The expansion of the neural crest cells is performed by culturing the neural crest cells in a culture solution containing a transforming growth factor β (TGFβ) inhibitor, an epidermal growth factor (EGF) and FGF2. , [27].
[29] The method according to [28], wherein the TGFβ inhibitor is SB431542.
[30] The method according to any one of [17] to [29], wherein the neural crest cells are cells induced to differentiate from pluripotent stem cells having a mutation in ACVR1.
[31] The method according to [30], wherein the differentiation induction of the neural crest cells is performed by culturing the pluripotent stem cells in a culture solution containing a TGFβ inhibitor and a glycogen synthase kinase 3β (GSK3β) inhibitor. The described method.
[32] The method according to [31], wherein the TGFβ inhibitor is SB431542 and the GSK3β inhibitor is CHIR99021.

  According to the screening method of the present invention, a pathological condition of ectopic ossification can be expressed with good reproducibility, so that it is possible to search for a preventive / therapeutic agent for ectopic ossification. In addition, the PAI1 inhibitor and the MMP1 inhibitor obtained by the screening method can suppress cartilage tissue hyperplasia, and thus can be an effective prophylactic / therapeutic agent for ectopic ossification.

FIG. 1 shows the results of preparing a gene-repaired FOP-iPSC (resFOP-iPSC) clone. (a) Construction of a BAC-based targeting vector for gene rescue. Arrows indicate the positions of the primers (Acvr1 (Ex7) F1, Acvr1 (Ex7) R1, Acvr1 (Ex7) R2, HR_Fow1 and neo_Rev). (b) shows the results of investigation on repair of the ACVR1 mutation (617 G> A). By determining the cDNA base sequence, it was confirmed that the gene was repaired. FOP is FOP-iPSC, and resFOP (cl1) and resFOP (cl2) are gene-repaired (rescue) FOP-iPSC clones. (c) shows the result of examining whether homologous recombination has occurred in the 5 ′ region of ACVR1. In the 5 'region of ACVR1, insertion of a 6.8 Kb PCR product was caused by homologous recombination. (d) shows the results of examining copy number variation of the pgk-neo resistance cassette. p.c. is the OSR1-GFP knock-in hiPSC clone before Cre-mediated excision of the pgk-neo cassette. (e) Representative images of FOP-iPSC and resFOP-iPSC ((cl1) and (cl2)) are shown. Scale bar is 200 μm. FIG. 2 shows the results of analyzing FOP-iPSC-derived iNCC (induced Neural Crest Cell) and iMSC (induced Mesenchymal Stromal Cell). (a) Representative images inducing differentiation of FOP-iPSC and resFOP-iPSC ((cl1) and (cl2)) into iNCC are shown. (b) The expression states of NCC (Neural Crest Cell) markers (TFAP2A, SOX10 and PAX3), and p75 (NGFR) in iNCCs derived from FOP-iPSC and resFOP-iPSC ((cl1) and (cl2)) were analyzed. The results are shown. All the gene markers were specifically expressed at the same level in iNCCs derived from FOP-iPSC and resFOP-iPSC ((cl1) and (cl2)). (c) Representative images that induced differentiation of FOP-iPSC and resFOP-iPSC ((cl1) and (cl2)) into iMSCs. (d) In iMSCs derived from FOP-iPSC and resFOP-iPSC ((cl1) and (cl2)), the expression states of cell surface markers (CD73, CD44 and CD105) of MSC (Mesenchymal Stromal Cell) and CD45 were analyzed. The results are shown. In iMSCs derived from FOP-iPSC and resFOP-iPSC ((cl1) and (cl2)), expression of MS73 cell surface markers CD73, CD44 and CD105 was observed, but CD45 was negative. FIG. 3 shows the results of analyzing the chondrocyte differentiation ability of FOP-iMSC. (a) Representative image of micromass (MM) stained with Alcian Blue on day 10 after induction of differentiation into chondrocytes from FOP-iMSC and resFOP-iMSC. (b) shows the results of quantifying the size of micromass in FOP-iMSC and resFOP-iMSC. Micromass size was increased in FOP-iMSCs than in resFOP-iMSCs. (c, d) shows the results of quantifying the GAG value (c) and the amount of DNA (d) in FOP-iMSC and resFOP-iMSC. The GAG value was higher in FOP-iMSC than in resFOP-iMSC, but no significant difference was observed in the amount of DNA. The results are shown as the mean ± standard deviation (SD), where N = 8 (FOP) and N = 4 (resFOP (cl1) and (cl2)). (e) shows the results of analyzing the expression state of chondrocyte differentiation markers (SOX9, COL2A1 and ACAN) in the micromass of FOP-iMSC and resFOP-iMSC. All chondrocyte differentiation markers were more highly expressed in FOP-iMSC than in resFOP-iMSC. The results are shown as the mean ± standard deviation (SD), and N = 3 (the value of resFOP is the average of cl1 (N = 2) and cl2 (N = 1)). In the Student's t test, **, P <0.01; ***, P <0.001. 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. (a) is the result of hierarchical clustering analysis in iPSC, iNCC and iMSC, and (b) is the result of PCA plot using different gene expression sets. FOP-iPSC and resFOP-iPSC showed similar gene expression profiles. All data for resFOP is the average of resFOP (cl1) and (cl2). N = 2 (resFOP-iPSC and resFOP-iNCC), N = 3 (resFOP-iMSC (N = 2, cl1; N = 1, cl2)), N = 2 (FOP-iPSC and FOP-iNCC), N = 3 (FOP-iMSC), N = 3 (PSC, iNCC and iMSC; average of each cell from 414C2, TIG120-4f1 and KhES1), and N = 2 (BMMSC; average of BM90 and BM91). (c-e) Scatter plots for global gene expression profiles in FOP-iPSC and resFOP-iPSC. iPSC and iNCC have N = 2, and iMSC has N = 3. The two lines above and below the diagonal indicate the boundary of the two-fold change between the two samples. (f) In FOP-iPSC and resFOP-iPSC, the top 20 genes showing statistically significant different expression status are shown (N = 3, p <0.05). FIG. 5 shows the results of analysis on the state of basic activity of the SMAD1 / 5/8, SMAD2 / 3, and ERK1 / 2 pathways in FOP-iMSC and resFOP-iMSC. (a) shows the results of Western blotting analysis on the phosphorylation status of SMAD1 / 5/8, SMAD2 / 3 and ERK1 / 2 in FOP-iMSC and resFOP-iMSC. The phosphorylation of SMAD1 / 5/8, SMAD2 / 3 and ERK1 / 2 was higher in FOP-iMSC than in resFOP-iMSC. (b) The graph which quantified the phosphorylation state in (a) is shown. (c) shows the results of evaluating the activity of a BMP-specific luciferase construct in FOP-iMSC and resFOP-iMSC. Luciferase activity was higher in FOP-iMSC than in resFOP-iMSC. (d) shows the results of analysis of the expression state of ID1 (BMP target gene) in FOP-iMSC and resFOP-iMSC. ID1 expression was higher in FOP-iMSCs than in resFOP-iMSCs. (e) shows the results of evaluating the activity of a TGFβ-specific luciferase construct in FOP-iMSC and resFOP-iMSC. Luciferase activity was higher in FOP-iMSC than in resFOP-iMSC. (f) shows the results of analyzing the expression state of PAI1 (target gene of TGFβ) in FOP-iMSC and resFOP-iMSC. PAI1 expression was higher in FOP-iMSCs than in resFOP-iMSCs. The results are shown as the mean ± standard deviation (SD), and N = 3 (the value of resFOP is the average of cl1 (N = 2) and cl2 (N = 1)). In the Student's t test, *, P <0.05; **, P <0.01; ***, P <0.001. FIG. 6 shows the results of analysis on the control pathway of PAI1 expression in iMSC. (a) In FOP-iMSC and resFOP-iMSC, treated with DMH1 (SMAD1 / 5/8 inhibitor, 2 μM), SB431542 (SMAD2 / 3 inhibitor, 10 μM) and U0126 (MEK1 / 2 inhibitor, 1 μM) 24 The result of analyzing the expression state of PAI1 after time is shown. PAI1 expression was reduced when treated with DMH1 or SB431542. (b) Results of analyzing the expression state of PAI1 in FOP-iMSC and resFOP-iMSC 24 hours after treatment with BMP4 (10 ng / ml), BMP7 (100 ng / ml) and TGFβ3 (10 ng / ml) Is shown. PAI1 expression was increased when treated with BMP4, BMP7 or TGFβ3. The results are shown as mean ± standard deviation (SD), N = 6 (the value of resFOP is the average of cl1 (N = 3) and cl2 (N = 3)). FIG. 7 shows the results of analyzing the functions of PAI1 and MMP1 during MSC induction. (a) Schematic representation of the time course of inhibitor treatment. (b) Representative images of micromass stained with Alcian Blue after treatment with Tiplaxtinin (PAI1 inhibitor, 10 μM). The result of quantifying the size (c) and GAG value (d) of the (c, d) positive region is shown. Results are shown as mean ± standard deviation (SD), N = 3. (e) Representative images of micromass stained with Alcian Blue after treatment with GM6001 (MMP1 inhibitor). The result of quantifying the size (f) and the GAG value (g) of the (f, g) positive region is shown. Results are shown as mean ± standard deviation (SD), N = 3. FIG. 8 shows the results of analyzing the function of SMAD1 / 5/8 during MSC induction. (a) Schematic representation of the time course of inhibitor treatment. (b) Representative images of micromass stained with Alcian Blue after treatment with DMH1 (SMAD1 / 5/8 inhibitor, 2 μM). The result of quantifying the size (c) and GAG value (d) of the (c, d) positive region is shown. Results are shown as mean ± standard deviation (SD), N = 3. FIG. 9 shows the results of analyzing the function of SMAD2 / 3 during MSC induction. (a) Schematic representation of the time course of inhibitor treatment. (b) Representative images of micromass stained with Alcian Blue after treatment with SB431542 (SMAD2 / 3 inhibitor, 10 μM). The result of quantifying the size (c) and GAG value (d) of the (c, d) positive region is shown. Results are shown as mean ± standard deviation (SD), N = 3.

  As used herein, “ectopic ossification” refers to a condition in which bone formation is observed in a tissue where bone formation does not naturally occur. Diseases associated with ectopic ossification include, for example, posterior longitudinal ligament ossification, yellow ligament ossification (yellow ligament hyperplasia), progressive ossifying fibrodysplasia (FOP), traumatic ossificative myositis Ectopic ossification, but is not limited thereto. FOP may involve a mutation in the gene, for example, a mutation in ACVR1. Mutations that occur in ACVR1 include, but are not limited to, for example, R206H, G356D, and the like. The mutation may have occurred alone or a plurality of mutations may have occurred simultaneously. The FVR ACVR1 mutation in the present invention may be R206H.

The present invention 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 the “prevention / treatment of the present invention”). Agent). In this specification, the term "prophylactic / therapeutic agent" refers to the active ingredient itself, and the term "prophylactic / therapeutic agent" refers to a pharmaceutical preparation to be used for 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 Inhibitor In the present invention, “PAI1” is an abbreviation for “plasminogen activator inhibitor 1” and includes tissue plasminogen activator (also referred to as “t-PA”) and urokinase-type plasminogen activator ( (Also referred to as "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 broadly inhibit PAI family members. Examples of PAI1 inhibitors include diketopiperazine (XR330, XR334, XR1853, XR5082, etc.), 11-keto-9 (E), 12 (E) -octadecadienoic acid, tipraxtinin, fosinopril, imidapril, captopril Enalapril, L158809, eprosartan, troglitazone, vitamin C, vitamin E, perindorpril, mifepristone (RU486), spironolactone and reactive central loop peptide, anti-PAI1 neutralizing antibody, siRNA against PAI1, and the like. It is not limited to these. The PAI1 inhibitor in the present invention is preferably tiplaxtinin. These substances can be produced by using a synthesis method described in a publicly known document or a general synthesis method, or can be obtained from a company that manufactures and sells these substances.

MMP1 inhibitor In the present invention, "MMP1" is an abbreviation for "matrix metalloprotease 1," and is one of metalloproteases that are proteolytic enzymes in which metal ions are coordinated in the active center. It is an enzyme involved in degradation. `` 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, even if it specifically inhibits MMP1, It may broadly inhibit MMP family members. As MMP1 inhibitors, for example, batimastat (Batimastat; BB-94), marimastat (Marimastat; BB-2516), purinomastat (Prinomastat; AG-3340), CGS-27023A (MMI-270B), neovastat (Neovastat) AE-941), BMS 275-291, tetracyclines, Matristatin, catechin, GM6001 (Galaldine, Ironomastat), Trocade (Ro-32-3555), anti-MMP1 neutralizing antibody, siRNA against MMP1, etc. But not limited thereto. The MMP1 inhibitor in the present invention is preferably GM6001. These substances can be produced by referring to synthetic methods described in known literature or by using ordinary synthetic methods, and can also be obtained from the manufacture, sale, development companies, etc. of these substances. it can.

  The PAI1 inhibitor and the MMP1 inhibitor of the present invention include not only the free form but also pharmacologically acceptable salts thereof. Pharmacologically acceptable salts vary depending on the type of inhibitor. For example, in the case of tiplaxtinin, alkali metal salts (sodium salt, potassium salt, etc.), alkaline earth metal salts (calcium salt, magnesium salt, etc.), aluminum salt Base addition salts such as inorganic base salts such as ammonium salts, and organic base salts such as trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, and N, N′-dibenzylethylenediamine; or Inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, phosphate, etc., citrate, oxalate, acetate, formate, propionate, benzoate , Trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfo Salts, acid addition salts such as organic acid salts such as p-toluenesulfonic acid salt. In the case of GM6001, the above-mentioned acid addition salts are exemplified.

Pharmaceutical formulation The prophylactic / therapeutic agent of the present invention is obtained by mixing a PAI1 inhibitor and / or an MMP1 inhibitor as it is or with a pharmacologically acceptable carrier, excipient, diluent, etc. It can be administered orally or parenterally as a pharmaceutical composition.

  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), syrups Agents, emulsions, suspensions and the like. On the other hand, as a composition for parenteral administration, for example, injections, suppositories, etc. are used, and injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections and the like. Dosage forms may be included. These formulations include excipients (e.g., sugar derivatives such as lactose, sucrose, glucose, 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, magnesium metasilicate aluminate; phosphates such as calcium hydrogen phosphate; Carbonates such as calcium; inorganic excipients such as sulfates such as calcium sulfate), and lubricants (eg, metal stearate such as stearic acid, calcium stearate, magnesium stearate; talc; Colloidal silica; beeswax, gay wax Borax; adipic acid; sulfates such as sodium sulfate; glycol; fumaric acid; sodium benzoate; DL leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic anhydride, silicic acid hydrate Such silicic acids; and the above-mentioned starch derivatives), binders (for example, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, and compounds similar to the above-mentioned excipients), disintegrants ( For example, cellulose derivatives such as low-substituted hydroxypropylcellulose, carboxymethylcellulose, carboxymethylcellulose calcium, internally crosslinked sodium carboxymethylcellulose; carboxymethylstarch, sodium carboxymethylstarch, Chemically modified starch and celluloses such as vinyl pyrrolidone), emulsifiers (colloidal clays such as bentonite and veegum; metal hydroxides such as magnesium hydroxide and aluminum hydroxide; sodium lauryl sulfate Anionic surfactants such as calcium stearate; cationic surfactants such as benzalkonium chloride; and nonionic surfactants such as polyoxyethylene alkyl ether, polyoxyethylene sorbitan fatty acid ester, and sucrose fatty acid ester. Activators), stabilizers (paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; phenol and cresol Phenols; thimerosal; dehydroacetic acid; and sorbic acid), flavoring agents (eg, commonly used sweeteners, sour agents, flavors, etc.), and additives such as diluents. It is manufactured by the method described above.

  The content of the PAI1 inhibitor and the MMP1 inhibitor in the prophylactic / therapeutic agent of the present invention can be about 0.01 to 100% by weight of the whole preparation, respectively. When the PAI1 inhibitor and the MMP1 inhibitor are used in combination in the prophylactic / therapeutic agent of the present invention, each inhibitor may be formulated individually or as a mixture. In the former case, each preparation can be administered to the same subject at the same time or at a time interval. The dose of the prophylactic / therapeutic agent of the present invention can be appropriately changed in consideration of various conditions such as the type of the drug, the species of the administration subject, the body weight, the age, the administration route, the severity of the symptoms, and the drug receptivity. However, for example, in the case of tiplaxtinin, 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 the amount is once or twice a day. Can be divided and administered.

The present invention relates to a method for screening a test substance in order to search for a prophylactic / therapeutic agent for ectopic ossification. Law).

In one embodiment of the screening method of the present invention, a prophylactic / therapeutic agent for ectopic ossification can be screened by including the following steps:
(A) a step of differentiating neural crest cells having a mutation in ACVR1 in the presence and absence of a test substance into mesenchymal stromal cells,
(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 as compared with the case where the step (a) is performed in the absence of the test substance, the test substance is transferred to the ectopic bone. Process of selection as a prophylactic / therapeutic agent.

Neural crest cells In the present invention, “neural crest cells” (also referred to as NCCs) mean cells equivalent to cells deepithelialized from neural crests, which have the ability to migrate to various sites in the embryo. That is, the neural crest cells of the present invention also include undifferentiated neural crest-derived cells in neural crest-derived tissues (eg, bone marrow, dorsal root ganglion, heart, cornea, iris, dental pulp, and olfactory mucosa). Neural crest cells can be cells that are preferably positive for at least one gene of TFAP2A, SOX10, PAX3 and p75 (NGFR). In the present invention, TFAP2A, as an NCBI accession number, in the case of humans, NM_001032280, NM_001042425 or NM_003220, in the case of mice, 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. In the present invention, SOX10 has, as an NCBI accession number, a gene having the nucleotide sequence described in NM_006941 in the case of human and NM_011437 in the case of mouse, and a protein encoded by the gene, and a function thereof in the case of mouse. Naturally occurring variants are included. In the present invention, PAX3 has, as an NCBI accession number, the nucleotide sequence described in NM_000438, NM_001127366, NM_013942, NM_181457, NM_181458, NM_181459, NM_181460 or NM_181461 in the case of human, and NM_001159520 or NM_008781 in the case of mouse. Includes genes and proteins encoded by the genes, as well as naturally occurring variants having these functions. In the present invention, p75 (NGFR) includes, as an NCBI accession number, a gene having a nucleotide sequence described in NM_002507 in the case of human and NM_033217 in the case of mouse, and a protein encoded by the gene, Naturally occurring variants having function are included.

  In the present invention, “having a mutation in ACVR1” means that in ACVR1, a mutation such as R206H (arginine at position 206 is substituted with histidine) and / or G356D (glycine at position 356 is substituted with aspartic acid) is present. Although exemplified, the mutation is not limited to this. Mutations may occur alone or a plurality of mutations may occur simultaneously. The mutation in ACVR1 may preferably be R206H. In the present invention, ACVR1 is a receptor for BMPs also called ALK-2 (activin receptor-like kinase-2), as an NCBI accession number, in the case of humans, NM_001105 or NM_001111067, in the case of mice, A gene having the nucleotide sequence described in NM_001110204, NM_001110205 or NM_007394, and a protein encoded by the gene.

  The neural crest cells used in the present invention can be used by expanding neural crest cells prepared in advance by cryopreservation or the like by expanding the culture. Examples of a method for expanding neural crest cells include a method for culturing neural crest cells in a culture solution containing a TGFβ inhibitor, EGF and FGF2.

  In the present invention, the culture solution used for expanding the neural crest cells can be prepared using a medium used for culturing animal cells as a basal medium. As the basal medium, for example, IMDM medium, Medium199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, StemPro34 (invitrogen), RPMI-base medium and a mixed medium thereof are included. In this step, an αMEM medium is preferably used. The medium may contain serum or may be serum-free. If necessary, the medium may be, for example, albumin, transferrin, Knockout Serum Replacement (KSR) (a serum substitute for FBS in ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acid, insulin, collagen It may contain one or more serum substitutes 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 molecules, antibiotics, antioxidants, pyruvate, buffers, inorganic salts and the like.

  In the present invention, a TGFβ inhibitor is a substance that inhibits signal transduction from binding of TGFβ to a receptor to SMAD, a substance that inhibits binding to the ALK family that is a receptor, or SMAD by the ALK family. There is no particular limitation as long as the substance inhibits phosphorylation. In the present invention, TGFβ inhibitors include, for example, Lefty-1 (NCBI Accession No. is exemplified by mouse: NM_010094, human: NM_020997), SB431542, SB202190 (all, RKLindemann et al., Mol. Cancer, 2003, 2:20), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories), A-83-01 (WO 2009/146408) and derivatives thereof Is exemplified. The TGFβ inhibitor used for expanding neural crest cells may preferably be SB431542.

  The concentration of a TGFβ inhibitor such as SB431542 in the culture solution is not particularly limited as long as it inhibits ALK5, and is preferably 1 nM to 50 μM, for example, 1 nM, 10 nM, 50 nM, 100 nM, 500 nM. nM, 750 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 40 μM, 50 μM, but not limited to these. More preferably, it is 10 μM.

  The concentration of EGF in the culture solution 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 expanding 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 is not limited thereto. More preferably, it is 20 ng / ml.

  In the expansion culture of neural crest cells of the present invention, when cells are separately separated and cultured, examples of the isolation method include mechanical separation and a separation solution having protease activity and collagenase activity (eg, trypsin And Accumase (TM) (Innovative Cell Technologies, Inc.) or a separation solution having only collagenase activity. When single cells are separated, 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). For example, Y-27632 (eg, Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000)) ; Narumiya et al., Methods Enzymol. 325,273-284 (2000)), Fasudil / HA1077 (eg, see Uenata et al., Nature 389: 990-994 (1997)), H-1152 (eg, Sasaki et al.) 93, 225-232 (2002)), Wf-536 (see, for example, Nakajima et al., Cancer Chemother Pharmacol. 52 (4): 319-324 (2003)) and derivatives thereof, And antisense nucleic acids against ROCK, RNA interference-inducing nucleic acids (eg, siRNA), dominant negative mutants, and expression vectors thereof. In addition, other known low molecular weight compounds can also be used as the ROCK inhibitor (for example, U.S. Patent Application Publication Nos. 2005/0209261, 2005/0192304, 2004/0014755, and 2004/0002508). No. 2004/0002507, No. 2003/0125344, No. 2003/0087919, and WO 2003/062227, No. 2003/059913, No. 2003/062225, No. 2002/076976 No., 2004/039796). In the present invention, one or more ROCK inhibitors may be used. Preferred ROCK inhibitors used in this step include 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 used.For example, when using Y-27632 as the ROCK inhibitor, 0.1 μM to 100 μM, preferably , 1 μM to 50 μM, more preferably 5 μM to 20 μM.

  The expansion culture of the neural crest cells of the present invention can be preferably performed by adhesion culture. In adhesion culture, a culture vessel can be coated and used in order to enhance the ability of neural crest cells to adhere to the culture vessel. Examples of the coating agent include Matrigel (BD Biosciences), Synthemax (Corning), collagen, gelatin, laminin, heparan sulfate proteoglycan, entactin, or fibronectin, and fragments or combinations thereof, and fibronectin is preferable.

Culture temperature is not limited to, about 30 to about 40 ° C., preferably about 37 ° C., incubated in an atmosphere of CO ₂ containing air is performed. CO ₂ concentration is from about 2 to about 5%, preferably about 5%.

  Neural crest cells used in the present invention may be derived from pluripotent stem cells and used. As a method of inducing neural crest cells from pluripotent stem cells, a method of culturing in a culture solution containing a TGFβ inhibitor and a GSK-3β inhibitor is exemplified. Neural crest cells induced by this method may be isolated and used using p75 as an index, or may be used as a cell population containing other cell types. As 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, for example, a method of labeling with a p75 antibody and isolating with a flow cytometer.

  In the present invention, a TGFβ inhibitor, a 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 expanding neural crest cells.

  In the present invention, a GSK-3β inhibitor used to induce neural crest cells from pluripotent stem cells is defined as a substance that inhibits the kinase activity of GSK-3β protein (eg, the ability to phosphorylate β-catenin), Although many are already known, for example, an indirubin derivative, BIO (also known as GSK-3β inhibitor IX; 6-bromoindirubin 3′-oxime), and a maleimide derivative, SB216763 (3- (2, 4-dichlorophenyl) -4- (1-methyl-1H-indol-3-yl) -1H-pyrrole-2,5-dione), a GSK-3β inhibitor VII (4-dibromo Acetophenone), L803-mts (also known as GSK-3β peptide inhibitor; Myr-N-GKEAPPAPPQSpP-NH2) which is a cell membrane-permeable phosphorylated peptide, and CHIR99021 (6- [2- [4- ( 2,4-Dichlorophenyl) -5- (4-methyl-1H-imidazol-2-yl) pyrimidin-2-ylamino] ethylamino] pyridine-3-c arbonitrile). These compounds are commercially available from, for example, Calbiochem, Biomol, and the like, and can be easily used. However, these compounds may be obtained from other sources, or may be prepared by themselves. Preferably, it may be CHIR99021.

  The concentration of a GSK-3β inhibitor such as CHIR99021 in the culture solution is not particularly limited as long as it is a concentration that inhibits the kinase activity of GSK-3β protein, and is preferably 1 nM to 5 μM, for example, 1 nM or 10 nM. , 50 nM, 100 nM, 500 nM, 750 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, but is not limited thereto. More preferably, it is 1 μM.

  In the present invention, the method for inducing neural crest cells from pluripotent stem cells can be preferably performed by adhesion culture. In adhesion culture, a culture vessel can be coated and used in order to enhance the ability of neural crest cells to adhere to the culture vessel. Examples of 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 Matrigel.

  In this step, the number of culture days for inducing neural crest cells from pluripotent stem cells is, for example, 10 days or less, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, The culture is carried out for 8, 9 and 10 days, preferably 3-9 days, particularly preferably 7 days.

Pluripotent stem cell In the present invention, a pluripotent stem cell is a stem cell having a pluripotency capable of differentiating into many cells existing in a living body and also having a proliferative ability, and is used in the present invention. Any cells that are induced into intermediate mesoderm cells are included. Examples of 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), and embryonic germ cells (EG cells), induced pluripotent stem (iPS) cells, cultured fibroblasts and pluripotent cells derived from bone marrow stem cells (Muse cells). Preferred pluripotent stem cells are iPS cells, and more preferably human iPS cells, from the viewpoint that they can be obtained without destroying embryos, eggs and the like in the production process.

  Methods for producing iPS cells are known in the art, and can be produced by introducing a reprogramming factor into any somatic cell. Here, 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 a gene product such as Glis1, and the like, these reprogramming factors may be used alone or in combination. Is also good. Examples of combinations of reprogramming factors include WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO2010 / 056831, WO2010 / 068955, WO2010 / 098419, WO2010 / 102267, WO2010 / 111409, WO2010 / 111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D, et al. (2008), Nat. Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26: 2467-2474, Huangfu D, et al. (2008), Nat.Biotechnol. 26: 1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008), Cell Stem Cell, 3: 475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat.Cell Biol. 11: 197-203, RL Judson et al., (2009), Nat.Biotechnol., 27: 459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci US A. 106: 8912-8917, Kim JB, et al. ( 2009), Nature.461: 649-643, Ichida JK, et al. (2009), Cell Stem Cell.5: 491-503, Heng JC, et al. (2010), Cell Stem Cell. 6: 167-74 , Han J, et al. (2010), Nature.463: 1096-100, Mali P, et al. (2010), Stem Cells.28: 713-720, Maekawa M, et al. (2011), Nature. 474: 225-9. Are exemplified.

  The somatic cells include, but are not limited to, fetal (pup) somatic cells, neonatal (pup) somatic cells, and mature, healthy or diseased somatic cells. , Subcultured cells, and cell lines. Specifically, somatic cells include, for example, (1) neural stem cells, hematopoietic stem cells, mesenchymal stem cells, tissue stem cells such as dental pulp stem cells (somatic stem cells), (2) tissue progenitor cells, (3) blood cells (peripheral cells) Blood cells, cord blood cells, etc.), lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, splenocytes, pancreatic cells (pancreatic exocrine cells) Etc.), differentiated cells such as brain cells, lung cells, kidney cells and fat cells.

  In the present invention, the mammal individual from which the somatic cells are collected is not particularly limited, but is preferably a human.

  In the present invention, pluripotent stem cells are produced from somatic cells derived from a patient with progressive ossifying fibrodysplasia (FOP) because ACVR1 desirably has a mutation such as R206H or G356D. iPS cells are preferred. Alternatively, it can also be obtained by introducing a mutation into the ACVR1 gene using a genetic modification technique for a pluripotent stem cell having no mutation in the ACVR1 gene. In this case, the gene modification technique can employ various techniques known in the art, for example, a method described in WO2013 / 042731, a method using ZFN, TALEN, CRISPR / Cas, etc. However, the present invention is not limited to these.

Step of differentiating neural crest cells into mesenchymal stromal cells In the present invention, “mesenchymal stromal cells” (also referred to as MSCs) refer to chondrocytes, osteoblasts, bone cells, muscle cells, fat cells, It refers to cells 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 cells in the present invention are, but not limited to, cells that are positive for CD73, CD44 and CD105 and negative for CD45. In the present invention, CD73 contains, as an NCBI accession number, a gene having a nucleotide sequence described in NM_001204813 or NM_002526 in the case of human and NM_011851 in the case of mouse, and a protein encoded by the gene, and a function thereof in the case of mouse. Naturally occurring variants having the following are included: In the present invention, the CD44 as accession numbers NCBI, in humans, NM_000610, NM_001001389, NM_001001390, NM_001001391, NM_001001392, NM_001202555, NM_001202556 or NM_001202557, the case of mice, NM_001039150, NM_001039151, NM_001177785, NM_001177786, NM_001177787 or NM_009851 And a protein encoded by the gene, and a naturally occurring variant having these functions. In the present invention, CD105, as an NCBI accession number, is encoded by a gene having the 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 human and the gene as the NCBI accession number. Included are proteins, as well as naturally occurring variants having these functions. In the present invention, CD45, as an NCBI accession number, in the case of a human, NM_001267798, NM_002838 or NM_080921, in the case of a mouse, NM_001111316, NM_001268286 or NM_011210 having a nucleotide sequence described in NM_011210 and encoded by the gene Included are proteins, as well as naturally occurring variants having these functions.

  In the present invention, 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, the cells are cultured by adhesion culture. When adhesion culture is performed, a culture vessel coated with a coating may be used, or culture may be performed on feeder cells.

  In the step of inducing mesenchymal stromal cells from neural crest cells, a medium for inducing mesenchymal stromal cells can be prepared using a medium used for culturing animal cells as a basal medium. As the basal medium, for example, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, StemPro34 (invitrogen) , RPMI-base medium and a mixed medium thereof. In this step, an αMEM medium is preferably used. The medium may contain serum or may be serum-free. If necessary, the medium may be, for example, albumin, transferrin, Knockout Serum Replacement (KSR) (a serum substitute for FBS in ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acid, insulin, collagen It may contain one or more serum substitutes 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 molecules, antibiotics, antioxidants, pyruvate, buffers, inorganic salts and the like. A preferred basal medium is an αMEM medium containing serum.

  The concentration of FGF2 in the culture solution used for inducing 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 culture days for the induction of mesenchymal stromal cells from neural crest cells does not affect the induction of mesenchymal stromal cells by culturing for a long period of time. 2 or more days, 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 of culture, preferably , 7 days or more, particularly preferably 13 days.

  Induction of mesenchymal stromal cells from neural crest cells can be preferably performed by adhesion culture. In adhesion culture, a culture vessel can be coated and used in order to enhance the ability of neural crest cells to adhere to the culture vessel. Examples of the coating agent include Matrigel (BD Biosciences), Synthemax (Corning), collagen, gelatin, laminin, heparan sulfate proteoglycan, entactin, or fibronectin, and fragments or combinations thereof, and fibronectin is preferable.

  In the present invention, during the induction period of mesenchymal stromal cells by adhesion culture, cells may be separated by a separation solution known per se, such as trypsin-EDTA, and subcultured by re-seeding under the same conditions. The agent may be changed. When changing the coating agent, for example, the initial culture can be performed in a culture vessel coated with fibronectin, and the subsequent culture can be performed by adhesion culture without using a coating agent. In this case, the number of culture days 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.

Culture temperature is not limited to, about 30 to about 40 ° C., preferably about 37 ° C., incubated in an atmosphere of CO ₂ containing air is performed. CO ₂ concentration is from about 2 to about 5%, preferably about 5%.

Step of Differentiating Mesenchymal Stromal Cells into Chondrocytes In the present invention, "chondrocytes" are cells that produce extracellular matrix constituting cartilage or cartilage tissue such as collagen, glycosaminoglycan (GAG), or It means a progenitor cell to be such a cell. In the present invention, chondrocytes may form a population solely by chondrocytes, or may be in the state of a culture (particles) consisting of chondrocytes and extracellular matrix produced from the chondrocytes (cartilage-like tissue). You may. Such a chondrocyte may be a cell that expresses a chondrocyte marker, and examples of the chondrocyte marker include type II collagen (COL2A1), SOX9 and AGGRECAN (ACAN). In the present invention, COL2A1, as an NCBI accession number, in the case of humans, NM_001844 or NM_033150 in the case of humans, in the case of mice, a gene having the nucleotide sequence described in NM_001113515 or NM_031163 and proteins encoded by the genes, Naturally occurring variants having the function of In the present invention, SOX9 has, as an NCBI accession number, a gene having a nucleotide sequence described in NM_000346 in the case of human and NM_011448 in the case of mouse, a protein encoded by the gene, and a function thereof in the case of mouse. Naturally occurring variants are included. In the present invention, AGGRECAN includes, as an NCBI accession number, a gene having a nucleotide sequence described in NM_001135 or NM_013227 in the case of human and NM_007424 in the case of mouse, and a protein encoded by the gene, and a function thereof. Naturally occurring variants having the following are included: Further, the chondrocytes in the present invention can be stained with Alcian Blue.

  In the present invention, as a method for inducing chondrocytes from mesenchymal stromal cells, a two-dimensional (2D) micromass culture method or a three-dimensional (3D) pellet culture method can be used.

(I) Two-dimensional (2D) micromass culture method
In the 2D micromass culture method, mesenchymal stromal cells can be separated by any method and cultured by adhesion culture. Here, as a separation method, a mechanical method or an enzymatic method can be used. In this step, preferably, the separation can be performed by TrypLE Select (Invitrogen).

  In the present invention, adhesion culture by 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 putting a solution containing an extracellular matrix into a culture vessel and then removing the solution as appropriate. In the present invention, it is preferably coated with fibronectin.

  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 culturing animal cells. TGFβ3, 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.

  Functional equivalents of PDGF-BB include, but are not limited to, for example, PDGF-AA, PDGF-AB, PDGF-CC, PDGF-DD, and the like. Functional equivalents of TGFβ3 include, but are not limited to, for example, TGFβ1, TGFβ2, and the like. Functional equivalents of BMP4 include, but are not limited to, for example, BMP2, BMP6, BMP7, and the like.

  As the basal medium used in the 2D micromass culture method, for example, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 A medium, a Fischer's medium, a mixed medium thereof and the like can be mentioned. The medium may contain serum (eg, FCS) or may be serum-free. If necessary, for example, albumin, transferrin, KnockOut Serum Replacement (KSR) (a serum substitute for FBS in ES cell culture) (Invitrogen), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acid, insulin, It may contain one or more serum substitutes such as sodium selenate, collagen precursors, 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, small molecules, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and the like. In one embodiment of this step, the basal medium is a serum-free chondrogenic medium in which DMEM medium and Ham's F12 medium are mixed at 1: 1.

  The concentration of PDGF-BB to be 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. 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 ng / ml, but not limited to these. Preferably, it is 40 ng / ml.

  When TGFβ3 is added, the concentration of TGFβ3 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. / 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 are not limited thereto. Preferably, it is 10 ng / ml.

  When BMP4 is added, 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. / 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 are not limited thereto. Preferably, it is 10 ng / ml.

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. CO ₂ concentration is from about 2 to about 5%, preferably about 5%. The O ₂ content in the air may be lower than usual 20%, for example, 15%, 10%, or 5%. The culture time in this step is, for example, culture for 20 days or less, and preferably 10 days.

  As described above, the addition of PDGF-BB, TGFβ3 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 order and in any combination depending on the stage of the culture process. In addition, PDGF-BB, TGFβ3 and BMP4 can be added to the basal medium directly to the medium during the culture or when the medium is replaced.

The addition of PDGF-BB, TGFβ3 and BMP4 to the basal medium in this step can be performed, for example, in the following order and combination.
(1) PDGF-BB,
(2) PDGF-BB and TGFβ3, and (3) BMP4.
The culturing period of the step (1) is, for example, 10 days or less, and preferably 3 days. The culture period in step (2) is, for example, 8 days or less, and preferably 7 days. The culturing in step (3) may be omitted, and the period of the culturing is, for example, 8 days or less, and preferably 4 days.

  The addition of PDGF-BB, TGFβ3 and BMP4 to the basal medium can be further performed in combination with other differentiation inducing factors. Other differentiation inducing factors include, but are not limited to, for example, Wnt3A, Activin, FGF2, Follistatin, GDF5, NT4, and the like.

(Ii) Three-dimensional (3D) Pellet Culture Method In the present invention, prior to the 3D pellet culture, a step of subculturing mesenchymal stromal cells in a medium supplemented with FGF2 and TGFβ3 may 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, for example, 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, These 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. Preferably, it is in the range of 1-5 ng / ml. The concentration of TGFβ3 in the 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 / 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, 100 ng / ml, but not limited to these. Preferably, it is 10 ng / ml.

In the present invention, prior to the 3D pellet culture, the method may further include a step of centrifuging the subcultured cells to form a pellet. Although the number of cells used in the centrifuge is not particularly limited, for example, 2.5 × 10 ⁵ cells can be used.

  The culture solution used in the 3D pellet culture method can be performed using the same medium as in the 2D micromass culture method.

In the 3D pellet 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. CO ₂ concentration is from about 2 to about 5%, preferably about 5%. The culture time in this step is, for example, 40 days or less, and preferably 28 days.

Screening step In the screening method of the present invention, a test substance having a reduced amount of cartilage tissue is used to prevent ectopic ossification, as compared to a case where the above-mentioned cells are brought into contact with the test substance and not contacted with the test substance.・ Select as a therapeutic drug.

  The measurement of the amount of cartilage tissue in the screening method of the present invention includes measuring the size of the chondrocyte tissue induced per unit cell of mesenchymal stromal cells, and measuring the amount of chondrocyte marker gene expression. Or, by measuring the amount of extracellular matrix of chondrocytes.

  In the screening method of the present invention, the contact between the test substance and the cells may be performed at least during the induction from the neural crest cells to the mesenchymal stromal cells, and the induction from the mesenchymal stromal cells to the chondrocytes. Do not prevent the test substance from contacting the cells.

  The measurement of the size of the chondrocyte tissue in the present invention can be performed by measuring the area or diameter stained with a substance that specifically stains chondrocytes. Examples of the substance that specifically stains chondrocytes include, but are not limited to, Alcian Blue, safranin O, and analogs thereof.

  Examples of the chondrocyte marker gene in the present invention include, but are not limited to, SOX9, ACAN, COL2A1, and the like. The chondrocyte marker gene is measured as the expression level or protein level of mRNA, and the measurement can be performed by a method well-known to those skilled in the art, for example, Northern blotting, RT-PCR, Western blotting, flow cytometry. And methods such as metrology.

  The measurement of the amount of extracellular matrix in the present invention can be performed using a method well known to those skilled in the art. For example, measurement of the amount of extracellular matrix can be performed using a Blyscan Glycosaminoglycan Assay (Biocolor) targeting glycosaminoglycan (GAG), but is not limited thereto.

  In the screening method of the present invention, any test substance can be used, and any known compounds and novel compounds may be used. For example, cell extracts, cell culture supernatants, microbial fermentation products, extraction from marine organisms Products, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic low-molecular compounds, natural compounds and the like. In the present invention, the test substance also includes (1) a biological library method, (2) a synthetic library method using deconvolution, and (3) a “one-bead one-compound” live. It can be obtained using any of a number of approaches to combinatorial library methods known in the art, including rallying 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 small molecule libraries of peptides, non-peptide oligomers, or compounds (Lam (1997) Anticancer Drug Des. 12: 145-67). Examples of methods for synthesizing 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. (1993) Science 261: 1303-5; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2061; Gallop et al. (1994) J. Med. Chem. 37: 1233-51). The compound library can be obtained from a solution (see Houghten (1992) Bio / Techniques 13: 412-21) or beads (Lam (1991) Nature 354: 82-4), chips (Fodor (1993) Nature 364: 555-). 6), bacteria (US Pat. No. 5,223,409), spores (US Pat. Nos. 5,571,698, 5,403,484 and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA) 89: 1865-9) or phage (Scott and Smith (1990) Science 249: 386-90; Devlin (1990) Science 249: 404-6; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87 : 6378-82; Felici (1991) J. Mol. Biol. 222: 301-10; U.S. Patent Application No. 2002103360).

  The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to these examples.

Example 1 Establishment of resFOP-iPSC clone by homologous recombination using BAC
The homologous recombination technique using BAC was used to prepare a highly reliable control iPSC having the same gene background as FOP-iPSC (having a 617G> A (R206H) mutation in exon 7). As previously described, targeting vectors were constructed using BAC to perform the BAC recombination method (Ikeya M., Int. J. Dev. Biol. 49: 807-823, 2005). Briefly, human BAC clone CTD-2025111 was purchased from life technologies (Carlsbad, CA, USA). Next, a targeting vector was constructed by inserting the floxed pgk-neo cassette into the sixth intron and shortening the 5 'region to 5 KB. The resulting targeting vector is a construct containing a 5 KB 5'-short arm and 120 Kb 3'-long arm, as shown in FIG. 1a.

  Subsequently, a targeting vector was introduced by electroporation by 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. Briefly, the ACVR1 targeting vector was linearized with the 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) was trypsinized, centrifuged, and resuspended in PBS. A total of 30 mg of linearized DNA was added to the resuspended FOP-iPSC, and the cells were subjected to electrical stimulation at room temperature with a single 250 V, 500 mF pulse (Gene pulser CE, Bio-Rad). Next, FOP-iPSC was seeded on a SNL feeder layer treated with mitomycin-C in a medium supplemented with ROCK inhibitor (Y-27632, 10 μM). Two days after electroporation, G418 (75 μg / ml) was added to select recombinants. Genomic DNA was extracted from the obtained colonies, and the gene repair clone was subjected to primary screening by determining the nucleotide sequence of the genomic DNA. Next, it was confirmed that homologous recombination had occurred using the 5'long PCR product (FIG. 1a, bottom) (FIG. 1c), and the base sequence of the cDNA was directly determined to confirm that the gene repair was successful. Confirmed (FIG. 1b). Genomic qPCR against the pgk-neo cassette confirmed insertion of a single copy (FIG. 1d).

As described above, two gene-repaired (rescue) FOP-iPSC clones were successfully established (resFOP-iPSC (cl1) and resFOP-iPSC (cl2)). At this time, two non-targeting G418-resistant clones were obtained. It was confirmed that these non-targeting clones exhibited the same phenotype as the parent FOP-iPSCs in the ability to differentiate into chondrocytes. Thereafter, one of the non-targeting G418 resistant clones was used as a FOP-iPSC clone.
resFOP-iPSC (cl1), resFOP-iPSC (cl2) and FOP-iPSC measure pluripotency marker gene expression, teratoma-forming ability, karyotype and morphology (FIG. 1e) and show no difference in pluripotency It was confirmed. In addition, it was confirmed that there was no difference between resFOP-iPSC and FOP-iPSC in the cartilage region in the teratoma.

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).

Induction of neural crest cells (NCC) was performed as follows. iPS cells were seeded on a 24-well plate coated with Matrigel at 1 × 10 ⁴ / well, cultured in mTeSR1 (STEMCELL Technologies) for 2 days, and then chemically defined medium (CDM) containing 10 μM SB431542 and 1 μM CHIR99021. For 7 days to induce NCC (hereinafter referred to as iNCC). CDM is 1x chemically defined lipid concentrate (GIBCO), 15 μg / ml apo-transferrin (Sigma), 450 μM monothioglycerol (Sigma), 5 mg / ml purified BSA (99% purified by crystallization; Sigma), 7 μg / ml Insulin (WAKO) and penicillin / streptomycin (Invitrogen) were added to Iscove's modified Dulbecco's medium / Ham's F-12 1: 1.

Subsequently, induction of differentiation from iNCC to mesenchymal stromal cells (MSC) was performed by the following method. From the cells after the above-mentioned induction, p75-positive cells were extracted by FACS using an anti-p75 antibody (BD, Cat No. 560326), and seeded at 2 × 10 ⁵ / well on a Fibronectin-coated 12-well plate, and 10 μM After culturing for 1 day in CDM supplemented with Y-27632 and 10 μM SB431542, the medium was changed to CDM supplemented with 10 μM SB431542, 20 ng / ml FGF2 and 20 ng / ml epidermal growth factor (EGF) (R & D). Maintenance culture was performed for 9 days. After maintenance culture, minimum essential medium alpha modification (αMEM; Invitrogen Co.) supplemented with MSC medium (5 ng / ml FGF2, 10% fetal bovine serum (FBS) (Nichirei Inc.) and 0.5% penicillin and streptomycin (Invitrogen)) ) And cultured for 2 days. Further, after separating the cells with trypsin, the cells were seeded at 3 × 10 ⁵ / well on a 6-well plate (Costar, # 3516) and subcultured in MSC medium for 11 days (hereinafter referred to as iMSC). .

Example 3 Analysis of iOPC / iMSC differentiation ability of FOP-iPSC
It has previously been reported that overexpression of ACVR1 (R206H) promotes cartilage induction in FOP-iPSCs (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 the chondrocyte differentiation characteristics of both were compared. On the 7th day after the start of iNCC induction, cells were sorted by FACS using an anti-p75 antibody, and the morphology of the iNCC was observed. As a result, no morphological difference was observed between FOP-iNCC and resFOP-iNCC (FIG. 2a). Furthermore, the expression status of NCC markers (TFAP2A, SOX10 and PAX3) and p75 (NGFR) in the obtained iNCC was analyzed by RT-PCR. In RT-PCR, total RNA was purified from each iNCC using an RNeasy kit (Qiagen, Valencia, CA) and treated with a DNase-one kit (Qiagen) to remove genomic DNA. 1 μg of total RNA was reverse transcribed into single-stranded cDNA using random oligos and Superscript III reverse transcriptase (Invitrogen) according to the manufacturer's instructions. PCR was performed using ExTaq (Takara, Shiga, Japan). Quantitative PCR was performed using Thunderbird SYBR qPCR Mix (TOYOBO, Osaka, Japan), and analysis was performed using a StepOne real-time PCR system (Applied Biosystems, Forester City, CA). As a result, for the NCC markers (TFAP2A, SOX10 and PAX3), and for all genes of p75 (NGFR), iNCCs derived from FOP-iPSC and resFOP-iPSC ((cl1) and (cl2)) were at similar levels. It was specifically expressed (FIG. 2b).

  At the third passage after the start of iMSC induction, cells having the same morphology as primary human cultured MSCs appeared, and no difference was observed in morphology between FOP-iPSC and resFOP-iPSC (FIG. 2c). On the 7th day after the start of iMSC induction, iMSCs derived from FOP-iPSC and resFOP-iPSCs ((cl1) and (cl2)) were analyzed using cell surface markers of MSCs (CD73, CD44 and CD105) and CD45 as indicators. Analysis was performed. FACS analysis was performed using AriaII (BD) according to the manufacturer's protocol. The antibodies used in FACS are described in Table 1.

  As a result, in iMSCs derived from FOP-iPSC and resFOP-iPSCs ((cl1) and (cl2)), expression of MS73 cell surface markers CD73, CD44 and CD105 was observed, but CD45 was negative. (FIG. 2d). As a result, it was confirmed that differentiation could be induced into MSC from any of the FOP-iPSC and resFOP-iPSC ((cl1) and (cl2)) iPS cells.

Example 4 Analysis of chondrocyte differentiation ability of FOP-iMSC
FOP-iMSC and resFOP-iMSC were differentiated into chondrocytes by 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)). The induced MSCs (1.5 × 10 ⁵ cells) were added to 5 μl 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 μM 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). One hour later, a cartilage medium was added to 1 ml. From day 3 to day 10, 10 ng / ml TGFβ3 (R & D system) was added to the medium and cultured. Micromass culture was performed at 5% CO ₂ at 37 ° C. for 10 days. Cartilage induction was evaluated by Alcian Blue staining, glycosaminoglycan (GAG) content, and DNA content. Briefly, Alcian Blue staining was performed by fixing the induced cells with 10% formalin (Sigma) for 30 minutes, washing with PBS, and then using an Alcian Blue solution (3% glacial acetic acid and 1% HCl, 1% Alcian pH 1). Blue (MUTO PURE CHEMICAL CO., LTD)) overnight, and destained with an acetic acid solution. The measurement of the GAG amount was performed by quantifying the GAG amount in the pellet using BLYSCAN Dye and Dissociation reagents (BIOCOLOR, Belfast, UK). On the other hand, the measurement of the DNA amount was performed using PicoGreen dsDNA Quantitation kit (Invitrogen).

  As a result, the size of the micromass induced to differentiate for 10 days was larger in FOP-iMSC than in resFOP-iMSC (FIGS. 3a and 3b). In addition, the GAG value was higher in FOP-iMSC than in resFOP-iMSC (FIG. 3c), but no significant difference was observed in the amount of DNA between them (FIG. 3d). From the above, it was shown that when FOP-iMSC was induced into chondrocytes, extracellular matrix was excessively produced.

  Subsequently, the expression of each chondrocyte differentiation marker of FOP-iMSC and resFOP-iMSC induced to differentiate into chondrocytes for 10 days was analyzed by RT-PCR. As a result, it was observed that all the chondrocyte differentiation markers (SOX9, COL2A1 and ACAN) were more highly expressed in FOP-iMSC-derived chondrocytes as compared to resFOP-iMSC-derived chondrocytes ( Figure 3e).

  Summarizing the above results, it was suggested that ACVR1 mutation contributes not to cell proliferation but to promotion of chondrocyte differentiation and chondrocyte maturation.

Example 5 Analysis of Gene Expression Profile of FOP-iPSC In Example 4, different differentiation ability to 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 the RNeasy Mini Kit (Qiagen), and then cDNA was synthesized using the GeneChip WT (Whole Transcript) Sense Target Labeling and Control Reagents kit as described by the manufacturer (Affymetrix). . Thereafter, hybridization, washing and scanning to GeneChip Human Gene 1.0 ST expression arrays were performed according to the protocol of the manufacturer (Affymetrix). Data analysis was performed by scatter plot and hierarchical clustering using GeneSpring GX 11.5.1 (Agilent Technologies). For hierarchical clustering, Chebyshev correlation was used for similarity measure and average linkage clustering. As a result, as for the gene expression profiles between FOP-iPSC and resFOP-iPSC, similarity was confirmed by hierarchical clustering (FIG. 4a), Primary Component Analysis (PCA) (FIG. 4b), and scatter diagram (FIG. 4c). Although the gene expression profiles were similar between FOP-iNCC and resFOP-iNCC (FIGS. 4a, b and d), the gene expression profiles between FOP-iMSC and resFOP-iMSC were hierarchical. Although no difference was observed between clustering and PCA (FIGS. 4a and b), there was a group of genes showing distinctly different gene expression states in the scatter diagram (FIG. 4e). MMP1 and PAI1 are shown in the figure as genes elevated in FOP-iMSC. In addition, 191 genes whose expression increased more than 2-fold and 110 genes whose expression decreased more than 2-fold in FOP-iMSC were confirmed, and the top 20 of these genes were shown in FIG. 4f. From the above, it was confirmed that FOP cells and resFOP cells were almost the same at the stage of iPS cells, NCC and MSC, but it was found that some gene expression was 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
The BMP signal is a canonical pathway by binding of transcription factor to SMAD binding element (SBE) of SMAD1 / 5/8 gene activated by BMP, and a non-canonical pathway by binding of transcription factor to AP-1 binding site. Conveyed by Therefore, the expression of the gene in FOP-iMSC, to confirm whether caused by BMP signaling, to examine whether the SBE and AP-1 binding site in the regulatory region of the up-regulated gene, in most genes, It was confirmed that there was one or more SBEs and AP-1 binding sites. On the other hand, it was also confirmed that some genes contain SAD of SMAD2 / 3 activated by TGFβ. Furthermore, the phosphorylation status of SMAD1 / 5/8, ERK1 / 2, p38, JNK and SMAD2 / 3 was examined by Western blotting. Western blotting was performed by SDS-PAGE and blotting using all the cell lysates of FOP-iMSC and resFOP-iMSC by a standard procedure. Protein bands were detected using Amercham ^™ ECL ^™ Prime Western Blotting Detection Reagent (GE Healthcare, Tokyo, Japan) and visualized using BIO-RAD Molecular Imager® Chemi-Doc ^™ XRS + with Image Lab ^™ software. . As a result, it was observed that the phosphorylation status of SMAD1 / 5/8, SMAD2 / 3 and ERK1 / 2 was higher in FOP-iMSC than in resFOP-iMSC (FIGS. 5a and 5b). On the other hand, no difference was seen between p38 and JNK.

  Subsequently, a reporter assay using a BMP-specific luciferase reporter construct (BRE-luciferase), and expression of ID1, which is a downstream gene of BMP, to examine that the BMP signal and TGFβ signal are activated in FOP-iMSC The state was analyzed. In the reporter assay, after dissolving FOP-iMSC and resFOP-iMSC, respectively, analysis was performed using a dual luciferase reporter assay system (Promega) according to the manufacturer's instructions. As a result, both luciferase activity and ID1 expression were observed to be higher in FOP-iMSC than in resFOP-iMSC (FIGS. 5c and d). As for the TGFβ signal, a reporter assay using a TGFβ-specific luciferase reporter construct (CAGA-lusiferase) and the expression state of PAI1, which is a downstream gene of TGFβ, were analyzed. 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 particular difference was found between the two. In addition, ELISA for BMP4, BMP6, BMP7 and TGFβ in the culture supernatant was performed, but no difference was observed between FOP and resFOP at any stage of iPS cells, NCC and MSC, and it was detected slightly. It was only. From the above results, it was suggested that the mutant ACVR1 (R206H) constantly activated the BMP-SMAD1 / 5/8 and TGFβ-SMAD2 / 3 pathways in FOP-iMSC.

Example 7 Analysis of control pathway of PAI1 expression in iMSC In Example 6, activation of SMAD1 / 5/8, SMAD2 / 3 and ERK1 / 2 pathways was observed in FOP-iMSC. It was examined whether expression was regulated. Cells (FOP-iMSC and resFOP-iMSC) differentiated into iMSCs in the same manner as in Example 3 were prepared using DMH1 (SMAD1 / 5/8 inhibitor, 2 μM), SB431542 (SMAD2 / 3 inhibitor, 10 μM) and U0126 ( (MEK1 / 2 inhibitor, 1 μM), and 24 hours later, the expression of PAI1 was analyzed by RT-PCR. As a result, the expression of PAI1 was reduced when treated with DMH1 or SB431542 (FIG. 6a). Subsequently, the cells were treated with BMP4 (10 ng / ml), BMP7 (100 ng / ml) and TGFβ3 (10 ng / ml), and after 24 hours, the expression state of PAI1 was analyzed by RT-PCR. , BMP4, BMP7 or TGFβ3 resulted in an increase in both FOP-iMSC and resFOP-iMSC (FIG. 6b). These results suggest that in iMSCs, PAI1 expression is positively regulated by BMP-SMAD1 / 5/8 and TGFβ-SMAD2 / 3 pathways, and furthermore, SMAD1 / 5/8 and SMAD2 / 3 Supports the result that the pathway is more enhanced in FOP-iMSC than in resFOP-iMSC.

Example 8 Analysis of functions of PAI1, MMP1, SMAD1 / 5/8 and SMAD2 / 3 in chondrocyte differentiation
Since the expression of PAI1 and MMP1 was higher in FOP-iMSCs than in resFOP-iMSCs (FIG. 4e), to determine whether PAI1 and MMP1 play an important role in chondrocyte differentiation, the induction of NCC to MSC induction During each phase (phase I), during induction of MSC to chondrocytes (phase II) and during induction of NCC to chondrocytes (phase I + II) (FIGS. 7a, 8a and 9a), PAI1, MMP1, SMAD1 / 5/8 and SMAD2 / 3 inhibitors (10 μM Tiplaxtinin (PAI1 inhibitor) (Axon Medchem), 10 nM GM6001 (MMP1 inhibitor) (Calbiochem), 2 μM DMH1 (SMAD1 / 5/8 inhibitor ) And 10 μM SB431542 (SMAD2 / 3 inhibitor)) were added to examine the size and GAG value of chondrocytes. Induction of differentiation into NCC was performed in the same manner as in Example 3, and differentiation induction into MSCs and chondrocytes was performed in the same manner as in Examples 3 and 4. As a result, in all cases where DMH1 and SB431542 were added at the stage of phase I, phase II or phase I + II, the size of the Alcian Blue positive region and the GAG value decreased as compared with the negative control (FIG. 8b, FIG. 8c, FIGS. 9b and 9c). This suggests that in the MSC induction step and the cartilage induction step, both SMAD1 / 5/8 and SMAD2 / 3 signals are involved in cartilage induction. On the other hand, GM6001 and Tiplaxtinin reduced cartilage induction in FOP-MSC when added at the phase I and phase I + II stages (FIGS. 7b to g). These results suggested that MMP1 and PAI1 enhance cartilage-inducing ability during the induction of FOP cells from NCC to MSC.

  Ectopic ossification in FOP patients is caused by endochondral ossification, where cartilage is first formed and then replaced by bones. Deemed useful. Furthermore, since it is thought that cartilage-forming ability is determined during the induction of MSCs in the ectopic ossification of FOP, PAI1 inhibitors and MMP1 inhibitors work during the induction of MSCs and suppress the cartilage differentiation of MSCs. It is considered to be effective in treating ectopic ossification of FOP.

Although the invention has been described with emphasis on preferred embodiments, it will be obvious to those skilled in the art that the preferred embodiments may be varied. The present invention contemplates that the present invention may be practiced other than as specifically described herein. Accordingly, the present invention includes all modifications that fall within the spirit and scope of the appended claims.
The contents of all publications, including the patents and patent application specifications mentioned herein, are hereby incorporated by reference, to the same extent as if all were expressly indicated. .

  This application is based on a patent application No. 2014-261086 filed in Japan on December 24, 2014, the entire contents of which are incorporated herein by reference.

  The present invention provides a novel screening method for a prophylactic / therapeutic agent for ectopic ossification. By using an appropriate differentiation inducing system, more effective screening can be performed. In addition, PAI1 inhibitors, MMP1 inhibitors, and the like newly discovered by the screening system are extremely useful for the prevention and treatment of ectopic ossification, particularly, FOP.

Claims (2)

An agent for preventing and / or treating ectopic ossification, comprising tiplaxtinin and / or gallardine (GM6001) . The agent according to claim 1, wherein the ectopic ossification is ectopic ossification in progressive ossifying fibrodysplasia (FOP).