Differentiation inducer for skeletal muscle type I cells

Abstract

To provide a novel differentiation inducer to skeletal muscle Type I cells, which uses a component that was not previously known to induce differentiation to Type I fibers.SOLUTION: A differentiation inducer to skeletal muscle Type I cells includes R-spondin 3.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an inducer for differentiating into skeletal muscle Type I cells, an activator of the β-catenin pathway, a method for determining whether a test cell is a skeletal muscle Type I cell, a pharmaceutical composition for treating diseases caused by atrophy of skeletal muscle Type I cells, and the like.

Background Art

[0002] Skeletal muscle is mainly composed of two different types of muscle fibers, namely Type I fibers and Type II fibers. The main differences in characteristics between Type I fibers and Type II fibers are as follows.

[0003]

Table 1

[0004] Although the characteristics of Type I fibers and Type II fibers have been clarified as described above, regarding which factors are involved in the differentiation of myoblasts into Type I fibers and Type II fibers, although many have been studied, most of them have not yet been clarified.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to provide a novel inducer for differentiating into skeletal muscle Type I cells, which uses a component whose effect of promoting differentiation into Type I fibers has not been known so far.

Means for Solving the Problems

[0007] Under these circumstances, the inventors searched for genes expressed in Type I and Type II fibers and investigated the functions of these genes. As a result, they found that R-spondin 3, in particular, which is highly expressed in Type I fibers, has a differentiation-inducing effect on skeletal muscle Type I cells. Based on this novel finding, the inventors studied the mechanisms by which R-spondin 3 is involved in skeletal muscle Type I cells, and after much trial and error, completed the present invention. Accordingly, the present invention provides the following: Item 1. A differentiation inducer for skeletal muscle type I cells containing R-spondin 3.

[0008] Item 2. A differentiation-inducing agent as described in Item 1, which is used by adding it to cells in vitro, or an injectable or capsule preparation for administration to a living organism.

[0009] Item 3. Activators of the β-catenin pathway, containing R-spondin 3.

[0010] Item 4. An activator as described in Item 3, which is an activator used by adding it to cells in vitro, or an injectable or capsule preparation for administration to a living organism.

[0011] Item 5. A method for determining whether test cells are skeletal muscle type I cells or not, using R-spondin 3 as an indicator.

[0012] Item 6. A pharmaceutical agent for treating conditions that can be treated by inducing differentiation of skeletal muscle type I cells, including R-spondin 3. [Effects of the Invention]

[0013] According to the present invention, it is possible to provide a novel differentiation inducer for skeletal muscle type I cells that uses a component whose differentiation-promoting effect on type I fibers has not been previously known. [Brief explanation of the drawing]

[0014] [Figure 1]The outline of the classification of muscle fibers and the RNA extraction method in Example 1 is shown in FIG. 1. [Figure 2] The RNA extraction results from muscle fibers in Example 1 are shown. [Figure 3] The outline of the test in Example 2 and the results of Western blotting of Rspo3 expression in the cell supernatant and inside the cells are shown (upper part of FIG. 3). Also, the information on the Rspo3 sequence cloned into pCAGGS used in the test is shown in the lower part of FIG. 3. Explanation of each sequence: EcoR I restriction enzyme site; GAATTC (boxed part) spacer sequence; GT (shaded part) Kozak sequence: CACCATG···TAG (underlined part) from A at position 13 to G at position 846 of the nucleotide sequence shown in FIG. 3: Mouse Rspo3 sequence (the start codon overlaps with a part of the Kozak sequence) [Figure 4] The results of Western blotting of Rspo3 expression in the cell supernatant and inside the cells in Example 2 are shown [Figure 5] The test results of Example 3 are shown (upper part of FIG. 5). The amino acid sequence of the recombinant mouse Rspo3 used in Example 3 is shown in the lower part of FIG. 5. [Figure 6] The test results of Example 3 are shown. [Figure 7] The test results of Example 3 are shown. [Figure 8] The test results of Example 3 are shown. [Figure 9] The test results of Example 3 are shown. [Figure 10] The test results of Example 3 are shown. [Figure 11] The test results of Example 4 are shown.

Mode for Carrying Out the Invention

[0015] Differentiation Inducer The present invention provides an inducer for differentiating into skeletal muscle Type I cells, which contains R-spondin 3.

[0016] The active ingredient of the present invention, R-spondin 3, is a protein belonging to the Rspo family. Examples of R-spondin 3 include those derived from humans, mice, monkeys, rats, pigs, chimpanzees, cattle, birds, zebrafish, African clawed frogs, etc. Examples of R-spondin 3 include those having the nucleotide sequence or amino acid sequence indicated by NCBI accession No. NM_028351.3 (Mouse RNA), No. NP_082627.3 (Mouse Protein); No. NM_032784.5 (Human RNA), and No. NP_116173.2 (Human Protein). In preferred embodiments of the present invention, examples of R-spondin 3 include those having the above-mentioned mouse amino acid sequence or an amino acid sequence with homology to the above-mentioned mouse amino acid sequence of 80% or more, preferably 85% or more, more preferably 90% or more. In the present invention, R-spondin 3 may also be referred to as Rspo3.

[0017] In one embodiment, the differentiation-inducing agent of the present invention induces differentiation from myoblasts to skeletal muscle type I cells in a subject when administered to the subject. In the present invention, R-spondin 3 itself may be used as the differentiation-inducing agent, or it may be used as a composition in combination with various pharmaceutically acceptable carriers (for example, isotonic agents, stabilizers, pH adjusters, antioxidants, solubilizers, viscosity enhancers, preservatives, etc.).

[0018] Examples of isotonic agents include sugars such as glucose, trehalose, lactose, fructose, mannitol, xylitol, and sorbitol; polyhydric alcohols such as glycerin, polyethylene glycol, and propylene glycol; and inorganic salts such as sodium chloride, potassium chloride, and calcium chloride.

[0019] Examples of chelating agents include edetates such as disodium edetate, disodium calcium edetate, trisodium edetate, tetrasodium edetate, and calcium edetate, as well as ethylenediaminetetraacetate, nitrilotriacetic acid or its salts, sodium hexametaphosphate, and citric acid.

[0020] Examples of stabilizers include sodium bisulfite.

[0021] Examples of pH adjusting agents include acids such as hydrochloric acid, carbonic acid, acetic acid, and citric acid, as well as alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates or bicarbonates such as sodium carbonate, alkali metal acetates such as sodium acetate, alkali metal citrates such as sodium citrate, and bases such as trometamol.

[0022] Examples of preservatives include sorbic acid, potassium sorbate, parahydroxybenzoic acid esters such as methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, and butyl parahydroxybenzoate, chlorhexidine gluconate, benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, and other quaternary ammonium salts, alkyl polyaminoethylglycine, chlorobutanol, polyquad, polyhexamethylene biguanide, and chlorhexidine.

[0023] Examples of antioxidants include sodium bisulfite, anhydrous sodium sulfite, sodium pyrosulfite, and concentrated mixed tocopherols.

[0024] Examples of solubilizers include sodium benzoate, glycerin, D-sorbitol, glucose, propylene glycol, hydroxypropyl methylcellulose, polyvinylpyrrolidone, macrogol, and D-mannitol. Examples of viscosity-concentrating agents include polyethylene glycol, methylcellulose, ethylcellulose, carmellose sodium, xanthan gum, chondroitin sulfate sodium, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, and polyvinyl alcohol.

[0025] In embodiments of the composition, the content of R-spondin 3 in the composition is not particularly limited and can be appropriately set from conditions such as 90% by mass or more, 70% by mass or more, 50% by mass or more, 30% by mass or more, 10% by mass or more, 5% by mass or more, 1% by mass or more, etc.

[0026] The formulation form is not particularly limited and can include various formulation forms such as oral preparations like capsules, tablets, pills, powders, granules, and syrups; and parenteral preparations such as injections (intramuscular injection, intravenous injection, local injection, etc.), mouthwashes, drips, topical preparations (ointments, creams, patches, inhalants), suppositories, and subcutaneously implantable capsules. Among the above formulation forms, preferred ones include injections (intramuscular injection, etc.) and subcutaneously implantable capsules.

[0027] Furthermore, the differentiation-inducing agent of the present invention may contain the R-spondin 3 protein, or it may contain a nucleic acid molecule that expresses the R-spondin 3 protein. Therefore, unless otherwise explicitly stated, in the present invention, "containing R-spondin 3" means both that the differentiation-inducing agent contains R-spondin 3 as a protein, and that it contains a nucleic acid molecule that expresses the R-spondin 3 protein.

[0028] Nucleic acid molecules that express the R-spondin 3 protein include nucleic acids (DNA, RNA, etc.) that encode the peptide of the present invention. These proteins or nucleic acid molecules may be incorporated into a suitable vehicle. For example, the differentiation-inducing agent of the present invention may contain the R-spondin 3 protein encapsulated in a vector such as a liposome. Alternatively, the differentiation-inducing agent of the present invention may contain the nucleic acid molecule that expresses the R-spondin 3 protein encapsulated in a vector such as a plasmid vector or a viral vector. In these embodiments, liposomes, plasmid vectors, viral vectors, etc., that are widely used in the pharmaceutical field can be used.

[0029] In this embodiment, the differentiation-inducing agent of the present invention is administered to a target such as a mammal. Examples of mammals include humans, monkeys, mice, rats, rabbits, cats, dogs, pigs, cattle, horses, and sheep.

[0030] In embodiments of the differentiation-inducing agent composition, the content of R-spondin 3 protein in the composition varies depending on the administration route, the patient's age, weight, symptoms, etc., and cannot be specified in general terms. However, it should be an amount that corresponds to a daily dose of approximately 0.1 to 5000 mg, more preferably 1 to 1000 mg, of peptide or nucleic acid for an adult (weighing 50 kg). If administered once a day, this amount should be contained in one formulation; if administered three times a day, one-third of this amount should be contained in one formulation.

[0031] Furthermore, in another embodiment, the differentiation-inducing agent of the present invention can also induce differentiation into skeletal muscle type I cells by adding it to cells such as stem cells, myoblasts, and myotubes in vitro. Accordingly, the present invention also provides a method for inducing differentiation into skeletal muscle type I cells, which includes the step of applying R-spondin 3 to cells such as stem cells, myoblasts, myotubes, and cultured cell lines (such as C2C12 cells, L6 cells, and Hu5 / KD3 cells, which are cultured skeletal muscle cells) in vitro.

[0032] Examples of stem cells include mesenchymal stem cells, satellite cells (skeletal muscle stem cells), iPS cells, and ES cells. Examples of myoblasts and myotubes include those differentiated from satellite cells. These cells may be known or produced according to known methods. Commercially available cells may also be used.

[0033] These cells may be isolated cells, cell aggregates, tissue fragments, or other forms. Examples of these cells include those derived from humans, mice, rats, guinea pigs, pigs, monkeys, dogs, cats, African clawed frogs, etc. The step of treating these cells with R-spondin 3 can be carried out, for example, by adding the R-spondin 3 protein or a nucleic acid molecule encoding it. The amount of R-spondin 3 to be added is not particularly limited, but can be set to, for example, 2-400 ng, preferably 100-400 ng, per 1 mL of solvent. Alternatively, R-spondin 3 may be added to achieve a final concentration of 0.0645-12.9 nM, preferably 3.23-12.9 nM, when added to the reaction system.

[0034] In the method of the present invention, the cells may be used in a state where they are placed in a solution such as a liquid medium. As the medium, any medium commonly used for culturing animal cells can be used as appropriate. Examples of such mediums include DMEM (Dulbecco's Modified Eagle Medium), RPMI (Roswell Park Memorial Institute) 1640 medium, MEM (Minimum Essential Medium), DMEM / F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12), and SkBM-2 basal medium. In addition, serum, buffers, antibiotics, amino acids, sodium pyruvate, inorganic salts, proteins that induce proliferation and differentiation such as insulin and FGF, vitamins, etc. may be added to the medium as appropriate.

[0035] Furthermore, in this step, the cells may be held in the presence of R-spondin 3. In this embodiment, the cells may be left undisturbed or shaken during the holding period. The holding time is not particularly limited, but can be set, for example, between 1 and 120 hours, preferably between 48 and 72 hours. The temperature in this step is also not limited, but can be set, for example, between 30 and 37.5°C, preferably between 37.0 and 37.2°C. Through this step, skeletal muscle type I cells are differentiated and proliferated from the cells. The method of the present invention may include a step of recovering the skeletal muscle type I cells differentiated and proliferated in the above step. The recovery step can use a wide range of methods used in the field of cell culture to which the present invention belongs. Methods used in the recovery step include centrifugation, filtration, washing, trypsin treatment, and centrifugation.

[0036] Pharmaceuticals As described above, in the present invention, R-spondin 3 exhibits a differentiation-inducing effect on skeletal muscle type I cells. Therefore, the differentiation-inducing agent for skeletal muscle type I cells of the present invention is useful for treating conditions that can be treated by inducing differentiation into skeletal muscle type I cells. Accordingly, in a preferred embodiment, the present invention provides a pharmaceutical agent comprising R-spondin 3 for treating conditions that can be treated by inducing differentiation into skeletal muscle type I cells. Conditions that can be treated by inducing differentiation into skeletal muscle type I cells include, for example, diseases that cause atrophy of skeletal muscle type I cells, such as disuse atrophy, diabetes, and damage to motor nerves that innervate type I cells. In addition, while both types of muscle cells decrease in cancer and chronic renal failure-related muscle loss (cachexia), at least type I cell atrophy can be prevented. Furthermore, conditions that can be treated by inducing differentiation into skeletal muscle type I cells include not only those diagnosed as diseases, but also conditions such as inactivity due to prolonged bed rest, immobilization due to cast fixation, and muscle atrophy due to prolonged stay in a weightless state. Furthermore, treatments that can be addressed by inducing differentiation into skeletal muscle type I cells include improving performance in sports requiring muscular endurance, such as marathons and long-distance swimming. Therefore, in the pharmaceutical invention of the present invention, "treatment" of conditions that can be addressed by inducing differentiation into skeletal muscle type I cells includes the prevention or treatment of the above-mentioned diseases, and the improvement of the above-mentioned conditions.

[0037] In the embodiments of the pharmaceutical, the active ingredient R-spondin 3, its method of use, dosage, target of administration, carrier, etc., are the same as those of the differentiation inducer described above. Furthermore, the pharmaceutical and differentiation inducer of the present invention may further contain components known to be useful in treating the above-mentioned diseases and conditions. Examples of components known to be useful in treating the above-mentioned diseases and conditions include myostatin inhibitors such as follistatin and its mimics, atrophy treatments such as myostatin antibodies and activin receptor II inhibitors, and diabetes treatments such as biguanides, DPP-4 inhibitors, and thiazolidinediones. In such embodiments, the pharmaceutical of the present invention may not only contain R-spondin 3 and components known to be useful in treating the above-mentioned diseases and conditions in a single formulation, but may also be a combination formulation containing R-spondin 3 and components known to be useful in treating the above-mentioned diseases and conditions in a single formulation.

[0038] Activators of the β-catenin pathway As described later, the inventors have found that R-spondin 3 induces differentiation into skeletal muscle type I cells through activation of the β-catenin pathway. Therefore, the present invention provides a β-catenin pathway activator containing R-spondin 3. In the embodiment of the β-catenin pathway activator, the active ingredient R-spondin 3, its method of use, dosage, target cells, carrier, etc., are the same as those of the differentiation inducer described above. The β-catenin pathway activator can also be used in vitro to activate the β-catenin pathway in target cells, etc., similar to the differentiation inducer.

[0039] Method for determining whether or not skeletal muscle cells are Type I cells. The inventors have found that R-spondin 3 is a Type I specific protein, and therefore, R-spondin 3 can be used as an indicator to determine whether or not a test cell is a Type I cell. Accordingly, the present invention provides a method for determining whether or not a test cell is a skeletal muscle Type I cell using R-spondin 3 as an indicator. The test cell may be either a skeletal muscle cell or a myotube cell. The method of the present invention can be carried out, for example, by measuring the gene expression level of R-spondin 3 in the test cell. In such embodiments, for example, the gene expression level of R-spondin 3 in positive control cells known to be Type I cells and / or negative control cells known to be Type II cells can also be measured and compared with the R-spondin 3 gene expression level in the test cell to determine whether or not the test cell is a Type I cell. Specifically, for example, if the R-spondin 3 gene expression level in the test cells is significantly higher than that in negative control cells known to be Type II cells (e.g., if there is a statistically significant difference, or if it is greater than a predetermined threshold), the test cells can be determined to be Type I cells. For example, if the R-spondin 3 gene expression level in the test cells is equal to or higher than that in positive control cells known to be Type I cells (e.g., if there is a statistically significant difference, or if it is greater than a predetermined threshold), the test cells can be determined to be Type I cells.

[0040] The present invention will be described more specifically below with reference to examples, but the present invention is not limited to these examples. [Examples]

[0041] Example 1 Classification of muscle fibers Figure 1 shows an overview of the muscle fiber classification and RNA extraction method used in this example. Specifically, the following steps were performed. Soleus muscle was extracted from 1.8-12 week old Myh7-CFP mice (Jackson Stock no. 016922), placed in 4% collagenase (Type I, Worthington, Lakewood, NJ, USA; the collagenase was dissolved in DMEM (Dulbecco's Modified Eagle Medium: high glucose GlutaMAX, Thermo Fisher Scientific, containing 1% anti-biotic) warmed at 37°C, and incubated with shaking for 1 hour. Using a Pasteur pipette coated with 2.5% BSA, the incubated muscle tissue was transferred to a petri dish containing DMEM + GlutaMAX + 1% AB, and then separated into individual muscle fibers. 3. Using a fluorescence microscope (Leica M165 FC), CFP fluorescence (blue) was classified as Type I fibers (CFP+), and all other fibers as Type II fibers (CFP-). 4. After collecting each muscle fiber, it was washed twice with PBS and then frozen with liquid nitrogen.

[0042] RNA extraction 1,500 μL of Trizol (ThermoScientific) was added, and muscle fibers were homogenized using a QIA shredder (Quiagen) (centrifugation at 13,000 × g, 2 min, room temperature). The pass-through fraction (suspension) was collected, and another 500 μL of Trizol was added to the QIA shredder, and the same procedure was repeated (second time). 2. The suspensions were collected and allowed to stand for 5 minutes. Then, 200 μL of chloroform was added, and the mixture was shaken vigorously for 15 seconds. After standing at room temperature for 3 minutes, the mixture was allowed to stand. Subsequently, it was centrifuged at 4°C, 12,000 xg for 15 minutes, and the upper aqueous layer was transferred to a 1.5 mL tube. 3,500 μL of isopropanol was added, vortexed, and allowed to stand at room temperature for 10 minutes. Then, the mixture was centrifuged at 4°C, 12,000 xg for 10 minutes (RNA precipitated). After washing with 4.1 mL of 70% ethanol, the RNA was centrifuged at 4°C, 7500 xg for 5 minutes, and the precipitated RNA was dissolved in RNA-free water to recover it.

[0043] Microarray (Figure 2) Total RNA was extracted from each muscle fiber (see RNA extraction). RNA concentration was analyzed using Nanodrop (ThermoScientific), and RNA purity was measured using an Agilent 2100 Bioanalyzer (reference values: absorbance (260 / 280) of 1.5 or higher, absorbance (260 / 230) of 1.0 or higher, RNA Integrity Number (RIN) of 7.0 or higher). The RNA extracted from each fiber was labeled using a single-color method, and DNA microarray analysis was performed (outsourced to DNA Chip Laboratory) (chip used: the Mouse Gene Arrays (Agilent), analysis software: Feature Extraction 11.5.1.1 (Agilent)). Gene expression levels in each sample were converted to log2 values ​​to normalize them, and the results were ranked in descending order of expression level in Type I fibers compared to Type II fibers. Among these, secreted proteins showing the highest expression ratio in Type I fibers compared to Type II fibers were searched for. The results are shown in the graph on the left of Figure 2. As shown in the graph in Figure 2, the expression ratio of the Rspo3 gene (Type I / Type II) was very high among the genes of Type I fibers.

[0044] Quantitative PCR Quantitative PCR was performed using the DyNAmo ColorFlash SYBR Green qPCR Kit (Thermo Fisher Scientific, MA, USA) on a 96-well PikoReal Real-Time PCR System according to the provided protocol. The results were corrected for the housekeeping gene TATA binding protein (Tbp). The primers used for quantitative PCR are listed below.

[0045] [Table 2]

[0046] The results are shown in Figure 2, the graph on the right.

[0047] Example 2 Verification of whether Rspo3 is secreted in muscle cells (Figures 3 and 4) 1. Soleus muscle from ICR mice was placed in DMEM containing 0.8% collagenase and incubated at 37°C for 2 hours. Single muscle fibers were then harvested from the muscle tissue. The muscle fibers were treated with Accutase (Innovative Cell Technologies, SAN, USA) for 10 minutes, then seeded in a petri dish and cultured for 7 days in growth medium (No glucose DMEM: containing 30% (v / v) FBS, 1% (v / v) GlutaMAX, 1% (v / v) chicken embryo extract, 10 ng / ml bFGF, 1% (v / v) penicillin-streptomycin) at 37°C and 5% CO2. Myoblasts derived from the proliferated satellite cells were placed in 12-well plates coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) in a 1.2 × 10⁶ well. 5 Seeds were seeded at a cell count of cells / well. 2. The following day, cells were transfected using Lipofectamine 3000 (Thermos Scientific) with either the pCAGGS-Rspo3 expression vector (the pCAGGS-Rspo3 vector was cloned by the inventors) or pCAGGS as a control group. *The information on the Rspo3 sequence cloned into pCAGGS is shown in Figure 3. 3. After proliferation, the cells were changed to differentiation medium (DMEM GlutaMAX, containing 5% horse serum and 1% penicillin-streptomycin). On day 3 of differentiation, the cells were harvested in a cell harvesting solution (50 mM Tris-HCl pH 7.5, 5 mM sodium pyrophosphate tetrabasic, 1 mM ethylenediaminetetraacetic acid pH 8.0, 1 mM sodium orthovanadate, 1% Nonidet P-40, 10 mM sodium fluoride, 150 mM sodium chloride, 10 mg / L leupeptin, 1 mM phenylmethylsulfonyl fluoride, 5 mg / mL aprotinin, 3 mM benzamidine, and 10 mM beta glycerophosphate). After sonication, the cells were centrifuged, and the supernatant was used as a cell suspension for expression analysis by Western blotting. *For the analysis of the culture medium, the serum-free DMEM medium was replaced on day 2 of differentiation, and the culture medium used for 24 hours was collected and concentrated by centrifugation. Centrifugal concentration was performed using the following protocol: The recovered Medium was centrifuged at 1,000Xg, 4°C for 15 minutes, and the supernatant was further centrifuged at 12,000Xg, 4°C for 35 minutes. The supernatant was then passed through a 0.22 μm filter (Millipore). 1,600 μL of the resulting solution was centrifuged and concentrated in an Amicon centrifugal concentration tube (Millipore) with a molecular weight cutoff of 3 kDa (2,900Xg, 180 min, 4°C). The solution remaining on the side that did not pass through the filter after centrifugation was collected and used for Western blotting. The results are shown in Figures 3 and 4. As shown in Figure 3, Rspo3 was detected not only in the cells but also in the culture supernatant of Rspo3-overexpressing cell samples transfected with the pCAGGS-Rspo3 expression vector. Therefore, it can be seen that myoblasts secrete Rspo3 extracellularly. Furthermore, as shown in Figure 4, Rspo3-overexpressing cells expressed MyHCI protein, a Type I fiber marker, at significantly higher levels than cells that did not overexpress Rspo3. Therefore, it can be seen that Rspo3-overexpressing cells were induced to differentiate into Type I fibers.

[0048] Example 3: Rspo3 addition experiment (Figures 5-10) 1. Soleus muscle was extracted from Myh7-CFP mice (Jackson Stock no. 016922), treated with 0.8% collagenase, and the muscle tissue was separated into single muscle fibers. After isolating the muscle fibers based on the presence or absence of CFP fluorescence (the method was the same as in Example 2), the fibers were cultured in growth medium for 6-7 days. 2. Cells 1.2 × 10 5 Cells were seeded in 12-well plates coated with Matrigel at a rate of 2 mL / well. The following day, recombinant mouse Rspo3 protein (R&D Systems, MN, USA; 4120-RS-025, sequence information shown in Figure 5 below) was added to the differentiation medium at a final concentration of 200 ng / mL (6.45 nM, dissolved in 200 ng / mL BSA) to induce differentiation. The control group received 200 ng / mL BSA. *When an inhibitor was added, XAV-939 (Chem Scene LLC, NJ, USA; 284028-89-3) 1.56 μg / mL (5 μM) was added simultaneously with Rspo3 or as a standalone inhibitor. The culture medium was changed daily. 3. Cells were harvested on day 3 of differentiation, and their expression levels were analyzed by Western blotting. *When separating the nuclear and cytoplasmic fractions, the NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Thermo Fisher Scientific) was used. Following the included protocol, the nuclear and cytoplasmic fractions were separated and then analyzed by Western blotting. The antibodies used for Western blotting are as follows.

[0049] [Table 3]

[0050] In addition to the above, we used a polyclonal antibody of Rspo3 manufactured by Cosmo Bio Inc. *The peptide sequence used to produce the antibody is shown below. A portion of the C-terminal amino acid sequence of mouse Rspo3 (CRARDKQQKSVSVSTVH) was used as the recognition site.

[0051] The results are shown in Figures 5-10. As shown in Figure 5, MyHCI protein expression, a Type I fiber marker, was significantly increased in myotubes differentiated in the presence of Rspo3 recombinant protein. As shown in Figure 6, when Myh7-CFP mouse skeletal muscle was isolated into Type I and Type II fibers, and their satellite cells were proliferated into myoblasts, and MyHCI protein expression was examined in myotubes differentiated in the presence of Rspo3 recombinant protein, MyHCI protein expression was significantly increased in the presence of Rspo3 regardless of the muscle fiber type from which the cells originated.

[0052] To elucidate the intracellular mechanism by which Rspo3 acts on skeletal muscle and induces MyHCI, we investigated the activation of the Wnt / β-catenin pathway, a known signaling pathway of Rspo3. Activation of the signaling pathway was verified by quantifying the amount of β-catenin in the cytoplasm and the amount of β-catenin translocated to the cell nucleus. As shown in Figure 7, regardless of fiber type, Rspo3 addition significantly increased the amount of β-catenin accumulated in the cytoplasm and the expression level of β-catenin in the cell nucleus, clearly demonstrating that Rspo3 activates the Wnt / β-catenin pathway.

[0053] We investigated the possibility that Rspo3 co-regulates other Wnt pathways besides the β-catenin pathway (Wnt / PCP pathway and Wnt / calcium pathway). Activation of the Wnt / PCP pathway was examined by JNK phosphorylation, and activation of the Wnt / calcium pathway was examined by NFATc1 expression levels. As shown in Figure 8, no increase in JNK phosphorylation was observed with the addition of Rspo3, indicating that the Wnt / PCP pathway is not involved. As shown in Figure 9, no change in NFATc1 expression levels was observed with the addition of Rspo3, clearly indicating that the Wnt / calcium pathway is not involved.

[0054] We used a Wnt / β-catenin pathway inhibitor (XAV939) to investigate whether inhibition of the Wnt / β-catenin pathway suppresses induction into Type I fibers. As shown in Figure 10, regardless of the originating muscle fiber type, inhibition of the Wnt / β-catenin pathway suppressed MHC I expression, and it became clear that Rspo3 was induced into Type I fibers via the Wnt / β-catenin pathway.

[0055] Example 4: Verification using live mice 1. Muscle damage was induced in anesthetized ICR mice by injecting 50 μL of 10 μM cardiotoxin (CTX, a muscle injury agent) into the tibialis anterior muscle. 2. Three days after CTX administration, 25 μL of 0.6 U / μL hyaluronidase was injected, followed by 25 μL of 2 μg / μL DNA (pCAGGS-Rspo3 or empty vector) (resulting in the administration of 50 μg of DNA). 3. Electroporation electrodes were inserted perpendicular to the long axis of the tibialis anterior muscle, and an electric current of 200 V / cm at 1 Hz was applied eight times. 4. Muscle tissue was collected 14 days after the onset of muscle injury, and expression levels were analyzed by Western blotting.

[0056] To investigate whether Rspo3 induces Type I fibers in vivo, we injected a muscle-damaging drug into mouse skeletal muscle to induce myoblast proliferation in the muscle tissue. Then, we overexpressed Rspo3 to verify whether Type I fibers were induced in the regenerated muscle.

[0057] As shown in Figure 11 (top), the tibialis anterior muscle of mice was injured with a muscle-injuring agent, and an Rspo3 expression vector was overexpressed in the muscle by electroporation on day 3 after injury. The muscle was harvested on day 14, when regeneration had progressed, and the expression level of MyHC I was quantified. As shown in Figure 11 (bottom), MyHC I expression was significantly increased in muscle tissue overexpressing Rspo3 after injury, demonstrating that Rspo3 induces Type I in vivo.

[0058] Although the above examples used mouse R-spondin 3, R-spondin 3 exhibits high amino acid sequence conservation across species (for example, mouse and human homology: 94% identical), so similar effects can be expected with R-spondin 3 from species other than mouse.

Claims

1. A differentiation-inducing agent for skeletal muscle Type I cells, containing R-spondin 3.

2. A differentiation-inducing agent according to claim 1, which is a differentiation-inducing agent used by adding it to cells in vitro, or an injectable or capsule preparation for administration to a living organism.

3. A method for determining whether test cells are skeletal muscle Type I cells or not, using R-spondin 3 as an indicator, comprising performing one or both of the following (1) and (2). （１） Step 1: Step to measure the gene expression level of R-spondin 3 in test cells. Step 2: A step to measure the gene expression level of R-spondin 3 in positive control cells known to be Type I cells. Step 3: If the R-spondin 3 gene expression level in the test cells measured in Step 1 is equal to or higher than the R-spondin 3 gene expression level in the positive control cells known to be Type I cells measured in Step 2, then the test cells are determined to be Type I cells. （２） Step 1: Step to measure the gene expression level of R-spondin 3 in test cells. Step 2: A step to measure the gene expression level of R-spondin 3 in negative control cells known to be Type II cells. Step 3: If the R-spondin 3 gene expression level in the test cells measured in Step 1 is significantly higher than the R-spondin 3 gene expression level in the negative control cells, which are known to be Type II cells and were measured in Step 2, then the test cells are determined to be Type I cells.

4. A pharmaceutical product containing R-spondin 3 for treating muscle injury.

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