Utilization of mesenchymal stem cells or culture supernatant thereof

Abstract

To provide a culture supernatant of mesenchymal stem cells or mesenchymal stem cells, containing components secreted from cells such as cytokines, useful for suppressing or improving disease; a pharmaceutical composition comprising mesenchymal stem cells; and a method for producing a pharmaceutical composition.SOLUTION: A culture supernatant of mesenchymal stem cells contains at least 500 pg / mL of G-CSF and at least 2700 pg / mL of IL-6. The culture supernatant preferably contains an imidazole dipeptide, and preferably further contains at least 670 pg / mL of TGF-β1, at least 3.5 pg / mL of IL-7, or at least 6500 pg / mL of IL-8.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The technical field of the present invention relates to mesenchymal stem cells or mesenchymal stem cell culture supernatants. [Background technology]

[0002] Mesenchymal stem cells (MSCs) and MSC culture supernatants are used in the field of regenerative medicine. MSC culture supernatants contain components secreted by cells, such as cytokines. MSC culture supernatants have the advantage of being cost-effective, as they can be administered to patients without the need for purification of the active ingredients.

[0003] Several studies have been reported on MSCs or MSC culture supernatants. For example, Patent Document 1 and Non-Patent Document 1 describe an investigation into the amount of cytokines in MSC culture supernatants (see the Examples in Patent Document 1 and the Abstract in Non-Patent Document 1). Patent Document 1 also describes that administration of an MSC cell suspension to a rat model of hind limb ischemia resulted in an increase in blood flow. Non-Patent Document 1 also describes that MSC culture supernatants have been shown to promote the repair of skin photodamage and promote vascular regeneration. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO2021 / 200744 [Non-patent literature]

[0005] [Non-Patent Document 1] "Cytoplasm or Supernatant: Where Is the Treasury of the Bioactive Antiaging Factor from Mesenchymal Stem Cells?" Luo et al., Stem Cells Dev. 2022 Sep;31(17-18):529-540. Summary of the Invention [Problem to be solved by the invention]

[0006] Considering the use of MSC culture supernatants for disease suppression or amelioration, there is room for improvement in the composition of conventional MSC culture supernatants.

[0007] Meanwhile, the present inventors conducted research into MSC culture methods. Surprisingly, they found that adding imidazole dipeptide to the culture medium during MSC culture yielded a culture supernatant useful for suppressing or ameliorating disease. Furthermore, the resulting cells were also useful for suppressing or ameliorating disease. Based on these findings, the present invention was completed. [Means for solving the problem]

[0008] According to one aspect of the present invention, there is provided a method for producing a culture supernatant, comprising the step of recovering a culture supernatant from a medium containing mesenchymal stem cells and an imidazole dipeptide. Using this method, a culture supernatant useful for suppressing or ameliorating a disease can be obtained. According to another aspect of the present invention, there is provided a method for producing cells, comprising the step of culturing mesenchymal stem cells in a medium containing an imidazole dipeptide to produce cultured cells. Using this method, it is possible to obtain cells useful for suppressing or ameliorating a disease.

[0009] According to another aspect of the present invention, there is provided a method for culturing cells, comprising the step of culturing mesenchymal stem cells in a serum-free medium containing an imidazole dipeptide to produce cultured cells. According to another aspect of the present invention, there is provided a medium for culturing mesenchymal stem cells, the medium containing an imidazole dipeptide and being serum-free. According to another aspect of the present invention, there is provided a method for inhibiting osteoblast production, comprising the step of culturing mesenchymal stem cells in a medium containing an imidazole dipeptide. According to another aspect of the present invention, there is provided a method for inhibiting osteoblast production, comprising the step of contacting mesenchymal stem cells with a culture supernatant obtained by culturing mesenchymal stem cells in a medium containing an imidazole dipeptide. According to another aspect of the present invention, there is provided a composition for inhibiting osteoblast production, comprising an imidazole dipeptide. According to another aspect of the present invention, there is provided a composition for inhibiting osteoblast production, comprising a culture supernatant obtained by culturing mesenchymal stem cells in a medium containing an imidazole dipeptide. Furthermore, another aspect of the present invention provides a method for promoting expression of G-CSF, MCP-1, VEGF-C, TGF-β1, IL-6, IL-7, IL-8, M-CSF, osteoprotegrin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, semaphorin 3B, or an exosome marker, the method comprising the step of culturing mesenchymal stem cells in a medium containing imidazole dipeptide. According to another aspect of the present invention, there is provided a composition for promoting the expression of G-CSF, MCP-1, VEGF-C, TGF-β1, IL-6, IL-7, IL-8, M-CSF, osteoprotegrin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, semaphorin 3B, or an exosome marker in mesenchymal stem cells, comprising an imidazole dipeptide. According to another aspect of the present invention, there is provided a method for promoting exosome secretion, comprising the step of culturing mesenchymal stem cells in a medium comprising an imidazole dipeptide. According to another aspect of the present invention, there is provided a composition for promoting exosome secretion from mesenchymal stem cells, comprising an imidazole dipeptide.According to another aspect of the present invention, there is provided a mesenchymal stem cell culture supernatant containing at least 500 pg / ml of G-CSF. According to another aspect of the present invention, there is provided a mesenchymal stem cell culture supernatant containing at least 2700 pg / ml of IL-6. According to another aspect of the present invention, there is provided an umbilical cord-derived mesenchymal stem cell that highly expresses or highly secretes G-CSF or IL-6. According to another aspect of the present invention, there is provided an IL-34-positive mesenchymal stem cell. According to another aspect of the present invention, there is provided an ADAM8-positive mesenchymal stem cell. According to another aspect of the present invention, there is provided a pharmaceutical composition for inhibiting bone differentiation, comprising cells obtained by culturing mesenchymal stem cells in a medium containing an imidazole dipeptide. According to another aspect of the present invention, there is provided a method for producing a pharmaceutical composition for inhibiting bone differentiation, comprising the step of culturing mesenchymal stem cells in a medium containing an imidazole dipeptide to produce cultured cells. According to another aspect of the present invention, there is provided a medium comprising an imidazole dipeptide and an LIF (leukemia inhibitory factor) component. According to another aspect of the present invention, a kit is provided which includes an imidazole dipeptide and an LIF component. [Brief explanation of the drawings]

[0010] [Figure 1] 1 shows micrographs of MSCs after culturing in carnosine-free medium. [Figure 2] 1 shows micrographs of MSCs after culturing in a medium containing 1 mM carnosine. [Figure 3] 1 shows micrographs of MSCs after culturing in a medium containing 10 mM carnosine. [Figure 4] 1 shows micrographs of MSCs after culturing in a medium containing 30 mM carnosine. [Figure 5] FIG. 1 shows the results of counting the number of MSC cells. [Figure 6] This figure shows the results of examining the amount of exosome markers in the culture supernatant. The four bar graphs shown for collection times 1 to 4 represent the results under conditions with 0 mM, 1 mM, 10 mM, and 30 mM carnosine added, from left to right. The same applies to Figures 7 to 14. [Figure 7] FIG. 1 shows the results of examining the amount of HGF in the culture supernatant. [Figure 8] FIG. 1 shows the results of examining the amount of G-CSF in the culture supernatant. [Figure 9] FIG. 1 shows the results of examining the amount of MCP-1 in the culture supernatant. [Figure 10] FIG. 1 shows the results of examining the amount of VEGF-C in the culture supernatant. [Figure 11] FIG. 1 shows the results of examining the amount of TGF-β1 in the culture supernatant. [Figure 12] This figure shows the results of examining the amount of IL-6 in the culture supernatant. "*>10,000" and "*>7,000" indicate that values ​​greater than the respective values ​​were the detection limit. [Figure 13] FIG. 1 shows the results of examining the amount of IL-7 in the culture supernatant. [Figure 14] FIG. 1 shows the results of examining the amount of IL-8 in the culture supernatant. [Figure 15] FIG. 1 shows the results of quantifying the amount of calcium deposition in bone marrow MSCs. [Figure 16] Alizarin Red S stained images of bone marrow MSCs. [Figure 17] FIG. 1 shows the results of quantifying the amount of calcium deposition during co-culture of bone marrow MSCs and umbilical cord MSCs. [Figure 18] 1 shows Alizarin Red S stained images of bone marrow MSCs and umbilical cord MSCs co-cultured. [Figure 19] This figure shows the culture conditions for umbilical cord MSCs. In the % increase calculation formula described in the Examples, all RPM values ​​are incremented by 1 before calculating each % increase. For example, even if the RPM value of M0 is 0, the % increase is calculated after incrementing by 1, so the % increase value for M0 is (M0 + 1) / (M0 + 1) = (0 + 1) / (0 + 1), which is 1. [Figure 20]Figures 20(a) to 20(j) show the changes in the % increase values ​​of 10 genes, respectively: gremlin 1, DAN family BMP antagonist, KIT ligand, R-spondin 2, semaphorin 3B, fibroblast growth factor 11, TNF receptor superfamily member 11b, ADAM metallopeptidase domain 8, interleukin 34, insulin-like growth factor binding protein 2, and Dickkopf WNT signaling pathway inhibitor 1. [Figure 21] FIG. 1 shows the RPM values ​​of interleukin 34 expression levels in umbilical cord MSCs. [Figure 22] FIG. 10 shows the RPM values ​​of ADAM metallopeptidase domain 8 expression levels in umbilical cord MSCs. [Figure 23] FIG. 1 shows the results of ELISA analysis of osteoprotegerin. [Figure 24] FIG. 1 shows the results of ELISA analysis of osteoprotegerin. [Figure 25] FIG. 1 shows the results of ELISA analysis of osteoprotegerin. [Figure 26] FIG. 1 shows the results of ELISA analysis of M-CSF. [Figure 27] FIG. 1 shows the results of ELISA analysis of M-CSF. [Figure 28] FIG. 1 shows the results of ELISA analysis of M-CSF. [Figure 29] FIG. 1 shows the results of qPCR analysis of osteoprotegerin. [Figure 30] FIG. 1 shows the results of qPCR analysis of M-CSF. [Figure 31]31 shows the results of ELISA analysis of osteoprotegerin, with (a) carnosine-added condition and (b) anserine-added condition. [Figure 32] These are the results of qPCR analysis of osteoprotegin. Figure 32(a) shows the results for UC_B_C, Figure 32(b) shows the results for BM_B_C, and Figure 32(c) shows the results for BM_C_C. [Figure 33] These are the results of qPCR analysis of osteoprotegin. Figure 33(a) shows the results for UC_B_A, Figure 33(b) shows the results for BM_B_A, and Figure 33(c) shows the results for BM_C_A. [Figure 34] 34(a) shows the results of ELISA analysis of M-CSF, with carnosine added and anserine added, respectively. [Figure 35] 35(a) and 35(b) show the results of qPCR analysis of M-CSF, under UC_B_C and BM_B_C conditions, respectively. [Figure 36] 36(a) and 36(b) show the results of qPCR analysis of M-CSF, under UC_B_A and BM_B_A conditions, respectively. [Figure 37] FIG. 1 shows the results of cell counting in groups 1 and 2. [Figure 38] FIG. 1 shows the results of cell counting in groups 3 and 4. [Figure 39] FIG. 1 shows the results of measuring the fluorescent area of ​​phalloidin in groups 1 to 4. [Figure 40] FIG. 1 shows the results of measuring the fluorescent area of ​​CD44 in groups 1 to 4. [Figure 41] FIG. 1 shows the results of capturing fluorescent images (cell nucleus staining) of groups 1 and 3. [Figure 42] FIG. 1 shows the results of capturing fluorescent images (phalloidin staining) of groups 1 and 3. [Figure 43] FIG. 1 shows the results of capturing fluorescent images (CD44 staining) of Groups 1 and 3. [Figure 44] FIG. 1 shows the results of cell counting in groups 5 and 6. [Figure 45]FIG. 1 shows the results of cell counting in groups 7 and 8. [Figure 46] FIG. 1 shows the results of measuring the fluorescent area of ​​phalloidin in groups 5 and 6. [Figure 47] FIG. 1 shows the results of measuring the fluorescence area of ​​phalloidin in groups 7 and 8. [Figure 48] FIG. 1 shows the results of measuring the CD44 fluorescence area in groups 5 and 6. [Figure 49] FIG. 1 shows the results of measuring the CD44 fluorescence area in groups 7 and 8. [Figure 50] FIG. 10 shows the results of capturing fluorescent images (phalloidin staining) of groups 5 and 7. [Figure 51] FIG. 1 shows the results of capturing fluorescent images (CD44 staining) of groups 5 and 7. [Figure 52] FIG. 10 shows the results of measuring the positive rate of MSC markers after culturing umbilical cord MSCs in MSC medium D. [Figure 53] FIG. 1 shows the results of cell counting for groups 9 and 10. [Figure 54] FIG. 1 shows the results of measuring the fluorescence area of ​​phalloidin in groups 9 and 10. [Figure 55] FIG. 10 shows the results of capturing fluorescent images (phalloidin staining) of Groups 9 and 10. DETAILED DESCRIPTION OF THE INVENTION

[0011] Hereinafter, embodiments of the present invention will be described in detail, with the same contents omitted as appropriate to avoid repetition.

[0012] (1) Method According to one embodiment of the present invention, a method is provided that includes a step of contacting mesenchymal stem cells (MSCs) with an imidazole dipeptide. This method can be used to obtain a culture supernatant useful for suppressing or ameliorating a disease (e.g., a culture supernatant containing a high concentration of at least one cytokine useful for suppressing or ameliorating a disease, or a culture supernatant useful for inhibiting bone differentiation). This method may include, for example, culturing MSCs in a medium containing an imidazole dipeptide to produce cultured MSCs, or recovering the culture supernatant from the medium containing the cultured MSCs. This method includes, for example, a method for producing or culturing cells, a method for producing a culture supernatant, a method for producing a pharmaceutical composition, a method for producing a cytokine-containing composition, a method for promoting cytokine or exosome secretion, a method for inhibiting osteoblast production, or a method for inhibiting bone differentiation. Furthermore, according to one embodiment of the present invention, there are provided cells, culture supernatants, pharmaceutical compositions, cytokine-containing compositions, or containers containing any of these obtained using this method.

[0013] (2) Method One embodiment of the present invention provides a method for producing a culture supernatant, comprising recovering the culture supernatant from a medium containing MSCs and an imidazole dipeptide. This method can be used to obtain a culture supernatant useful for suppressing or ameliorating a disease (e.g., a culture supernatant containing a high concentration of at least one cytokine useful for suppressing or ameliorating a disease, or a culture supernatant useful for suppressing bone differentiation).

[0014] (3) Method According to one embodiment of the present invention, a cell culture method or production method is provided, which includes a step of culturing MSCs in a medium containing imidazole dipeptide to produce cultured cells. This method can produce MSCs useful for suppressing or ameliorating disease (e.g., MSCs secreting high concentrations of at least one cytokine useful for suppressing or ameliorating disease, or MSCs useful for inhibiting bone differentiation). Furthermore, this method can produce safe MSCs by culturing MSCs in a serum-free, serum albumin-free, insulin-free, IGF (insulin-like growth factor)-free (e.g., IGF-1-free), FGF (fibroblast growth factor)-free (e.g., FGF-1 or -2-free), growth factor-free, cytokine-free, or protein-free medium. By recovering a culture supernatant from the medium containing the cells obtained by this method, a culture supernatant useful for suppressing or ameliorating disease (e.g., a culture supernatant containing high concentrations of at least one cytokine useful for suppressing or ameliorating disease, or a culture supernatant useful for inhibiting bone differentiation) can be obtained.

[0015] (4) Culture supernatant One embodiment of the present invention provides a culture supernatant obtained by culturing MSCs in a medium containing an imidazole dipeptide. Use of this culture supernatant can provide, for example, the effects of MSC culture supernatant or effects useful for suppressing or ameliorating a disease (e.g., the effects of containing at least one cytokine at high concentrations useful for suppressing or ameliorating a disease, or the effect of suppressing bone differentiation).

[0016] (5) Method One embodiment of the present invention provides a method for suppressing a disease, comprising administering to a subject a culture supernatant obtained by culturing MSCs in a medium containing an imidazole dipeptide. This method can be used to suppress or ameliorate bone differentiation (e.g., suppress or ameliorate a disease associated with bone differentiation). Another aspect provides use of a pharmaceutical composition comprising a culture supernatant obtained by culturing MSCs in a medium containing an imidazole dipeptide in the manufacture of a pharmaceutical composition for suppressing a disease.

[0017] (6) Composition According to one embodiment of the present invention, a pharmaceutical composition is provided, comprising a culture supernatant obtained by culturing MSCs in a medium containing an imidazole dipeptide. Use of this composition can suppress or ameliorate bone differentiation (e.g., suppress or ameliorate diseases associated with bone differentiation). In another aspect, a pharmaceutical composition for use in suppressing disease is provided, comprising a culture supernatant obtained by culturing MSCs in a medium containing an imidazole dipeptide.

[0018] (7) Method One embodiment of the present invention provides a method for suppressing a disease, comprising administering to a subject cells obtained by culturing MSCs in a medium containing an imidazole dipeptide. This method can be used to suppress or ameliorate bone differentiation (e.g., suppress or ameliorate diseases associated with bone differentiation). Another aspect provides use of a pharmaceutical composition comprising cells obtained by culturing MSCs in a medium containing an imidazole dipeptide in the manufacture of a pharmaceutical composition for suppressing a disease.

[0019] (8) Composition According to one embodiment of the present invention, a pharmaceutical composition is provided comprising cells obtained by culturing MSCs in a medium containing an imidazole dipeptide. This composition can be used to inhibit or ameliorate bone differentiation (e.g., inhibit or ameliorate diseases associated with bone differentiation). Another aspect provides a pharmaceutical composition for use in inhibiting a disease, comprising cells obtained by culturing MSCs in a medium containing an imidazole dipeptide.

[0020] (9) Method According to one embodiment of the present invention, there is provided a method for inhibiting bone differentiation of cells in a subject, comprising the step of administering to the subject a culture supernatant obtained by culturing MSCs in a medium containing an imidazole dipeptide. This method can be used to inhibit bone differentiation of cells in the subject. According to another aspect, there is provided use of a pharmaceutical composition comprising a culture supernatant obtained by culturing MSCs in a medium containing an imidazole dipeptide in the manufacture of a pharmaceutical composition for inhibiting bone differentiation of cells in a subject. According to another aspect, there is provided a pharmaceutical composition comprising a culture supernatant obtained by culturing MSCs in a medium containing an imidazole dipeptide, for use in inhibiting bone differentiation of cells in a subject.

[0021] (10) Method According to one embodiment of the present invention, there is provided a method for inhibiting bone differentiation of cells in a subject, comprising the step of administering to the subject cells obtained by culturing MSCs in a medium containing imidazole dipeptide. This method can be used to inhibit bone differentiation of cells in the subject. According to another aspect, there is provided use of a pharmaceutical composition comprising cells obtained by culturing MSCs in a medium containing imidazole dipeptide in the manufacture of a pharmaceutical composition for inhibiting bone differentiation of cells in a subject. According to another aspect, there is provided a pharmaceutical composition comprising cells obtained by culturing MSCs in a medium containing imidazole dipeptide, for use in inhibiting bone differentiation of cells in a subject.

[0022] (11) Culture medium According to one embodiment of the present invention, there is provided a medium for culturing MSCs, which contains imidazole dipeptide and is serum-free, serum albumin-free, insulin-free, IGF-free, FGF-free, growth factor-free, cytokine-free, or protein-free. Culturing MSCs using this medium can yield cells useful for suppressing or ameliorating disease (e.g., cells secreting high concentrations of at least one cytokine useful for suppressing or ameliorating disease, or cells useful for inhibiting bone differentiation). Alternatively, a culture supernatant useful for suppressing or ameliorating disease (e.g., a culture supernatant containing high concentrations of at least one cytokine useful for suppressing or ameliorating disease, or a culture supernatant useful for inhibiting bone differentiation) can be obtained. Culturing MSCs in this medium can yield MSCs or culture supernatants with excellent safety.

[0023] (12) Method According to one embodiment of the present invention, there is provided a method for inhibiting osteoblast formation, comprising the step of contacting MSCs with an imidazole dipeptide. This method can be used to inhibit osteoblast formation. According to another aspect, there is provided use of a composition comprising an imidazole dipeptide in the manufacture of a composition for inhibiting osteoblast formation. According to another aspect, there is provided a method for inhibiting osteoblast formation, comprising the step of administering an imidazole dipeptide to a subject.

[0024] (13) Method According to one embodiment of the present invention, there is provided a method for inhibiting osteoblast formation, which comprises culturing MSCs in a medium containing an imidazole dipeptide.

[0025] (14) Composition According to one embodiment of the present invention, there is provided a composition for inhibiting osteoblast formation, comprising an imidazole dipeptide. When this composition is contacted with MSCs, osteoblast formation can be inhibited. According to another aspect, there is provided a composition for use in inhibiting osteoblast formation, comprising an imidazole dipeptide.

[0026] (15) Method According to one embodiment of the present invention, there is provided a method for inhibiting osteoblast formation, comprising the step of contacting MSCs with a culture supernatant or MSC secretion product obtained by culturing MSCs in a medium containing an imidazole dipeptide. This method can be used to inhibit osteoblast formation. According to another aspect, there is provided use of a composition comprising a culture supernatant or MSC secretion product obtained by culturing MSCs in a medium containing an imidazole dipeptide in the manufacture of a composition for inhibiting osteoblast formation. According to another aspect, there is provided a method for inhibiting osteoblast formation, comprising the step of administering to a subject a culture supernatant or MSC secretion product obtained by culturing MSCs in a medium containing an imidazole dipeptide.

[0027] (16) Composition According to one embodiment of the present invention, there is provided a composition for inhibiting osteoblast formation, comprising a culture supernatant or an MSC secretion product obtained by culturing MSCs in a medium containing an imidazole dipeptide. Contacting this composition with MSCs can inhibit osteoblast formation. According to another aspect, there is provided a composition for use in inhibiting osteoblast formation, comprising a culture supernatant or an MSC secretion product obtained by culturing MSCs in a medium containing an imidazole dipeptide.

[0028] (17) Method According to one embodiment of the present invention, there is provided a method for promoting expression of G-CSF (granulocyte colony-stimulating factor), MCP-1 (monocyte chemotactic protein), VEGF-C (vascular endothelial growth factor-C), TGF-β1 (transforming growth factor beta 1), IL-6 (interleukin 6), IL-7 (interleukin 7), IL-8 (interleukin 8), M-CSF, osteoprotegin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, semaphorin 3B, or an exosome marker, the method comprising the step of culturing MSCs in a medium containing an imidazole dipeptide. This method can promote the expression or secretion of G-CSF, MCP-1, VEGF-C, TGF-β1, IL-6, IL-7, IL-8, M-CSF, osteoprotegrin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, semaphorin 3B, or exosome markers from MSCs.

[0029] (18) Method One embodiment of the present invention provides a method for promoting the expression of G-CSF, MCP-1, VEGF-C, TGF-β1, IL-6, IL-7, IL-8, M-CSF, osteoprotegerin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, semaphorin 3B, or an exosome marker, comprising contacting MSCs with an imidazole dipeptide. This method can promote the expression or secretion of G-CSF, MCP-1, VEGF-C, TGF-β1, IL-6, IL-7, IL-8, or an exosome marker from MSCs. In another aspect, there is provided use of a composition comprising an imidazole dipeptide in the manufacture of a composition for promoting expression of G-CSF, MCP-1, VEGF-C, TGF-β1, IL-6, IL-7, IL-8, M-CSF, osteoprotegrin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, semaphorin 3B, or an exosome marker in MSCs. In another aspect, there is provided a method for promoting expression of G-CSF, MCP-1, VEGF-C, TGF-β1, IL-6, IL-7, IL-8, M-CSF, osteoprotegrin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, semaphorin 3B, or an exosome marker in MSCs, the method comprising the step of administering an imidazole dipeptide to a subject.

[0030] (19) Composition One embodiment of the present invention provides a composition for promoting the expression of G-CSF, MCP-1, VEGF-C, TGF-β1, IL-6, IL-7, IL-8, M-CSF, osteoprotegin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, semaphorin 3B, or an exosome marker in MSCs. Contacting this composition with MSCs can promote the expression or secretion of G-CSF, MCP-1, VEGF-C, TGF-β1, IL-6, IL-7, IL-8, M-CSF, osteoprotegin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, semaphorin 3B, or an exosome marker from MSCs.

[0031] (20) Method One embodiment of the present invention provides a method for promoting exosome secretion, comprising culturing MSCs in a medium containing an imidazole dipeptide. This method can promote exosome secretion from MSCs.

[0032] (21) Method According to one embodiment of the present invention, there is provided a method for promoting exosome secretion, comprising the step of contacting MSCs with an imidazole dipeptide. This method can be used to promote exosome secretion from MSCs. According to another aspect, there is provided use of a composition comprising an imidazole dipeptide in the manufacture of a composition for promoting exosome secretion from MSCs. According to another aspect, there is provided a method for promoting exosome secretion, comprising the step of administering an imidazole dipeptide to a subject.

[0033] (22) Composition One embodiment of the present invention provides a composition for promoting exosome secretion from MSCs, comprising an imidazole dipeptide. Contacting this composition with MSCs can promote exosome secretion from MSCs.

[0034] (23) Method According to one embodiment of the present invention, there is provided a method for producing a composition, comprising the step of recovering a culture supernatant from a medium containing MSCs and an imidazole dipeptide. The composition may be a culture supernatant or a cytokine-containing composition. Use of this composition can provide the effects of MSC culture supernatant or effects useful for suppressing or ameliorating a disease (e.g., the effects of at least one or more cytokines at high concentrations useful for suppressing or ameliorating a disease, or the effect of suppressing bone differentiation).

[0035] (24) Method One embodiment of the present invention provides a method for producing a composition containing a secretion product of MSCs, comprising the step of contacting MSCs with an imidazole dipeptide. The composition can be used to obtain the effects of the secretion product of MSCs or effects useful for suppressing or ameliorating a disease (e.g., the effects of a high concentration of at least one cytokine useful for suppressing or ameliorating a disease, or the effect of suppressing bone differentiation).

[0036] (25) Culture supernatant According to one embodiment of the present invention, there is provided a MSC culture supernatant containing a protein. The culture supernatant may be, for example, an MSC culture supernatant containing at least 500 pg / ml of G-CSF. Use of this culture supernatant can achieve the disease-suppressing or ameliorating effect of a high concentration of G-CSF. The culture supernatant may be, for example, an MSC culture supernatant containing at least 2700 pg / ml of IL-6. Use of this culture supernatant can achieve the disease-suppressing or ameliorating effect of a high concentration of IL-6.

[0037] (26) Cell According to one embodiment of the present invention, MSCs (preferably umbilical cord-derived MSCs) that highly express or secrete G-CSF or IL-6 are provided. The use of this culture supernatant provides a disease-suppressing or ameliorating effect due to high concentrations of IL-6. The use of these cells provides a disease-suppressing or ameliorating effect due to high concentrations of G-CSF or IL-6. Alternatively, a culture supernatant containing high concentrations of G-CSF or IL-6 can be obtained by recovering a culture supernatant from a medium containing these cells. According to another aspect, a composition is provided that contains an MSC cell population, wherein 60% or more of the cells in the composition are MSCs (preferably umbilical cord-derived MSCs) that highly express or secrete G-CSF or IL-6. The use of this composition provides a disease-suppressing or ameliorating effect due to high concentrations of G-CSF or IL-6. The recovery of a culture supernatant from this composition provides a culture supernatant containing high concentrations of G-CSF or IL-6.

[0038] (27) Cell According to one embodiment of the present invention, there are provided MSCs that are positive for G-CSF, MCP-1, VEGF-C, TGF-β1, IL-6, IL-7, IL-8, M-CSF, osteoprotegin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, or semaphorin 3B. These MSCs may be purified or isolated. According to another aspect, there is provided a composition comprising purified or isolated MSCs. According to another aspect, there is provided MSCs that exhibit elevated expression of G-CSF, MCP-1, VEGF-C, TGF-β1, IL-6, IL-7, IL-8, M-CSF, osteoprotegin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, or semaphorin 3B. The increased expression may be, for example, an increase compared to MSCs cultured in the absence of imidazole dipeptide. In another aspect, a pharmaceutical composition comprising the MSCs is provided. In another aspect, a method for treating a disease is provided, comprising administering the MSCs to a subject. Positivity can be assessed, for example, by immunoassay (e.g., ELISA) or PCR (e.g., quantitative PCR).

[0039] (28) Cell According to one embodiment of the present invention, IL-34-positive or ADAM8-positive MSCs are provided. These MSCs are particularly suitable for use in, for example, inhibiting bone differentiation. These MSCs may be purified or isolated. According to another aspect, a composition comprising purified or isolated MSCs is provided. According to another aspect, a pharmaceutical composition comprising these MSCs is provided. According to another aspect, a method for treating a disease is provided, comprising the step of administering these MSCs to a subject.

[0040] (29) Method According to one embodiment of the present invention, a method for modifying the properties of a medium during MSC culture to promote secretion of G-CSF, MCP-1, VEGF-C, TGF-β1, IL-6, IL-7, IL-8, M-CSF, osteoprotegrin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, semaphorin 3B, or exosome markers is provided. This method can be used to prepare a medium suitable for producing a culture supernatant or MSCs useful for suppressing or ameliorating disease. According to another aspect, according to one embodiment of the present invention, a method for preparing a medium is provided, comprising the step of adding an imidazole dipeptide to the medium.

[0041] (30) Culture medium According to one embodiment of the present invention, there is provided a medium containing an imidazole dipeptide and an LIF component, which can promote the proliferation of MSCs.

[0042] (31) Method According to one embodiment of the present invention, a method for producing cells or a culture supernatant is provided, which comprises culturing MSCs in a medium containing an imidazole dipeptide and an LIF component to produce cultured cells. The resulting cells can be used to obtain the effect of suppressing bone differentiation. The resulting culture supernatant can be used to obtain the effects of MSC culture supernatant or effects useful for suppressing or ameliorating disease (e.g., the effect of containing at least one or more cytokines at high concentrations that are useful for suppressing or ameliorating disease, or the effect of suppressing bone differentiation).

[0043] (32) Kit According to one embodiment of the present invention, a kit is provided that includes an imidazole dipeptide and an LIF component. This kit may be, for example, a kit for preparing a medium (e.g., a medium for MSC culture). The components included in this kit can be mixed to prepare a medium. The obtained medium can be used to promote MSC cell proliferation. In another aspect, a method for preparing a medium is provided, which includes a step of mixing an imidazole dipeptide and an LIF component.

[0044] (33) Culture medium According to one embodiment of the present invention, there is provided a medium comprising an imidazole dipeptide and a laminin fragment or a variant thereof, which can promote the proliferation of MSCs.

[0045] (34) Method According to one embodiment of the present invention, there is provided a method for producing cells or a culture supernatant, comprising culturing MSCs in a medium containing an imidazole dipeptide and a laminin fragment or a modified form thereof to produce cultured cells. The resulting cells can be used to obtain the effect of suppressing bone differentiation. The resulting culture supernatant can be used to obtain the effects of MSC culture supernatant or effects useful for suppressing or ameliorating disease (e.g., the effect of containing at least one or more cytokines at high concentrations that are useful for suppressing or ameliorating disease, or the effect of suppressing bone differentiation).

[0046] (35) Kit According to one embodiment of the present invention, a kit is provided comprising an imidazole dipeptide and a laminin fragment or a variant thereof. This kit may be, for example, a kit for preparing a medium (e.g., a medium for MSC culture). The components contained in this kit can be mixed to prepare a medium. The obtained medium can be used to promote MSC cell proliferation. In another aspect, a method for preparing a medium is provided, comprising the step of mixing an imidazole dipeptide and LIF.

[0047] (36) Method According to one embodiment of the present invention, there is provided a method for producing a cytokine-containing composition, comprising the step of processing a culture supernatant (e.g., containing the above-mentioned (4) or (25)) or composition (e.g., containing the above-mentioned (6), (8), (14), (16), (19), or (22)) according to an embodiment of the present invention. The composition obtained by this method can be used to obtain the effects of MSC culture supernatant, the effects of at least one or more cytokines at high concentrations that are useful for suppressing or ameliorating a disease, or the effect of suppressing bone differentiation. The processing may be, for example, formulation or filling into a container.

[0048] (37) Composition According to one embodiment of the present invention, there is provided a cytokine-containing composition obtained by carrying out a method according to any of the embodiments of the present invention (e.g., including the above-mentioned (1) to (3), (5), (7), (9), (10), (12), (13), (15), (17), (18), (20), (21), (23), (24), (29), (31), (34), or (36)). Use of this composition can provide the effects of MSC culture supernatant, the effects of at least one or more cytokines at high concentrations that are useful for suppressing or ameliorating disease, or the effect of suppressing bone differentiation.

[0049] (38) Cell According to one embodiment of the present invention, there is provided a cell obtained by carrying out a method according to an embodiment of the present invention (including, for example, the above-mentioned (1) to (3), (31), or (34)). The cell is capable of secreting at high concentrations at least one or more cytokines useful for suppressing or ameliorating a disease. The cell can be used to suppress or ameliorate a disease. By recovering a culture supernatant from a medium containing the cell, a culture supernatant containing at high concentrations at least one or more cytokines useful for suppressing or ameliorating a disease, or a culture supernatant useful for suppressing bone differentiation, can be obtained.

[0050] (39) Cell According to one embodiment of the present invention, there is provided a cell population comprising the cells of any of the embodiments of the present invention (e.g., including the above (26) to (28) or (38)). Use of these cells can achieve the effect of suppressing or ameliorating a disease or the effect of suppressing bone differentiation due to high concentrations of G-CSF or IL-6. Alternatively, recovery of a culture supernatant from a medium containing these cells can provide a culture supernatant containing high concentrations of G-CSF or IL-6, or a culture supernatant useful for suppressing or ameliorating a disease. In another aspect, there is provided a composition comprising a cell population comprising the cells of the above (25) or (28). Use of this composition can achieve the effect of suppressing or ameliorating a disease or the effect of suppressing bone differentiation due to high concentrations of G-CSF or IL-6. Alternatively, recovery of a culture supernatant from a medium containing this composition can provide a culture supernatant containing high concentrations of G-CSF or IL-6, or a culture supernatant useful for suppressing or ameliorating a disease.

[0051] (40) Method According to one embodiment of the present invention, there is provided a method for treating a disease, comprising the step of administering to a subject a supernatant (e.g., comprising (4) or (25) above) or cells (e.g., comprising (26) to (28) or (38)) according to an embodiment of the present invention. The subject to be treated may be, for example, a patient in need of inhibition of bone differentiation. According to another aspect, there is provided a pharmaceutical composition for use in treating a disease, comprising the supernatant or cells according to an embodiment of the present invention. According to another aspect, there is provided use of the supernatant or cells according to an embodiment of the present invention for producing a pharmaceutical composition for treating a disease.

[0052] (41) Container According to one embodiment of the present invention, there is provided a container containing a culture supernatant (e.g., containing the above-mentioned (4) or (25)) or a composition (e.g., containing the above-mentioned (6), (8), (14), (16), (19), (22), or (37)) according to an embodiment of the present invention. Use of this container can provide the effects of an MSC culture supernatant, the effects of at least one or more cytokines at high concentrations that are useful for suppressing or ameliorating a disease, or the effect of suppressing bone differentiation.

[0053] The methods of the present invention (e.g., methods (1) to (3), (5), (7), (9), (10), (12), (13), (15), (17), (18), (20), (21), (23), (24), (29), (31), (34), (36), or (40) above) may include one or more of the following steps (i) to (ii): (i) culturing MSCs in a medium containing an imidazole dipeptide to produce cultured MSCs, or (ii) recovering a culture supernatant from the cultured MSCs and the medium containing an imidazole dipeptide. Furthermore, the method according to the embodiment of the present invention (e.g., the method according to the above (1) to (3), (5), (7), (9), (10), (12), (13), (15), (17), (18), (20), (21), (23), (24), (29), (31), (34), (36), or (40)) may include one or more of the following steps (iii) to (xvi):(iii) suspending MSCs in a medium and seeding the resulting cell suspension in a culture vessel; (iv) adding a medium containing an adhesion factor to the medium after seeding and culturing MSCs; (v) mixing the imidazole dipeptide and medium components; (vi) replacing the medium in which MSCs were cultured with a medium containing the imidazole dipeptide; (vii) culturing MSCs in a medium containing the imidazole dipeptide; (viii) culturing MSCs by adhesion culture to proliferate the cells; (ix) centrifuging or filtering the cultured MSCs and the medium containing the imidazole dipeptide; (x) recovering the culture supernatant or the cultured MSCs; and (xi) recovering G-CSF, MCP-1, VEGF-C, TGF-β1, IL-6, IL-7, IL-8, M-CSF, osteoprotegin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34 in the culture supernatant. (xii) measuring the amount of IGFBP2, DKK1, semaphorin 3B, or exosome markers; (xiii) formulating the culture supernatant or cultured MSCs; (xiv) administering a therapeutically effective amount of the culture supernatant or cultured MSCs to a subject; (xv) transferring the culture supernatant or cultured MSCs into a medical container; or (xvi) transferring the culture supernatant or cultured MSCs from the medical container to a syringe, vial, or medical bag. Using one or more of these steps is useful for obtaining a culture supernatant containing high concentrations of at least one cytokine useful for suppressing or ameliorating disease, or a culture supernatant or MSCs useful for suppressing bone differentiation. When two or more steps are used, the order can be determined according to the desired procedure.

[0054] In embodiments of the present invention (including, for example, (1) to (41) above), MSCs include, for example, umbilical cord-derived MSCs, adipose-derived MSCs, bone marrow-derived MSCs, placenta-derived MSCs, or umbilical cord blood-derived MSCs. MSCs may be mammalian MSCs, preferably human MSCs. Mammals include, for example, humans, monkeys, rodents (e.g., mice, hamsters), rabbits, dogs, cats, horses, cows, sheep, pigs, goats, marmosets, and the like. MSCs may exist as a cell population. Unless otherwise specified, references to MSCs herein include MSCs existing as a cell population. A cell population includes a plurality of cells generated by cell division. The proportion of MSCs of embodiments of the present invention (including, for example, (25) or (28) above) in a cell population may be, for example, 30, 40, 50, 60, 70, 80, 90, 100%, or more, or may be within a range of any two of these values. MSCs include MSCs before culturing using the above-described method and MSCs obtained by culturing using the above-described method. MSCs include MSCs cultured in a medium containing imidazole dipeptide. MSCs include HLA-ABC-positive MSCs, CD105-positive MSCs, HLA-ABC-negative MSCs, and CD105-negative MSCs. MSCs include MSCs that are CD44, CD73, CD90, or CD105-positive, or CD45, CD34, CD31, or HLA-DR-negative. MSCs may be isolated or purified MSCs. Isolated or purified MSCs include, for example, a cell population that is substantially free of cells other than MSCs, or MSCs in a preparation from which impurities have been removed. Impurities may include, for example, medium components, and removal may include, for example, partial removal.

[0055] In an embodiment of the present invention (including, for example, the above (1) to (41)), MSCs that highly express or secrete G-CSF or IL-6 have a cell density of at least 5.56 × 10 when cultured for 72 hours in a medium supplemented with carnosine (for example, 10 mM). -3 pg / cell of G-CSF-secreting cells or at least 3 x 10 -2The amount of G-CSF secreted may be, for example, at least 5.56 × 10 -3 , 6×10 -3 , 8×10 -3 , 1×10 -2 , 1.2 × 10 -2 , 1.7×10 -2 , 2 × 10 -2 , 3×10 -2 , 1×10 -1 , 1, or 3 pg / cell, or may be within a range of any two of these values. -3 ~3, 5.56×10 -3 ~3×10 -2 , 5.56 x 10 -3 ~2×10 -2 , 1.2 × 10 -2 ~3×10 -2 , or 1.2 × 10 -2 ~1.7×10 -2 The amount of IL-6 secreted may be, for example, at least 3 × 10 pg / cell. -2 , 4×10 -2 , 5×10 -2 , 6×10 -2 , 7×10 -2 , 8×10 -2 , 1×10 -1 , 1.5×10 -1 , 2 × 10 -1 , 1, 10, or 20 pg / cell, or within a range of any two of these values. -2 ~20, 3×10 -2 ~2×10 -1 , 3×10 -2 ~8×10 -2 , 4×10 -2 ~8×10 -2 , or 4 x 10 -2 ~7×10 -2 The secretion amount can be calculated by multiplying the secretion concentration in the medium after culturing the cells (unit: pg / mL, for example) by the amount of medium used (unit: mL, for example) divided by the number of cells after culturing.

[0056] In embodiments of the present invention (including, for example, the above (1) to (41)), the imidazole dipeptide includes a compound having a structure in which an amino acid having an imidazole group is bonded to another amino acid. The imidazole dipeptide includes, for example, a compound having a structure of the following formula (1) or (2): [ka] [ka]

[0057] In the above formula (1) and formula (2), R 1 ~R 6 The meaning of R is as follows: 1 , R 2 , R 3 and R 4 are each independently H or C 1-6 It is alkyl. R 5 and R 6 are each independently -NHR 7 or -CH2NHR 7 where R 7 is H or -COR 8 where R 8 is H, C 1-6 Alkyl, optionally substituted phenyl, -OCH2R 9 , or -CH=CHR 9 where R 9 is H, C 1-6 It is alkyl, or optionally substituted phenyl. The compound having the structure of formula (1) or (2) may have an effect of promoting cytokine secretion from MSCs.

[0058] From the viewpoint of more efficiently promoting cytokine secretion from MSCs, R in formula (1) 1 and R 2 is H on one side and C on the other side 1-6 Preferably, they are alkyl, and more preferably, they are both H. Here, R in formula (1) 1 and R 2 One of them is C 1-6For alkyl, C 1-6 The alkyl is preferably methyl. From the viewpoint of promoting cytokine secretion from MSCs, R 3 and R 4 is H on one side and C on the other side 1-6 Preferably, they are alkyl, and more preferably, they are both H. Here, R in formula (2) 2 and R 3 One of them is C 1-6 For alkyl, C 1-6 The alkyl is preferably methyl. From the viewpoint of promoting cytokine secretion from MSCs, R 8 is H, C 1-6 Preferably, R in formula (1) or (2) is alkyl or -OCH3, and more preferably methyl. From the viewpoint of promoting cytokine secretion from MSCs, 9 is H or C 1-6 Preferably, R in formula (1) or (2) is alkyl. 7 The optionally substituted phenyl of formula (1) or (2) may be, for example, unsubstituted, or may be substituted with —OH, C 1-6 It may be phenyl substituted with alkyl or -OCH3. The substitution position may be the 2-, 3-, 4-, 5- or 6-position of the phenyl.

[0059] From the viewpoint of more efficiently promoting cytokine secretion from MSCs, it is desirable to use R 1 , R 2 , R 3 and R 4 are each independently H or C 1-6 may be alkyl, R 1 and R 2 may be H, and R 3 and R 4 may be H, and R 5 and R 6 are each independently -NHR 7 or -CH2NHR 7 R may be 7may be H or —COCH3.

[0060] The alkyl group mentioned above includes straight or branched hydrocarbon chains. 1-6 is a hydrocarbon having 1, 2, 3, 4, 5, or 6 carbon atoms. 1-6 The alkyl is an alkyl having 1, 2, 3, 4, 5, or 6 carbon atoms. 1-6 Alkyl includes, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl groups and the like.

[0061] Regarding the method for producing the compounds represented by formula (1) and formula (2), the method and principle for producing imidazole dipeptides described in JP-A-2020-022433, JP-A-2019-131532, JP-A-2010-31004, JP-A-2006-232686, or JP-A-2006-504701, etc., can be used.

[0062] In embodiments of the present invention (including, for example, the above (1) to (41)), the imidazole dipeptide includes carnosine, a methylated form of carnosine (e.g., anserine or balenine), or homocarnosine. The imidazole dipeptide includes a compound having a structure of formula (1) or (2) and having antioxidant activity. For example, carnosine, anserine, balenine, and homocarnosine are known to have antioxidant activity (see, for example, Boldyrev et al., Physiol Rev. 2013 Oct;93(4):1803-45). The imidazole dipeptide may be in the L-form or the D-form.

[0063] In embodiments of the present invention (including, for example, the above (1) to (41)), carnosine includes L-carnosine or D-carnosine unless otherwise specified. L-carnosine can also be referred to as beta-alanyl-L-histidine. L-carnosine can be represented by the CAS registry number 305-84-0.

[0064] In the embodiments of the present invention (including, for example, the above (1) to (41)), anserine includes L-anserine or D-anserine unless otherwise specified. L-anserine can also be referred to as beta-alanyl-3-methyl-L-histidine. L-anserine can be represented by the CAS Registry Number 584-85-0.

[0065] In the embodiments of the present invention (including, for example, the above (1) to (41)), balenine includes L-balenine or D-balenine unless otherwise specified. L-balenine can also be referred to as beta-alanyl-1-methyl-L-histidine. L-balenine can be represented by the CAS Registry Number 331-38-4.

[0066] In embodiments of the present invention (including, for example, the above (1) to (41)), homocarnosine includes L-homocarnosine or D-homocarnosine unless otherwise specified. L-homocarnosine can also be referred to as gamma-aminobutyryl-L-histidine. L-homocarnosine can be represented by the CAS registry number 3650-73-5.

[0067] In embodiments of the present invention (including, for example, the above (1) to (41)), the effects brought about by high concentrations of cytokines include, for example, the effects brought about by high concentrations of G-CSF, IL-6, MCP-1, VEGF-C, TGF-β1, IL-7, IL-8, M-CSF, osteoprotegrin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, or semaphorin 3B. Because G-CSF is a cytokine that suppresses bone formation, it is believed that high concentrations of G-CSF can suppress bone formation. Because IL-6 is a cytokine that suppresses bone formation, it is believed that high concentrations of IL-6 can suppress bone formation. Because IL-6, VEGF, MCP-1, VEGF-C, TGF-β1, and IL-8 are angiogenic factors, it is believed that high concentrations of these cytokines can promote angiogenesis. Because G-CSF, MCP-1, TGF-β1, and IL-7 are immunoregulatory factors, it is thought that these cytokines can suppress autoimmune diseases, etc. Because M-CSF, osteoprotegerin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, and semaphorin 3B are factors involved in the inhibition of bone formation, it is thought that a combination of these cytokines can suppress bone formation.

[0068] In embodiments of the present invention (including, for example, the above (1) to (41)), diseases or symptoms to be inhibited or ameliorated include, for example, bone differentiation, diseases associated with bone differentiation (e.g., heterotopic ossification), ischemic diseases (e.g., lower limb ischemia, myocardial infarction, cerebral infarction, spinal cord infarction, or chronic arterial occlusion), wounds (e.g., epithelial wounds or burns), sarcopenia associated with aging, arthritis (e.g., rheumatism, herniated disc, osteoarthritis), inflammatory diseases (e.g., nephritis, keratitis, cytokine storm), psychiatric disorders (e.g., autism or insomnia thought to be partly caused by neuroinflammation), immune diseases (e.g., GVHD (graft-versus-host disease), Sjögren's syndrome, atopic dermatitis, collagen diseases, multiple sclerosis, or autoimmune diseases), or cancer. This disease or symptom may be suppressed or ameliorated, for example, by administering to a subject the culture supernatant, cells, or composition according to an embodiment of the present invention (including, for example, (1) to (41) above). This disease or symptom may be suppressed or ameliorated, for example, by the effect of a high concentration of cytokines. The above-mentioned ectopic ossification includes a phenomenon in which abnormal bone formation occurs in sites where bone formation does not normally occur. Ectopic ossification includes, for example, ossification occurring in soft tissues (e.g., tendons, membranes, ligaments, muscles, joint capsules, etc.). Ectopic ossification may be in a state with a trabecular bone structure. Ectopic ossification includes, for example, ossification of the posterior longitudinal ligament, ossification of the ligamentum flavum, fibrodysplasia ossificans progressiva, myositis ossificans progressiva, myositis ossificans traumatic, and diffuse idiopathic hyperostosis.

[0069] In embodiments of the present invention (including, for example, the above (1) to (41)), inhibition of bone differentiation is useful, for example, for inhibiting diseases associated with bone differentiation. Diseases associated with bone differentiation include, for example, ossification (e.g., ectopic ossification). Inhibition of bone differentiation includes, for example, inhibiting abnormal bone differentiation. Furthermore, uses of inhibition of bone differentiation include uses for inhibiting or ameliorating diseases associated with bone differentiation. Pharmaceutical compositions for inhibiting bone differentiation include pharmaceutical compositions for diseases associated with bone differentiation, such as ectopic ossification. Methods for inhibiting bone differentiation include methods for inhibiting or ameliorating diseases associated with bone differentiation, such as ectopic ossification.

[0070] In embodiments of the present invention (including, for example, the above (1) to (41)), culturing includes incubating cells under conditions suitable for cell growth or maintenance. Incubation may be performed at approximately 37°C in an atmosphere of approximately 5% CO2. The culture may be performed in a serum-free, serum-free, serum albumin-free, insulin-free, IGF-free, FGF-free, growth factor-free, cytokine-free, or protein-free medium. "Free" includes a state in which the target component is not added to the medium, a state in which the medium is completely free of the target component, or a state in which the medium does not contain the component at a concentration above the detection limit. "Free" includes a state in which the target component is substantially free or substantially free. The culture may be performed by adherent culture in the presence of a cell adhesion factor (e.g., mixed in the medium or in a pre-coated container). When the culture involves cell growth, the culture may include cell production. The culture time may be, for example, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, or 50 days or more, or may be within a range of any two of these values. Culture may be performed until the cells reach a confluent state. A confluent state includes a state in which the cells cover 80% or more of the bottom area of ​​the culture vessel. More preferably, culture may be performed until the cells cover 80-90% of the bottom area of ​​the culture vessel. MSC culture may be performed by alternately using medium X, where medium X is a medium not containing imidazole dipeptide (e.g., a protein-containing medium) and medium Y is a medium containing imidazole dipeptide (e.g., a protein-free medium). Alternating use includes starting culture with medium X or Y, then culturing with medium X followed by medium Y, or culturing with medium X followed by medium X. For example, the following sequence may be performed: culture with X, replacement with medium Y and culture, replacement with medium X and culture, replacement with medium Y and culture, replacement with medium X and culture, replacement with medium Y and culture, replacement with medium X and culture, replacement with medium Y and culture, replacement with medium X and culture, and replacement with medium Y. Under such conditions in which MSCs are cultured alternately using X and Y, the culture of Y may be performed, for example, at least the first, second, third, fourth, fifth, sixth, or seventh time. The culture supernatant may be collected at any time after the culture in Y.Under the conditions for culturing MSCs using X and Y alternately as described above, the culture supernatant may be collected, for example, after the first, second, third, fourth, fifth, sixth, or seventh culture of Y. Alternatively, the supernatant may be collected at two or more of these times, or at all times, and the resulting mixture of all supernatants may be used as the desired supernatant. The time from the start of culture to replacement can be the same as the culture time described above.

[0071] In embodiments of the present invention (including, for example, the above (1) to (41)), MSCs may be cultured in a medium containing, for example, 0.1 to 100 mM imidazole dipeptide. This concentration may be, for example, 0.1, 0.2, 0.5, 1, 2, 5, 7, 10, 15, 20, 25, 30, 35, 40, 50, 60, 80, or 100 mM, or may be equal to or greater than these values, or may be within a range of any two of these values. From the viewpoint of more efficiently obtaining a culture supernatant containing a high concentration of at least one cytokine useful for suppressing or ameliorating a disease, this concentration is preferably 1 to 50 mM, more preferably 3 to 30 mM, even more preferably 5 to 25 mM, particularly preferably 5 to 20 mM, and most preferably 10 to 20 mM. Culture may be adherent culture. Those skilled in the art can easily confirm whether cells are attached to a container by, for example, tilting the container to confirm that no cell migration occurs, or by confirming that no cell migration occurs when changing the medium in the container. Adherent culture may be performed, for example, by culturing cells in the presence of a cell adhesion factor (e.g., contacting cells with the adhesion factor in a container, mixing a cell suspension containing cells and medium with the adhesion factor, placing the resulting mixture in a container and culturing, or coating (e.g., pre-coating) the container with the adhesion factor and then seeding the cells in the container). Herein, the terms "cell adhesion factor" and "adhesion factor" refer to the same entity. Examples of adhesion factors include laminin, fibronectin, vitronectin, tenascin, cadherin, poly-L-lysine, poly-D-lysine, collagen, thrombospondin, galectin, or nidogen-1, or fragments thereof that have cell adhesion activity. Cell adhesion may also include adhesion between cells and a container via an extracellular matrix (e.g., an adhesion factor). Adherent cells may include cells that can be cultured as adherent cells or cells that can grow while attached.

[0072] In the above-mentioned adhesion culture, the concentration of the adhesion factor in the medium may be, for example, 0.01, 0.1, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 2, or 3 μg / ml, or may be equal to or greater than these values, or may be within a range of any two of these values. This value may be, for example, 0.01 to 2, 0.1 to 1.5, or 0.5 to 1 μg / ml. The concentration of the adhesion factor in the medium is, for example, 0.01 to 2, 0.1 to 1.5, or 0.5 to 1 μg / ml per 1 cm of culture area of ​​the culture vessel. 2 The concentration may be 0.005, 0.01, 0.1, 0.25, 0.5, 1, 1.5, or 2 μg per cm of culture area of ​​the culture vessel, or may be equal to or greater than these values, or may be within a range of any two of these values. 2The amount of the adhesive factor may be 0.01 to 1.5, 0.1 to 1, or 0.1 to 0.5 μg per cell. The adhesive factor is a factor having adhesive activity, such as laminin, fibronectin, vitronectin, tenascin, cadherin, poly-L-lysine, poly-D-lysine, collagen, thrombospondin, galectin, nidogen-1, fragments thereof, or modified forms thereof. The adhesive factor may be a laminin fragment or a modified form thereof. The laminin fragment may have integrin-binding activity and may be derived from a human. The laminin fragment may be a laminin E8 fragment. The laminin fragment may be a laminin-511 E8 fragment, a laminin-521 E8 fragment, a laminin-411 E8 fragment, a laminin-421 E8 fragment, a laminin-332 E8 fragment, a laminin-311 E8 fragment, a laminin-321 E8 fragment, a laminin-211 E8 fragment, a laminin-221 E8 fragment, a laminin-213 E8 fragment, a laminin-111 E8 fragment, or a laminin-121 E8 fragment. The laminin-511 E8 fragment can be prepared, for example, by the method described in WO2011 / 043405A1. Laminin-511 includes laminins composed of α5, β1, and γ1 subunit chains. The laminin fragment variant may be a known complex composed of a laminin fragment having integrin-binding activity and another functional molecule (e.g., a complex between a laminin fragment having integrin-binding activity and a cell adhesion molecule, or a complex between a laminin fragment having integrin-binding activity and a growth factor-binding molecule (e.g., heparan sulfate)) (see, for example, WO2012 / 137970, WO2014 / 103534, and WO2016 / 010082). The laminin fragment variant can be produced as a recombinant protein using known genetic recombination techniques.The laminin fragment or variant thereof may have integrin-binding activity, and may include, for example, (a) a trimer comprising a polypeptide having the amino acid sequence shown in SEQ ID NO: 1, a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, and a polypeptide having the amino acid sequence shown in SEQ ID NO: 3; (b) a trimer comprising a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 1, a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 2, and a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 3; or (c) a trimer comprising a polypeptide having an amino acid sequence with one to several amino acid deletions, substitutions, insertions, or additions relative to the amino acid sequence shown in SEQ ID NO: 1, a polypeptide having an amino acid sequence with one to several amino acid deletions, substitutions, insertions, or additions relative to the amino acid sequence shown in SEQ ID NO: 2, and a polypeptide having one to several amino acid deletions, substitutions, insertions, or additions relative to the amino acid sequence shown in SEQ ID NO: 3. The laminin fragments, variants, or subunit chains described above may optionally have a tag at the N- or C-terminus (see, e.g., Mishra, Curr Protein Pept Sci. 2020;21(8):821-830). The tag may be, for example, a purification tag. The purification tag may be, for example, a peptide tag (e.g., a His tag). The purification tag includes, for example, a biochemically inert tag. The cell seeding density may be, for example, 1 x 10e1, 1 x 10e2, 5 x 10e2, 1 x 10e3, 1.5 x 10e3, 1 x 10e4, 1 x 10e5, or 1 x 10e6 cells / mL, or may be equal to or greater than these values, or may be within a range of any two of these values. This value may be, for example, 1x10e1 to 1x10e5, 1x10e2 to 1x10e4, 5x10e2 to 5x10e4, or 5x10e2 to 1x10e4 cells / mL. The cell seeding density may be, for example, 0.002, 0.01, 0.1, 1, 50, 100, 300, 500, 1000, 1500, or 2000 cells / cm. 2The cell density may be, for example, 0.01 to 2000, 0.1 to 1000, 1 to 1000, 1 to 500, or 1 to 100 cells / cm. 2 may be.

[0073] In embodiments of the present invention (including, for example, the above (1) to (41)), the culture supernatant includes a culture supernatant obtained by culturing cells. The culture supernatant includes, for example, a cytokine-containing culture supernatant. The culture supernatant may contain, for example, cell metabolites (e.g., amino acids, lipids, sugars, etc.), secreted proteins (e.g., hormones, peptides, cytokines, extracellular matrix, etc.), or exosomes. When MSCs are cultured in a medium containing imidazole dipeptide, the culture supernatant may contain imidazole dipeptide. The culture supernatant also includes supernatants separated from cellular components and subjected to various treatments (e.g., centrifugation, filtration, freezing, lyophilization, storage, sterilization, etc.). The culture supernatant may be a culture supernatant obtained using a medium containing only MSCs as cultured cells. From the viewpoints of reducing production costs and ensuring uniformity between lots, it is preferable that the cytokines contained in the culture supernatant are only cytokines derived from the cultured cells. From the viewpoint of reducing production costs, it is preferable that the culture supernatant is non-concentrated. The culture supernatant may contain, for example, HGF, G-CSF, MCP-1, VEGF-C, TGF-β1, IL-6, IL-7, IL-8, M-CSF, osteoprotegrin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, semaphorin 3B, or exosomes.

[0074] In embodiments of the present invention (including, for example, the above (1) to (41)), the culture supernatant may contain at least 500 pg / mL of G-CSF or at least 2700 pg / mL of IL-6. Use of this culture supernatant allows the effects of high concentrations of G-CSF or IL-6 to be obtained. This culture supernatant may contain, for example, 500 to 2000, 700 to 1700, 800 to 1600, or 1000 to 1600 pg / mL of G-CSF. This concentration may be, for example, at least 500, 600, 700, 800, 900, 1000, 1250, 1500, 1600, 1700, 1800, 1900, or 2000 pg / mL, or may be within a range of any two of these values. The culture supernatant may contain IL-6 at a concentration of, for example, 2700 to 10,000, 3,000 to 10,000, 4,000 to 10,000, or 3,000 to 4,000 pg / mL. This concentration may be, for example, at least 2700, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, or 12,000 pg / mL, or may be within a range of any two of these values. In addition to the above concentrations of G-CSF and IL-6, this culture supernatant may contain at least 6,000 pg / mL of MCP-1, at least 150 pg / mL of VEGF-C, at least 670 pg / mL of TGF-β1, at least 3.5 pg / mL of IL-7, or at least 6,500 pg / mL of IL-8. Use of this culture supernatant allows for the effects of high concentrations of G-CSF, IL-6, MCP-1, VEGF-C, TGF-β1, IL-7, or IL-8 to be achieved. This culture supernatant may contain, for example, at least 6,000, 7,000, 8,000, 9,000, or 10,000 pg / mL of MCP-1, or may contain MCP-1 within a range between any two of these values. The culture supernatant may contain, for example, at least 150, 200, 250, 300, 350, or 400 pg / mL of VEGF-C, or within a range between any two of these values.The culture supernatant may contain, for example, at least 670, 700, 750, 800, 850, 900, 950, or 1000 pg / mL of TGF-β1, or within a range between any two of these values.This culture supernatant may contain IL-7 at, for example, at least 3.5, 4, 5, 6, 7, 8, 9, or 10 pg / mL, or within a range between any two of these values. This culture supernatant may contain IL-8 at, for example, at least 6,500, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, or 15,000 pg / mL, or within a range between any two of these values. From the viewpoint of enhancing the effects of these cytokines, it is preferable that the culture supernatant contains the above seven cytokines in the above concentration ranges. Furthermore, the above culture supernatant may contain M-CSF at, for example, at least 1,350, 1,370, 1,390, 1,400, 1,420, 1,440, 1,450, 1,470, 1,490, or 1,500 pg / mL, or within a range between any two of these values. Furthermore, the culture supernatant may contain osteoprotegins at, for example, at least 95, 100, 110, 120, 130, or 140 pg / mL, or within a range between any two of these values.

[0075] In embodiments of the present invention (including, for example, (1) to (41) above), the culture supernatant may contain at least 100 pg / mL of G-CSF, at least 550 pg / mL of IL-6, at least 6000 pg / mL of MCP-1, at least 150 pg / mL of VEGF-C, at least 670 pg / mL of TGF-β1, at least 3.5 pg / mL of IL-7, at least 6500 pg / mL of IL-8, or at least 120 pg / mL of CD9 / CD63 fusion protein. The culture supernatant may contain, for example, at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1600, 1700, 1800, 1900, or 2000 pg / mL of G-CSF, or within a range between any two of these values. The culture supernatant may contain, for example, at least 550, 800, 1000, 2000, 2500, 2700, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10000, 11000, or 12000 pg / mL of IL-6, or within a range between any two of these values. The culture supernatant may contain, for example, at least 6,000, 7,000, 8,000, 9,000, or 10,000 pg / mL of MCP-1, or within a range between any two of these values. The culture supernatant may contain, for example, at least 150, 200, 250, 300, 350, or 400 pg / mL of VEGF-C, or within a range between any two of these values. The culture supernatant may contain, for example, at least 670, 700, 750, 800, 850, 900, 950, or 1,000 pg / mL of TGF-β1, or within a range between any two of these values. The culture supernatant may contain, for example, at least 3.5, 4, 5, 6, 7, 8, 9, or 10 pg / mL of IL-7, or within a range between any two of these values. The culture supernatant may contain IL-8 at, for example, at least 6500, 7000, 8000, 9000, 10000, 11000, 12000, or 15000 pg / mL, or within a range between any two of these values.The culture supernatant may contain, for example, at least 120, 140, 160, 180, 200, 250, or 300 pg / mL of CD9 / CD63 fusion protein, or within a range between any two of these values. Furthermore, the culture supernatant may contain, for example, at least 1350, 1370, 1390, 1400, 1420, 1440, 1450, 1470, 1490, or 1500 pg / mL of M-CSF, or within a range between any two of these values. Furthermore, the culture supernatant may contain, for example, at least 95, 100, 110, 120, 130, or 140 pg / mL of osteoprotegin.

[0076] In embodiments of the present invention (including, for example, the above (1) to (41)), the culture supernatant may contain at least 1350 pg / mL of M-CSF or at least 95 pg / mL of osteoprotegerin. Use of this culture supernatant allows for the effects of high concentrations of M-CSF or osteoprotegerin to be obtained. This culture supernatant may contain, for example, 1350 to 1500, 1350 to 1450, 1350 to 1420, or 1350 to 1400 pg / mL of M-CSF. This concentration may be, for example, at least 1350, 1370, 1390, 1400, 1420, 1440, 1450, 1470, 1490, or 1500 pg / mL, or may be within a range of any two of these values. The culture supernatant may contain osteoprotegins at concentrations of, for example, 95 to 140, 95 to 130, 95 to 120, or 95 to 110 pg / mL. This concentration may be, for example, at least 95, 100, 110, 120, 130, or 140 pg / mL, or may be within a range of any two of these values. In addition to the above concentrations of M-CSF and osteoprotegins, the culture supernatant may contain G-CSF at least 500 pg / mL, IL-6 at least 2700 pg / mL, MCP-1 at least 6000 pg / mL, VEGF-C at least 150 pg / mL, TGF-β1 at least 670 pg / mL, IL-7 at least 3.5 pg / mL, or IL-8 at least 6500 pg / mL. Use of this culture supernatant allows for the effects of high concentrations of M-CSF, osteoprotegin, G-CSF, IL-6, MCP-1, VEGF-C, TGF-β1, IL-7, or IL-8 to be achieved. In another embodiment, the culture supernatant may contain G-CSF at a concentration of, for example, at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1600, 1700, 1800, 1900, or 2000 pg / mL, or within a range between any two of these values.The culture supernatant may contain, for example, at least 550, 800, 1000, 2000, 2500, 2700, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000, or 12,000 pg / mL of IL-6, or within a range between any two of these values. The culture supernatant may contain, for example, at least 6,000, 7000, 8000, 9000, or 10,000 pg / mL of MCP-1, or within a range between any two of these values. The culture supernatant may contain, for example, at least 150, 200, 250, 300, 350, or 400 pg / mL of VEGF-C, or within a range between any two of these values. The culture supernatant may contain, for example, 670, 700, 750, 800, 850, 900, 950, or 1000 pg / mL of TGF-β1, or within a range between any two of these values. The culture supernatant may contain, for example, at least 3.5, 4, 5, 6, 7, 8, 9, or 10 pg / mL of IL-7, or within a range between any two of these values. The culture supernatant may contain, for example, at least 6500, 7000, 8000, 9000, 10000, 11000, 12000, or 15000 pg / mL of IL-8, or within a range between any two of these values.

[0077] In embodiments of the present invention (including, for example, (1) to (41) above), the medium comprises a medium used for culturing cells. The medium may be a liquid medium or a solid medium. The medium comprises a culture solution. Any common cell culture medium may be used as the medium, and the composition is not limited. The medium may contain, for example, amino acids, inorganic salts, vitamins, minerals, or a carbon source (e.g., glucose). The medium may be, for example, a serum medium (e.g., FBS, etc.), a basal medium (e.g., MEM, etc.), a complex medium, a serum-free medium, etc. The medium may be commercially available as a medium for growing human MSCs. Basal cell culture media available from manufacturers include, for example, MEM (e.g., Thermo Fisher Scientific Inc.), DMEM (Dulbecco's Modified Eagle Medium) (e.g., Sigma-Aldrich), IMDM (e.g., Sigma-Aldrich), Ham's F-12 (e.g., Fujifilm Wako Pure Chemical Industries, Ltd.), DMEM / F12 (e.g., Sigma-Aldrich), and RPMI 1640 (e.g., Nacalai Tesque). For example, a medium containing DMEM / F12 supplemented with amino acids can be used. The amino acids to be added include commercially available amino acid solutions for medium supplementation, such as MEM essential amino acid solution (Fujifilm Wako Pure Chemical Industries, Ltd.) and MEM non-essential amino acid solution (Fujifilm Wako Pure Chemical Industries, Ltd.). This DMEM / F12-based medium does not contain any xenogeneic components, cytokines, insulin, proteins, or human serum. Considering that the cultured cells and culture supernatants will be used in regenerative medicine and other therapeutic applications, it is desirable that the medium be serum-free, serum albumin-free, insulin-free, IGF-free, FGF-free, growth factor-free, cytokine-free, protein-free, or xeno-free. This has the advantage of eliminating the risk of exogenous active components other than those derived from the cultured cells entering the patient's body. The medium may contain, for example, an LIF component, an adhesion molecule, an FGF component, an insulin component, an albumin component, or a transferrin component. From the perspective of particularly promoting MSC proliferation, it is preferable that the medium contain an LIF component.From the viewpoint of particularly promoting cell proliferation of MSCs, the medium preferably further contains an adhesion molecule.

[0078] In embodiments of the present invention (including, for example, the above (1) to (41)), LIF includes, for example, a component also referred to as leukemia inhibitory factor. Details of the amino acid sequence of LIF can be confirmed on websites such as NCBI or UniProt. The primary accession number of LIF listed in UniProt is, for example, P15018. The amino acid sequence of LIF may be, for example, the amino acid sequence shown in SEQ ID NO: 4 or 5. LIF includes, for example, human-derived LIF. In embodiments of the present invention (including, for example, the above (1) to (41)), the LIF component includes a molecule having LIF activity and derived from wild-type human LIF. The LIF component may be, for example, (a) a protein having the amino acid sequence set forth in SEQ ID NO: 4 or 5 or a biologically active fragment thereof; (b) a protein having an amino acid sequence with 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 4 or 5 or a biologically active fragment thereof; or (c) a protein having an amino acid sequence with one to several amino acid deletions, substitutions, insertions, or additions relative to the amino acid sequence set forth in SEQ ID NO: 4 or 5 or a biologically active fragment thereof. LIF activity includes, for example, the activity of promoting LIF signal transduction, the activity of binding to a LIF receptor, or the activity of inhibiting the proliferation of leukemia cells. Biologically active fragments include, for example, polypeptides having LIF activity but lacking a portion of LIF not involved in LIF activity. The LIF component may optionally have a tag at the N- or C-terminus (see, for example, Mishra, Curr Protein Pept Sci. 2020;21(8):821-830). The tag may be, for example, a purification tag. The purification tag may be, for example, a peptide tag (e.g., a His tag). The purification tag may include, for example, a biochemically inactive tag. The concentration of the LIF component in the medium may be, for example, 0.01 ng / mL or more, e.g., 0.01, 0.05, 0.1, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 8, or 10 ng / mL, or any of these values ​​or more, or within a range of any two of these values.This concentration may be, for example, 0.1 to 4, 0.1 to 3, 0.5 to 2, or 0.8 to 1.2 ng / ml. From the viewpoint of promoting MSC proliferation, the concentration is preferably 0.1 ng / ml or more, more preferably 0.5 ng / ml or more, and even more preferably 0.8 ng / ml or more.

[0079] In embodiments of the present invention (including, for example, the above (1) to (41)), FGF includes, for example, a component also referred to as fibroblast growth factor. FGF includes, for example, bFGF. bFGF includes, for example, a component also referred to as basic fibroblast growth factor. Details of the amino acid sequence of bFGF can be confirmed on websites such as NCBI or UniProt. The primary accession number of bFGF listed in UniProt is, for example, P09038. The amino acid sequence of bFGF may be, for example, the amino acid sequence shown in SEQ ID NO: 6, 7, or 8. bFGF includes, for example, bFGF derived from human. In embodiments of the present invention (including, for example, the above (1) to (41)), the bFGF component has bFGF activity and includes a molecule derived from wild-type human bFGF. The bFGF component may be, for example, (a) a protein having the amino acid sequence set forth in SEQ ID NO: 6, 7, or 8, or a biologically active fragment thereof; (b) a protein having an amino acid sequence with 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 6, 7, or 8, or a biologically active fragment thereof; or (c) a protein having an amino acid sequence with one to several amino acid deletions, substitutions, insertions, or additions relative to the amino acid sequence set forth in SEQ ID NO: 6, 7, or 8, or a biologically active fragment thereof. bFGF activity includes, for example, the activity of promoting bFGF signal transduction, the activity of binding to a bFGF receptor, or the activity of promoting fibroblast proliferation. Biologically active fragments include, for example, polypeptides having bFGF activity, in which a portion of bFGF not involved in bFGF activity has been deleted. The FGF component may optionally have a tag at the N- or C-terminus (see, for example, Mishra, Curr Protein Pept Sci. 2020;21(8):821-830). The tag may be, for example, a purification tag. The purification tag may be, for example, a peptide tag (e.g., a His tag). Purification tags include, for example, biochemically inert tags. The concentration of the bFGF component in the medium may be, for example, 0.1 ng / mL or higher.This concentration may be, for example, 0.1, 0.5, 1, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 80, or 100 ng / mL, or may be any of these values ​​or greater, or may be within a range of any two of these values. This concentration may be, for example, 1 to 40, 1 to 30, 5 to 20, or 8 to 12 ng / mL. From the viewpoint of promoting MSC proliferation, a concentration of 1 ng / mL or greater is preferred, 5 ng / mL or greater is more preferred, and 8 ng / mL or greater is even more preferred.

[0080] In embodiments of the present invention (including, for example, the above (1) to (41)), insulin includes a type of hormone. Details of the amino acid sequence of insulin, etc., can be confirmed on websites such as NCBI or UniProt. The primary accession number of insulin listed in UniProt is, for example, P01308. The amino acid sequence of human insulin includes, for example, the amino acid sequence shown in SEQ ID NO: 9. Insulin includes, for example, insulin derived from human. In embodiments of the present invention (including, for example, the above (1) to (41)), the insulin component includes a molecule that has insulin activity and is derived from wild-type human insulin. The insulin component may be, for example, (a) a protein having the amino acid sequence shown in SEQ ID NO: 9 or a biologically active fragment thereof; (b) a protein having an amino acid sequence that is 90% or more homologous to the amino acid sequence shown in SEQ ID NO: 9 or a biologically active fragment thereof; or (c) a protein having an amino acid sequence with one to several amino acid deletions, substitutions, insertions, or additions relative to the amino acid sequence shown in SEQ ID NO: 9 or a biologically active fragment thereof. Insulin activity includes, for example, the activity of promoting insulin signaling or the activity of activating insulin receptor tyrosine kinase. Biologically active fragments include, for example, polypeptides having insulin activity, lacking portions of insulin that are not involved in insulin activity. The insulin component may optionally have a tag at the N- or C-terminus (see, for example, Mishra, Curr Protein Pept Sci. 2020;21(8):821-830). The tag may be, for example, a purification tag. The purification tag may be, for example, a peptide tag (e.g., a His tag). The purification tag includes, for example, a biochemically inert tag. The concentration of the insulin component in the medium may be, for example, 0.1 μg / mL or higher. This concentration may be, for example, 0.1, 0.5, 1, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 80, or 100 μg / mL, or may be equal to or higher than any of these values, or may be within a range between any two of these values. This concentration may be, for example, 1 to 40, 1 to 30, 5 to 20, or 8 to 12 μg / ml.From the viewpoint of promoting the proliferation of MSCs, the concentration is preferably 1 μg / ml or more, more preferably 5 μg / ml or more, and even more preferably 8 μg / ml or more.

[0081] In embodiments of the present invention (including, for example, the above (1) to (41)), albumin includes a type of protein that is abundant in serum. Details of the amino acid sequence of albumin, etc., can be confirmed on websites such as NCBI or UniProt. The primary accession number of albumin listed in UniProt is, for example, Q56G89. The amino acid sequence of human albumin includes, for example, the amino acid sequence shown in SEQ ID NO: 10. Albumin includes, for example, albumin derived from human. In embodiments of the present invention (including, for example, the above (1) to (41)), the albumin component includes a molecule that has albumin activity and is derived from wild-type human albumin. The albumin component may be, for example, (a) a protein having the amino acid sequence shown in SEQ ID NO: 10 or a biologically active fragment thereof; (b) a protein having an amino acid sequence that is 90% or more homologous to the amino acid sequence shown in SEQ ID NO: 10 or a biologically active fragment thereof; or (c) a protein having an amino acid sequence with one to several amino acid deletions, substitutions, insertions, or additions relative to the amino acid sequence shown in SEQ ID NO: 10 or a biologically active fragment thereof. Albumin activity includes, for example, blood osmolarity regulation activity or binding activity to fatty acids or bilirubin. Biologically active fragments include, for example, polypeptides having albumin activity, in which a portion of albumin not involved in albumin activity has been deleted. The albumin component may optionally have a tag at the N- or C-terminus (see, for example, Mishra, Curr Protein Pept Sci. 2020;21(8):821-830). The tag may be, for example, a purification tag. The purification tag may be, for example, a peptide tag (e.g., a His tag). The purification tag includes, for example, a biochemically inactive tag. The albumin component concentration in the medium may be, for example, 10 μg / mL or more. This concentration may be, for example, 10, 50, 100, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000, 3000, 4000, 5000, 8000, or 10000 μg / mL, or may be any of these values ​​or greater, or within a range of any two of these values.This concentration may be, for example, 100 to 4000, 10 to 3000, 500 to 2000, or 800 to 1200 ng / ml. From the viewpoint of promoting MSC proliferation, the concentration is preferably 100 μg / ml or more, more preferably 500 μg / ml or more, and even more preferably 800 μg / ml or more.

[0082] In embodiments of the present invention (including, for example, the above (1) to (41)), transferrin comprises a type of protein present in serum. Details of the amino acid sequence of transferrin can be confirmed on websites such as NCBI or UniProt. The primary accession number of transferrin listed in UniProt is, for example, P02787. The amino acid sequence of human transferrin comprises, for example, the amino acid sequence shown in SEQ ID NO: 11. Transferrin includes, for example, transferrin derived from human. In embodiments of the present invention (including, for example, the above (1) to (41)), the transferrin component has transferrin activity and comprises a molecule derived from wild-type human transferrin. The transferrin component may be, for example, (a) a protein having the amino acid sequence set forth in SEQ ID NO: 11 or a biologically active fragment thereof; (b) a protein having an amino acid sequence with 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 11 or a biologically active fragment thereof; or (c) a protein having an amino acid sequence with one to several amino acid deletions, substitutions, insertions, or additions relative to the amino acid sequence set forth in SEQ ID NO: 11 or a biologically active fragment thereof. Transferrin activity includes, for example, binding activity to a transferrin receptor or binding activity to iron ions. Bioactive fragments include, for example, polypeptides having transferrin activity, in which a portion of transferrin not involved in transferrin activity is deleted. The transferrin component may optionally have a tag at the N- or C-terminus (see, for example, Mishra, Curr Protein Pept Sci. 2020;21(8):821-830). The tag may be, for example, a purification tag. The purification tag may be, for example, a peptide tag (e.g., a His tag). The purification tag may include, for example, a biochemically inactive tag. The transferrin component concentration in the medium may be, for example, 0.1 μg / mL or more, e.g., 0.1, 0.5, 1, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 80, or 100 μg / mL, or any of these values ​​or more, or within a range of any two of these values.This concentration may be, for example, 1 to 40, 1 to 30, 5 to 20, or 8 to 12 μg / ml. From the viewpoint of promoting MSC proliferation, the concentration is preferably 1 μg / ml or more, more preferably 5 μg / ml or more, and even more preferably 8 μg / ml or more.

[0083] In the embodiments of the present invention (including, for example, the above (1) to (41)), the vessel used for culture may be, for example, a flat-bottom vessel. The flat-bottom vessel may be, for example, a plate-type, petri dish-type, or flask-type. The shape of the bottom or top of the vessel may be square or round.

[0084] In the embodiments of the present invention (including, for example, the above (1) to (41)), the container for containing the culture supernatant may be a medical container. This container includes, for example, a syringe, a vial, a bottle, or a bag. The medical container may be sterile (for example, the solution-containing portion is sterile), a container for intravenous drip, or a container for cryopreservation. The medical bag includes a soft bag and a bag connected to a tube.

[0085] In embodiments of the present invention (including, for example, (1) to (41) above), the recovering step may include, for example, a step of separating a cell fraction from a culture supernatant, or a step of transferring the culture supernatant into a container. The separating step may include, for example, a step of centrifuging or filtering a medium containing MSCs and an imidazole dipeptide to separate the cell fraction from the culture supernatant. The recovering step may include a step of centrifuging or filtering the supernatant to remove dead cells or impurities from the supernatant, a step of sterilizing the supernatant, or a step of transferring the supernatant to a medical container. The recovering step may include a step of removing medium components from a composition containing cells and medium. The recovering step may be performed in a sterile environment.

[0086] The methods of the present invention (including, for example, the above (1) to (41)) may include a step of sterilizing the collected culture supernatant to produce a sterilized culture supernatant. The sterilization may include, for example, a step of passing the culture supernatant through a sterilizing filter.

[0087] In embodiments of the present invention (including, for example, the above (1) to (41)), the contacting step may be carried out by culturing MSCs in a medium containing imidazole dipeptide. The contacting step may be carried out in vitro or in vivo.

[0088] In embodiments of the present invention (including, for example, the above (1) to (41)), suppressing a disease includes treating the disease. Suppression includes, for example, exerting an effect of suppressing, suppressing recurrence, improving symptoms, or preventing the disease or one or more symptoms associated with the disease in a patient. Suppression also includes, for example, suppressing ossification in a patient, suppressing osteoblast production, or suppressing bone differentiation of cells (for example, suppressing differentiation of MSCs into osteoblasts or chondrocytes).

[0089] In embodiments of the present invention (including, for example, (1) to (41) above), the pharmaceutical composition may comprise a culture supernatant or cultured MSCs. When the pharmaceutical composition comprises a culture supernatant, the pharmaceutical composition includes a composition consisting of the culture supernatant. The pharmaceutical composition may comprise, for example, cytokines or exosomes. In this case, the concentrations of the cytokines and exosome markers may be within the concentration ranges described in the above-mentioned embodiment of the culture supernatant. The pharmaceutical composition may comprise, for example, imidazole dipeptide. The concentration of the imidazole dipeptide may be within the concentration range described in the above-mentioned embodiment of the medium. The pharmaceutical composition includes a composition used for suppressing or ameliorating the above-mentioned diseases or a composition used for preventing the onset of the above-mentioned diseases. The pharmaceutical composition may be produced, for example, by mixing an active ingredient with one or more pharmaceutically acceptable carriers using any method known in the technical field of pharmaceuticals. Furthermore, the pharmaceutical composition may be in any form used as long as it is used for treatment, and may comprise the active ingredient alone or a mixture of the active ingredient with any ingredient. Furthermore, the form of the carrier is not particularly limited, and may be, for example, a solid or liquid (e.g., a buffer solution). The content of the carrier may be, for example, a pharmaceutically effective amount. An effective amount may be, for example, an amount sufficient for pharmaceutical stability or delivery of the active ingredient. For example, a buffer is effective for stabilizing the active ingredient in a container. The pharmaceutical composition may also contain a stabilizer, buffer, or pH adjuster. The dosage, administration interval, administration method, and administration route are not particularly limited and can be selected appropriately depending on the patient's age, weight, symptoms, target organ, etc. Furthermore, the pharmaceutical composition preferably contains a therapeutically effective amount, or an effective amount that exerts the desired effect, of the active ingredient. In one embodiment of the present invention, a therapeutically effective amount includes an amount necessary to clinically observe improvement or suppression of symptoms in a patient (e.g., an amount sufficient to suppress bone differentiation (e.g., suppression of heterotopic ossification) in a subject). In one embodiment of the present invention, "pharmaceutically acceptable" includes a state that is suitable for use within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio. There are no particular limitations on the components other than the culture supernatant or MSCs in the pharmaceutical composition, as long as they do not impair the effects of the present invention, and they can be selected appropriately depending on the purpose.

[0090] In embodiments of the present invention (including, for example, (1) to (41) above), the subject (including a patient) includes humans and non-human mammals (e.g., one or more species of mice, guinea pigs, hamsters, rats, mice, rabbits, pigs, sheep, goats, cattle, horses, cats, dogs, marmosets, monkeys, chimpanzees, etc.). The patient may also be a patient in need of inhibition of bone differentiation, a patient diagnosed with ossification, a patient in need of treatment for ossification, or a patient in need of increased expression of G-CSF, MCP-1, VEGF-C, TGF-β1, IL-6, IL-7, IL-8, M-CSF, osteoprotegrin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, semaphorin 3B, or an exosome marker.

[0091] In embodiments of the present invention (including, for example, (1) to (41) above), the route of administration of the culture supernatant, MSCs, or pharmaceutical composition to a subject is preferably one that is effective for the treatment, and may be, for example, intravenous, intraarterial, subcutaneous, intralymph node, intramuscular, intraperitoneal, or oral. The dosage form is preferably one that is effective for the treatment, and may be, for example, an injection (e.g., intravenous injection), a liquid, or a solid preparation.

[0092] In embodiments of the present invention (including, for example, the above (1) to (41)), the dose, administration interval, and administration method of the culture supernatant, MSCs, or pharmaceutical composition to a subject can be appropriately selected depending on the age, weight, symptoms, target organ, etc. of the patient. The dose may be, for example, 0.01 to 1000 mL for the supernatant, or 1 x 10 per administration for the cells. 3 ~1×10 11 The administration interval may be, for example, once or twice every 1 to 28 days or every 1 to 4 weeks.

[0093] In embodiments of the present invention (including, for example, the above (1) to (41)), the inhibition of osteoblast production includes, for example, inhibition caused by inhibition of MSC differentiation into osteoblasts or inhibition caused by inhibition of osteoblast proliferation. For example, the inhibition of MSC differentiation into osteoblasts may occur by contacting MSC with an imidazole dipeptide.

[0094] In embodiments of the present invention (including, for example, the above (1) to (41)), the method for inhibiting osteoblast production may include, for example, the steps of culturing MSCs in a medium containing an imidazole dipeptide, recovering an MSC secretion product, and contacting the MSC secretion product with another MSC, wherein the MSCs during culture and the MSCs during contact are different MSCs.

[0095] In embodiments of the present invention (including, for example, (1) to (41) above), the MSC secretions include, for example, G-CSF, MCP-1, VEGF-C, TGF-β1, IL-6, IL-7, IL-8, M-CSF, osteoprotegin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, semaphorin 3B, or exosomes, which may be derived from humans.

[0096] In embodiments of the present invention (including, for example, the above (1) to (41)), the percentage of the composition or cell population containing the cells of the above (26) to (28) or (38) may be, for example, 30, 40, 50, 60, 70, 80, 90, 95, 98, 99, or 100%, or may be equal to or greater than these values, or may be within a range of any two of these values. This percentage may be, for example, 30 to 100%, 60 to 100%, 80 to 100%, 60 to 90%, 80 to 90%, or 90 to 100%.

[0097] In one embodiment of the present invention, the homology (%) may be calculated by, for example, calculating the percentage of homologous amino acids between multiple amino acid sequences according to methods known in the art. Before calculating the percentage, the sequences to be compared are aligned, and gaps are introduced into the sequences, if necessary, to maximize the percentage of homologous / identical amino acids. Alignment methods, percentage calculation methods, and related computer programs are well known in the art. Homology is calculated, for example, using global or local alignment. The former may be determined using the Needleman-Wunsch algorithm (Needleman et al., J. Mol. Biol. 1970 Mar;48(3):443-53.) or EMBOSS Needle (Rice et al., EMBOSS User's Guide: Practical Bioinformatics, 25 July 2011.). The default settings for EMBOSS Needle may include, for example, MATRIX: BLOSUM62, Gap Open Penalty: 10, Gap Extend Penalty: 0.5, Output formats: pair, End Gap Penalty: false, End Gap Open Penalty: 10, End Gap Extend Penalty: 0.5. The latter may be determined using the BLAST algorithm (Altschul et al., J Mol Biol. 1990 Oct 5;215(3):403-10.) or calculated using blastp (The BLAST Sequence Analysis Tool. The NCBI Handbook, 2nd edition, March 15, 2013.). The default settings for blastp may include, for example, MATRIX: BLOSUM62, Gap Open Penalty: 11, Extension: 1, Compositional adjustments: Conditional compositional score matrix adjustment.Homology may be expressed as (number of homologous amino acids / number of amino acids in the amino acid sequence to be compared) × 100. Homology is preferably calculated using global alignment. Unless otherwise specified, a test homology of a particular value or greater includes a value calculated by at least one of the above algorithms of a particular value or greater. Any of the above algorithms may be used with default settings.

[0098] In one embodiment of the present invention, the homology value of 90% or more may be, for example, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or may be any of these values ​​or more, or within a range of any two of these values. From the viewpoint of maintaining functional equivalence, this percentage is preferably 95% or more, more preferably 97% or more, even more preferably 98% or more, and particularly preferably 99% or more. In one embodiment of the present invention, the number of homologous sequences may be, for example, 15, 10, 8, 6, 5, 4, 3, 2, 1, or 0, or may be any of these values ​​or less, or within a range of any two of these values. From the viewpoint of maintaining functional equivalence, this number is preferably 15 or less, more preferably 10 or less, and particularly preferably 5 or less.

[0099] In one embodiment of the present invention, when substitutions are made to the original sequence, conservative amino acid substitutions (see, for example, French et al., J Mol Evol (1983) 19, 171-175) are preferred. In one embodiment of the present invention, conservative amino acid substitutions involve replacing an amino acid residue with an amino acid residue having a side chain with similar properties. Amino acid residues are classified into several families based on their side chains, such as basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan). Conservative amino acid substitutions are preferably substitutions between amino acid residues within the same family.

[0100] In one embodiment of the present invention, the above-mentioned amino acid is a general term for organic compounds having an amino group and a carboxyl group. When a protein according to an embodiment of the present invention comprises a "specific amino acid sequence," any of the amino acids in the amino acid sequence may be chemically modified. Any of the amino acids in the amino acid sequence may form a salt or a solvate. Any of the amino acids in the amino acid sequence may be in the L- or D-form. Even in such cases, the protein according to an embodiment of the present invention can be said to comprise the above-mentioned "specific amino acid sequence." Examples of chemical modifications that amino acids contained in a protein undergo in vivo include N-terminal modifications (e.g., acetylation, myristoylation, etc.), C-terminal modifications (e.g., amidation, glycosylphosphatidylinositol addition, etc.), and side chain modifications (e.g., phosphorylation, glycosylation, etc.).

[0101] In one embodiment of the present invention, significance may be evaluated as statistically significant using a Student's t-test (one-tailed or two-tailed) where p<0.05 or p<0.01, or may be evaluated as significant when a substantial difference occurs.

[0102] All publications cited herein are incorporated by reference in their entirety. In this specification, "or" is used when "at least one or more" of the items listed in the text can be employed. The same applies to "alternative." In this specification, when it is stated that "within a range of two values," the range also includes the two values ​​themselves. In this specification, "A to B" includes A and B. As used herein, "the above (1) to (41)" includes reference to any one or more of (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21), (22), (23), (24), (25), (26), (27), (28), (29), (30), (31), (32), (33), (34), (35), (36), (37), (38), (39), (40), or (41).

[0103] An embodiment of the present invention may include, for example, the following embodiments. 1. A method for producing a culture supernatant, comprising the step of recovering a culture supernatant from a medium containing mesenchymal stem cells and an imidazole dipeptide. 2. The production method described in 1 above, wherein the mesenchymal stem cells are mesenchymal stem cells cultured in a medium containing imidazole dipeptide. 3. The production method described in 1 or 2 above, which comprises the step of culturing mesenchymal stem cells in a medium containing imidazole dipeptide to produce cultured mesenchymal stem cells. 4. The production method described in any one of 1 to 3 above, wherein the recovery step includes a step of centrifuging or filtering a medium containing mesenchymal stem cells and imidazole dipeptide. 5. The production method according to any one of 1 to 4 above, comprising the step of sterilizing the collected culture supernatant to produce a sterilized culture supernatant. 6. The production method according to any one of 1 to 5 above, wherein the culture supernatant is a culture supernatant obtained by adherent culture. 7. The production method described in any one of 1 to 6 above, wherein the culture supernatant contains at least 500 pg / mL of G-CSF or at least 2700 pg / mL of IL-6. 8. The production method described in any one of 1 to 7 above, wherein the culture supernatant contains G-CSF, MCP-1, VEGF-C, TGF-β1, IL-6, IL-7, IL-8, and exosomes. 9. The production method described in any one of 1 to 8 above, wherein the imidazole dipeptide is a compound having the structure of the aforementioned formula (1) or (2). 10. The production method described in any one of 1 to 9 above, wherein the imidazole dipeptide is carnosine, anserine, balenine, or homocarnosine. 11. The production method described in any one of 1 to 10 above, wherein the imidazole dipeptide is carnosine. 12. The production method described in any one of 1 to 10 above, wherein the imidazole dipeptide is anserine. 13. A culture supernatant obtained by the production method described in any one of 1 to 12 above. 14. A pharmaceutical composition comprising a culture supernatant obtained by the production method described in any one of 1 to 12 above. 15. The culture supernatant according to 13 above or the pharmaceutical composition according to 14 above, for use in inhibiting bone differentiation. 16. A syringe, vial, or medical bag containing the culture supernatant described in 13 above or the pharmaceutical composition described in 14 or 15 above. 17. A method for culturing cells, comprising the step of culturing mesenchymal stem cells in a serum-free medium containing imidazole dipeptide to produce cultured cells. 18. The culture method described in 17 above, which comprises a step of recovering the cultured cells from the medium. 19. The culture method described in 17 or 18 above, wherein the culture comprises a step of culturing in the presence of an adhesion factor. 20. The culture method according to any one of 17 to 19 above, wherein the medium is insulin-free, IGF-free, or FGF-free. 21. The culture method described in any one of 17 to 19 above, wherein the culture medium is a growth factor-free culture medium. 22. The culture method according to any one of 17 to 19 above, wherein the medium is a protein-free medium. 23. A method for producing cells, comprising a step of carrying out the culture method described in any one of 17 to 22 above. 24. Cells obtained by the production method described in 23 above. 25. A composition comprising a cell population of cells according to 24 above. 26. A pharmaceutical composition comprising cells obtained by the production method described in 23 above. 27. The pharmaceutical composition described in 26 above for inhibiting bone differentiation. 28. A medium for culturing mesenchymal stem cells, the medium containing imidazole dipeptide and being serum-free. 29. The medium according to claim 28, which is an insulin-free, IGF-free, or FGF-free medium. 30. The medium according to 28 or 29 above, which is growth factor-free. 31. The medium according to any one of 28 to 30 above, which is protein-free. 32. A medium according to any one of 28 to 31 above, which contains mesenchymal stem cells. 33. A method for inhibiting osteoblast formation, comprising the step of culturing mesenchymal stem cells in a medium containing imidazole dipeptide. 34. A method for inhibiting osteoblast production, comprising the step of contacting mesenchymal stem cells with a culture supernatant obtained by culturing mesenchymal stem cells in a medium containing an imidazole dipeptide. 35. A composition for inhibiting osteoblast formation, comprising an imidazole dipeptide. 36. A composition for inhibiting osteoblast formation, comprising a culture supernatant obtained by culturing mesenchymal stem cells in a medium containing imidazole dipeptide. 37. A method for promoting the expression of G-CSF, MCP-1, VEGF-C, TGF-β1, IL-6, IL-7, IL-8, M-CSF, osteoprotegrin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, semaphorin 3B, or an exosome marker, comprising the step of culturing mesenchymal stem cells in a medium containing imidazole dipeptide. 38. A composition for promoting the expression of G-CSF, MCP-1, VEGF-C, TGF-β1, IL-6, IL-7, IL-8, M-CSF, osteoprotegrin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, semaphorin 3B, or an exosome marker in mesenchymal stem cells, comprising an imidazole dipeptide. 39. A method for promoting exosome secretion, comprising the step of culturing mesenchymal stem cells in a medium containing imidazole dipeptide. 40. A composition for promoting exosome secretion from mesenchymal stem cells, comprising an imidazole dipeptide. 41. A mesenchymal stem cell culture supernatant containing at least 500 pg / ml of G-CSF. 42. A mesenchymal stem cell culture supernatant containing at least 2700 pg / ml of IL-6. 43. Umbilical cord-derived mesenchymal stem cells that highly express or secrete G-CSF or IL-6. 44. A composition comprising a cell population of cells according to 43 above. 45. IL-34 positive mesenchymal stem cells. 46. ​​A purified mesenchymal stem cell according to 45 above. 47. ADAM8-positive mesenchymal stem cells. 48. A purified mesenchymal stem cell according to 47 above. 49. Mesenchymal stem cells according to any one of 45 to 48 above, which are positive for osteoprotegerin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, and semaphorin 3B. 50. A purified mesenchymal stem cell according to 49 above. 51. A pharmaceutical composition comprising the mesenchymal stem cells described in any one of 45 to 50 above. 52. A pharmaceutical composition described in 51 above for inhibiting bone differentiation. 53. A pharmaceutical composition for inhibiting bone differentiation, comprising cells obtained by culturing mesenchymal stem cells in a medium containing imidazole dipeptide. 54. The pharmaceutical composition according to claim 53, further comprising a pharmaceutically acceptable carrier. 55. The pharmaceutical composition described in 53 or 54 above, wherein the cells are positive for osteoprotegerin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, and semaphorin 3B. 56. The pharmaceutical composition according to any one of 53 to 55 above, wherein the cells are obtained by culturing in a medium containing imidazole dipeptide and LIF. 57. The pharmaceutical composition described in any one of 53 to 56 above, wherein the culture is an adherent culture. 58. A method for producing a pharmaceutical composition for inhibiting bone differentiation, comprising the step of culturing mesenchymal stem cells in a medium containing imidazole dipeptide to produce cultured cells. 59. The production method described in 58 above, which includes a step of recovering the cultured cells from the medium. 60. A production method described in 58 or 59 above, which comprises a step of mixing the cultured cells with a pharmaceutically acceptable carrier. 61. A medium containing an imidazole dipeptide and a LIF (leukemia inhibitory factor) component. 62. The medium according to claim 61, further comprising an adhesion factor. 63. A medium according to 61 or 62 above, for culturing mesenchymal stem cells. 64. A medium according to any one of 61 to 63 above, containing mesenchymal stem cells. 65. A method for producing cells, comprising the step of culturing mesenchymal stem cells in the medium described in any one of 61 to 64 above to generate cultured cells. 66. The production method according to claim 65, wherein the culture is an adherent culture. 67. Cells obtained by the production method described in 65 or 66 above. 68. A composition comprising a cell population of cells described in 67 above. 69. A pharmaceutical composition comprising cells obtained by the production method described in 65 or 66 above. 70. A pharmaceutical composition described in 69 above for inhibiting bone differentiation. 71. A kit comprising an imidazole dipeptide and an LIF component. 72. A kit according to claim 71, for use in preparing a medium for culturing mesenchymal stem cells.

[0104] Although the embodiments of the present invention have been described above, these are merely examples of forms that may be included in the present invention, and the present invention is not limited to these, and various configurations other than those described above may also be adopted. Furthermore, the present invention may be adopted by combining or independently adopting each of the configurations or features described in the above embodiments. [Example]

[0105] The present invention will be further explained below with reference to examples, but is not limited to these.

[0106] Example 1: Analysis of culture supernatant 1.1 Experimental method: Cultivation of MSCs using imidazole dipeptide-containing medium and collection of culture supernatant In this experiment, carnosine was used as the imidazole dipeptide. The experimental procedure was as follows: Human umbilical cord-derived mesenchymal stem cells were suspended at a cell count of 0.3 x 10e5 in 20 ml of MSC Expansion XSFM B (Fujifilm Wako Pure Chemical Industries, Ltd.) (hereinafter referred to as MSC medium B). 10 μl of iMatrix-511 laminin fragment (0.5 μg / μl) (Nippi Corporation) was added to the 20 ml cell suspension, and the resulting cell suspension was seeded into a T150 flask (Sumitomo Bakelite Co., Ltd.).

[0107] Seven days after seeding, 10 ml of MSC medium B containing 5 μl of iMatrix-511 laminin fragment (0.5 μg / μl) (Nippi Corporation) was added. 14 days after seeding (day 14), the medium was replaced twice with PBS to wash away any remaining medium. Then, 30 ml of protein-free medium (hereinafter referred to as MSC medium A) based on DMEM / F12 medium supplemented with amino acids was added (hereinafter, 30 ml was used when replacing with MSC medium A). The amino acids added here were MEM essential amino acid solution (Fujifilm Wako Pure Chemical Corporation) and MEM non-essential amino acid solution (Fujifilm Wako Pure Chemical Corporation). Furthermore, the imidazole dipeptide L-carnosine (Fujifilm Wako Pure Chemical Corporation) was added to MSC medium A at concentrations of 0 mM, 1 mM, 10 mM, or 30 mM. The cells were added to MSC medium A supplemented with carnosine and cultured for 3 days (production culture). This culture is referred to as the "first production culture."

[0108] After the first production culture, the culture supernatant of MSC medium A was collected. The amount of cytokines contained in the culture supernatant was analyzed using an ELISA analysis kit (R&D Systems) or a Milliplex kit (Merck Millipore). The cytokines analyzed were hepatocyte growth factor (HGF), granulocyte colony stimulating factor (G-CSF), monocyte chemotactic protein 1 (MCP-1), vascular endothelial growth factor-C (VEGF-C), transforming growth factor-β1 (TGF-β1), interleukin-6 (IL-6), interleukin-7 (IL-7), and interleukin-8 (IL-8). The amount of exosomes contained in the culture supernatant was also analyzed using a CD9 / CD63 ELISA kit (Cosmo Bio) using the exosome marker protein (CD9 / CD63 fusion protein) as an indicator. In this example, the amounts of cytokines and exosome marker proteins refer to values ​​measured by ELISA using specific antibodies.

[0109] After the first production culture, the medium was returned to MSC medium B and cultured for two days (recovery culture). After the two-day recovery culture, the medium was replaced with MSC medium A supplemented with carnosine as described above and cultured for three days (the carnosine concentration was the same as in the first production culture). This culture is referred to as the "second production culture."

[0110] After the second production culture, the culture supernatant of MSC medium A supplemented with carnosine was collected. Cytokines contained in the culture supernatant were analyzed using an ELISA analysis kit (R&D Systems).

[0111] After the second production culture, the medium was returned to MSC medium B and cultured for two days (recovery culture). After the two-day recovery culture, the medium was replaced with MSC medium A supplemented with carnosine as described above and cultured for three days (the carnosine concentration was the same as in the first production culture). This culture is referred to as the "third production culture."

[0112] After the third production culture, the culture supernatant of MSC medium A supplemented with carnosine was collected. Cytokines contained in the culture supernatant were analyzed using an ELISA analysis kit (R&D Systems).

[0113] After the third production culture, the medium was returned to MSC medium B and cultured for two days (recovery culture). After the two-day recovery culture, the medium was replaced with MSC medium A supplemented with carnosine as described above and cultured for three days (the carnosine concentration was the same as in the first production culture). This culture is referred to as the "fourth production culture."

[0114] After the fourth production culture, the culture supernatant of MSC medium A supplemented with carnosine was collected. Cytokines contained in the culture supernatant were analyzed using an ELISA analysis kit (R&D Systems).

[0115] As described above, a total of four production cultures were performed, and for MSC medium A supplemented with various concentrations of carnosine, a total of four culture supernatant collections and cytokine analysis were performed. After the fourth collection of culture supernatant, cell morphology was observed. Furthermore, after the fourth collection of culture supernatant, the cells were enzymatically treated with TrypLE™ Select (Thermo Fisher Scientific) for 20 minutes, dispersed, and then counted. The results of these experiments are described below.

[0116] 1.2 Cell morphology observation 1 to 4 are micrographs of MSCs cultured in carnosine-containing or -free media. Cell death was not observed, and no clear differences in cell morphology were observed at each carnosine concentration.

[0117] 1.3 Cell counting Figure 5 shows the results of counting MSC cell numbers after culturing in a medium containing or not containing carnosine. Addition of carnosine to the medium did not significantly change the cell number.

[0118] 1.4 Exosome marker amount Figure 6 shows the results of examining the amount of exosome markers in the culture supernatant. Addition of carnosine to the medium showed a tendency for the exosome markers to increase.

[0119] 1.5 Cytokine levels Figures 7 to 14 show the results of examining the amounts of HGF (HGF, 3082), G-CSF (CSF3, 1440), MCP-1 (CCL2, 6347), VEGF-C (VEGFC, 7424), TGF-β1 (TGFB1, 7040), IL-6 (IL6, 3569), IL-7 (IL7, 3574), and IL-8 (CXCL8, 3576) in the culture supernatant. The addition of carnosine to the medium did not significantly change the amount of HGF. On the other hand, the amounts of G-CSF, MCP-1, VEGF-C, TGF-β1, IL-6, IL-7, and IL-8 tended to increase (the numbers in parentheses indicate the official NCBI symbol and gene ID). The increase rates of G-CSF and IL-6 were particularly significant. When comparing the results at the fourth collection of culture supernatant, the G-CSF concentration increased 14.7-fold (approximately 1621 pg / mL) and the IL-6 concentration increased 22-fold (over 7000 pg / mL) when 10 mM carnosine was added.

[0120] Furthermore, in the same experimental method as above, umbilical cord-derived MSCs were replaced with adipose-derived MSCs for culture. The amounts of G-CSF and MCP-1 in the culture supernatant were then measured. The results showed that the addition of carnosine to the culture medium increased both levels.

[0121] Example 2: Inhibitory effect of culture supernatant on bone differentiation 2.1 Experimental method 2.1.1 Cultivation of MSCs using imidazole dipeptide-containing medium and collection of culture supernatant Using human umbilical cord-derived mesenchymal stem cells (CET03 line) established by the present inventors, culture supernatants were collected according to the experimental method up to the third production culture in Example 1 described above. However, this time, the MSC medium A used in Example 1 was replaced with DMEM (Sigma), DMEM / F12 (Sigma), or IMDM (Sigma). Furthermore, the concentration of carnosine added was changed to 0 or 10 mM. As a result, first, second, and third production culture supernatants were obtained. Furthermore, equal amounts of the first to third production culture supernatants were mixed to obtain umbilical cord MSC supernatant. The six types of umbilical cord MSC supernatants obtained are referred to as DMEM_supernatant, DMEM_C_supernatant, DMEM / F12_supernatant, DMEM / F12_C_supernatant, IMDM_supernatant, and IMDM_C_supernatant (the names of these supernatants are indicated by the name of the medium used on the left side. Supernatants obtained under carnosine-supplemented conditions are indicated by a C to the right of the medium name).

[0122] 2.1.2 MSC osteogenic differentiation induction and calcium deposition analysis Procedure 1. Human bone marrow MSCs (primary cultured human bone marrow MSCs) were cultured in α-MEM medium containing 10% FBS (Hyclone) and treated with trypsin-EDTA solution to dissociate into single cells. The resulting single cells were seeded onto fibronectin-coated 24-well plates at a density of 8 x 10e4 cells / well in basal medium (DMEM (Sigma) supplemented with 0.6 mM CaCl2) containing 10% FBS. The day after seeding, the medium was replaced with basal medium (DMEM (Sigma) supplemented with 0.6 mM CaCl2) supplemented with osteogenic differentiation components (10% FBS, 0.1 μM dexamethasone, 50 μM ascorbic acid, and 10 mM β-glycerophosphate) and carnosine at a final concentration of 0 or 10 mM (hereinafter referred to as osteogenic differentiation medium). The medium used for this medium replacement was replaced every 2–3 days, and the cells were cultured for 10–14 days until calcium deposition was observed on the cells. After culturing, the amount of intracellular calcium deposition in the cells was quantified by calcium assay. The cells obtained are designated as DMEM_medium_cells and DMEM_C_medium_cells (the name of the cell is indicated by the name of the medium used on the left side of the cell name. Cells obtained under carnosine-supplemented conditions are indicated by C to the right of the medium name).

[0123] Step 2: Step 1 was repeated except that the basal medium was replaced with DMEM / F12 (Sigma) supplemented with 0.6 mM CaCl2. The resulting cells are designated DMEM / F12 medium cells and DMEM / F12 C medium cells (the names of these cells are indicated by the name of the medium used on the left. Cells obtained under carnosine-supplemented conditions are designated by a C to the right of the medium name).

[0124] Step 3. Step 1 was repeated except that the basal medium was replaced with IMDM (Sigma) supplemented with 2 mM L-glutamin. The resulting cells are designated IMDM_medium_cells and IMDM_C_medium_cells (the names of these cells are indicated by the name of the medium used on the left. Cells obtained under carnosine-supplemented conditions are indicated by a C to the right of the medium name). The same procedure as Step 3 was also performed separately, and calcium deposition images were taken of the cells after osteogenic differentiation induction culture using an Alizarin Red S staining kit (Cosmo Bio).

[0125] Step 4. The osteogenic differentiation medium used in Step 1 was replaced with the umbilical cord MSC culture supernatant (DMEM_supernatant or DMEM_C_supernatant) obtained in 2.1.1 above, supplemented with components for osteogenic differentiation (10% FBS, 0.1 μM dexamethasone, 50 μM ascorbic acid, and 10 mM β-glycerophosphate). The resulting cells are referred to as DMEM_supernatant_cells and DMEM_C_supernatant_cells (the names of these cells are indicated by the name of the medium used on the left. Cells obtained under carnosine-supplemented conditions are indicated by a letter C to the right of the medium name).

[0126] Step 5. The osteogenic differentiation medium used in Step 2 was replaced with the umbilical cord MSC culture supernatant (DMEM / F12 supernatant or DMEM / F12_C_supernatant) obtained in Step 2.1.1 above, supplemented with components for osteogenic differentiation (10% FBS, 0.1 μM dexamethasone, 50 μM ascorbic acid, and 10 mM β-glycerophosphate). The resulting cells are designated DMEM / F12_supernatant_cells and DMEM / F12_C_supernatant_cells (the names of these cells are indicated by the name of the medium used on the left. Cells obtained under carnosine-supplemented conditions are indicated by a C to the right of the medium name).

[0127] Step 6. The osteogenic differentiation medium used in Step 3 was replaced with the umbilical cord MSC culture supernatant (IMDM_supernatant or IMDM_C_supernatant) obtained in Step 2.1.1 above, supplemented with components for osteogenic differentiation (10% FBS, 0.1 μM dexamethasone, 50 μM ascorbic acid, and 10 mM β-glycerophosphate). The resulting cells are designated IMDM_supernatant_cells and IMDM_C_supernatant_cells (the names of these cells are indicated by the medium name on the left. Cells obtained under carnosine-supplemented conditions are indicated by a C to the right of the medium name). The same procedure as Step 6 was also performed separately, and calcium deposition images were taken of the cells after osteogenic differentiation induction culture using an Alizarin Red S staining kit (Cosmo Bio).

[0128] 2.2 Results Figure 15 shows the results of quantifying the amount of calcium deposition in bone marrow MSCs. The names of the media in the figure indicate the names of the basal media used. Quantifying the osteogenic differentiation of bone marrow MSCs by calcium content revealed that the cells obtained using the supernatant of MSCs cultured in carnosine-supplemented medium (supernatant_C_10mM in the figure; corresponding samples are DMEM_C_supernatant_cells, DMEM / F12_C_supernatant_cells, and IMDM_C_supernatant_cells) had significantly lower calcium levels than the cells obtained using a medium without carnosine (medium_C_0mM in the figure; corresponding samples are DMEM_medium_cells, DMEM / F12_C_supernatant_cells, and IMDM_medium_cells) and the cells obtained using the supernatant of MSCs cultured in a medium without carnosine (supernatant_C_0mM in the figure; corresponding samples are DMEM_supernatant_cells, DMEM / F12_supernatant_cells, and IMDM_supernatant_cells). Furthermore, the calcium content was significantly lower than that of cells obtained using carnosine-supplemented medium (medium C 10 mM in the figure; corresponding samples are DMEM C medium cells, DMEM / F12 C medium cells, and IMDM C medium cells). The supernatant of MSCs obtained by culturing in carnosine-supplemented medium was found to have a significant inhibitory effect on bone differentiation.

[0129] Figure 16 shows images of Alizarin Red S staining of bone marrow MSCs. When bone marrow MSCs were stained with Alizarin Red S after osteogenic differentiation induction, cells obtained using a medium without carnosine (DMEM_medium_cells), cells obtained using the supernatant of MSCs cultured in a medium without carnosine (DMEM_supernatant_cells), and cells obtained using a medium containing carnosine (DMEM_C_medium_cells) were stained with Alizarin Red. On the other hand, cells obtained using the supernatant of MSCs cultured in a medium containing carnosine (DMEM_C_supernatant_cells) were not stained with Alizarin Red. The supernatant of MSCs cultured in a medium containing carnosine was found to have a significant inhibitory effect on osteogenic differentiation.

[0130] Example 3: Inhibitory effect of MSCs on bone differentiation 3.1 Experimental method 3.1.1 Cultivation of umbilical cord MSCs using imidazole dipeptide-containing medium Human umbilical cord MSCs established by the present inventors were cultured in DMEM / F12 medium (hereinafter referred to as MSC medium C) containing 10 ng / ml bFGF (Peprotech, AF-100-18C), 10 μg / ml insulin (Nacalai, 12878-44), 1000 μg / ml albumin (Sigma, A9511), 10 μg / ml transferrin (Nacalai, 12879-34), and 1 ng / ml LIF (Peprotech, AF-300-05). After treatment with trypsin-EDTA solution, single cells were dissociated. Carnosine was added to the same medium at a final concentration of 0 or 5 mM, and iMatrix-511 laminin fragments (Nippi Corporation) were added to a final concentration of 0.25 μg / ml, and the cells were seeded into T25 flasks (Corning). After 2 weeks of culture, the cells were washed with PBS to remove carnosine, then treated with trypsin-EDTA solution to dissociate them into single cells, which were then used for co-culture with bone marrow MSCs as described below.

[0131] 3.1.2 Co-culture of bone marrow MSCs and umbilical cord MSCs Bone marrow MSCs (primary cultured human bone marrow MSCs) were cultured in αMEM medium containing 10% FBS (Hyclone) and treated with trypsin-EDTA solution to dissociate into single cells. These bone marrow MSCs were mixed with umbilical cord MSCs obtained by culturing in the medium containing 0 or 5 mM carnosine (as described in 3.1.1 above) at a ratio of 1:0, 0:1, 1:1, or 3:1, respectively. Furthermore, the combined MSCs were seeded onto a 24-well plate in αMEM medium containing 10% FBS at a total cell count of 8.0 x 10e4 cells / well. The day after seeding, the medium in the wells for each co-culture cell condition was replaced with either (1) 10% FBS αMEM medium supplemented with osteogenic differentiation components (0.1 μM dexamethasone, 50 μM ascorbic acid, and 10 mM β-glycerophosphate) or (2) 10% FBS αMEM medium without osteogenic differentiation components. The medium was replaced with the same medium every 2–3 days for 9 days. Osteogenic differentiation was assessed by calcium assay and Alizarin Red S staining.

[0132] 3.2 Results Figure 17 shows the quantitative results of calcium deposition during coculture of bone marrow MSCs and umbilical cord MSCs. While significant calcium deposition was observed in bone differentiation conditions involving a 1:1 coculture of bone marrow MSCs and umbilical cord MSCs (BM:UC (1:1)), calcium deposition was significantly suppressed in bone differentiation conditions involving a 1:1 coculture of bone marrow MSCs with umbilical cord MSCs precultured with carnosine (BM:UC_preC (1:1)). Umbilical cord MSCs precultured with carnosine were shown to have the effect of suppressing MSC osteogenic differentiation. Because carnosine was removed from the umbilical cord MSCs by washing them with PBS after preculture and because carnosine was not added to the medium during osteogenic differentiation induction, the effect was observed solely in the cells (umbilical cord MSCs precultured with carnosine). The following terms are used in the figure: BM: Non-co-culture conditions using bone marrow MSCs 8x10^4 cells / well. αMEM medium conditions with osteogenic differentiation inducers. BM_NC: Non-co-culture conditions using bone marrow MSCs 8x10^4 cells / well. αMEM medium conditions without osteogenic differentiation inducers. UC: Non-co-culture conditions using umbilical cord MSCs (8x10^4 cells / well) pre-cultured for 2 weeks in MSC medium C without carnosine. αMEM medium condition with osteogenic differentiation inducer. UC_preC: Non-co-culture conditions using umbilical cord MSCs (8x10^4 cells / well) pre-cultured for 2 weeks in MSC medium C containing 5mM carnosine. αMEM medium conditions with osteogenic differentiation induction. BM:UC (1:1): Co-culture conditions in which bone marrow MSCs and umbilical cord MSCs pre-cultured for 2 weeks in MSC medium C without carnosine were mixed and seeded at 4x10^4 cells / well. αMEM medium condition with osteogenic differentiation inducer. BM:UC_preC (1:1): Bone marrow MSCs and umbilical cord MSCs pre-cultured for 2 weeks in CET original medium containing 5mM carnosine were mixed and seeded at 4x10^4 cells / well in a co-culture condition. αMEM medium condition with osteogenic differentiation inducer. BM:UC (3:1): Co-culture conditions in which bone marrow MSCs (6x10^4 cells / well) were mixed and seeded with umbilical cord MSCs (2x10^4 cells / well) that had been pre-cultured for 2 weeks in MSC medium C without carnosine. αMEM medium conditions with osteogenic differentiation inducers. BM:UC_preC (3:1): Co-culture conditions in which bone marrow MSCs (6x10^4 cells / well) and umbilical cord MSCs (2x10^4 cells / well) pre-cultured for 2 weeks in CET original medium containing 5mM carnosine were mixed and seeded. αMEM medium conditions with osteogenic differentiation inducers.

[0133] Figure 18 shows images of Alizarin Red S staining during co-culture of bone marrow MSCs and umbilical cord MSCs. In the osteogenic differentiation conditions (BM:UC (1:1)) of bone marrow MSCs and umbilical cord MSCs, a large amount of calcium was deposited. However, in the osteogenic differentiation conditions (BM:UC_preC (1:1)) of bone marrow MSCs and umbilical cord MSCs pre-cultured with carnosine, calcium deposition was significantly suppressed. Umbilical cord MSCs pre-cultured with carnosine were shown to have the effect of suppressing MSC osteogenic differentiation.

[0134] Example 4: Analysis of MSCs 4.1 Experimental method 4.1.1 RNA collection from MSCs cultured in imidazole dipeptide-containing medium Using human umbilical cord-derived mesenchymal stem cells (CET03 line) established by the present inventors, culture supernatants were collected according to the experimental method up to the third production culture in Example 1. However, the proliferation medium (MSC medium B), supernatant collection medium (MSC medium A), carnosine concentration, and culture vessel used in Example 1 were changed as shown in Figure 19. The supernatant collection medium was αMEM (Sigma), DMEM (Sigma), or DMEM / F12 (Sigma). The proliferation medium was MSC medium B or MSC medium C (see Example 3 for composition). Carnosine was added at concentrations of 0 mM, 10, 20, or 30 mM. The resulting first, second, and third production culture supernatants were used in ELISA tests. RNA was extracted from the cells after the first culture and used for RNAseq analysis, as described below.

[0135] 4.1.2 RNAseq analysis On day 27 after supernatant collection, cells were harvested, and RNA was extracted using the miRNeasy Mini Kit (Qiagen) and used for RNAseq analysis. The sequencing analysis equipment used was the NextSeq500 (Illumina). The sequence read length was 75 bp for a single read, equivalent to 1 million reads. The analysis program was StrandNGS ver. 4.0 (Strand Life Sciences Pvt Ltd). Fastq reads after removal of unreliable bases were used to map to the human reference genome hg38, and RPM expression level correction was performed. RPM correction values ​​for each gene were calculated for a total of 19 samples.

[0136] 4.1.3 Listing of genes with increased expression levels by RNAseq analysis The average percentage increase in gene expression (comparing 0 mM carnosine with 10-30 mM carnosine) was calculated for a total of 13 cell samples obtained under carnosine-supplemented conditions. Genes with the highest average percentage increase in cells obtained under carnosine-supplemented conditions were listed in descending order. Of these, the top 21 genes encoding secreted proteins were selected as follows: From top to bottom: gremlin 1, DAN family BMP antagonist, inhibitor subunit beta E, thymic stromal lymphopoietin, hyaluronan and proteoglycan link protein 3, KIT ligand, R-spondin 2, semaphorin 3B, ADAM metallopeptidase with thrombospondin type 1 motif 13, fibroblast growth factor 11, TNF receptor superfamily member 11b, angiopoietin like 7, ADAM metallopeptidase domain 8, fibroblast growth factor binding protein 3, hyaluronan and proteoglycan link protein 1, transforming growth factor beta regulator 4, interleukin 34, neurotrophin 3, insulin like growth factor binding protein 2, growth differentiation factor 6, amphiregulin, dickkopf WNT signaling pathway inhibitor 1.

[0137] 4.2 Results RNA sequencing analysis identified the top 21 secreted protein-encoding genes whose expression levels were increased by carnosine. Of these, the following 10 genes have previously been reported to be involved in the inhibition of bone formation: gremlin 1, DAN family BMP antagonist (GREM1, 26585, Gremlin 1), KIT ligand (KITLG, 4254, SCF), R-spondin 2 (RSPO2, 340419), semaphorin 3B (SEMA3B, 7869), fibroblast growth factor 11 (FGF11, 2256), TNF receptor superfamily member 11b (TNFRSF11B, 4982, Osteoprotegerin), ADAM metallopeptidase domain 8 (ADAM8, 101), interleukin 34 (IL34, 146433, IL-34), insulin like growth factor binding protein 2 (IGFBP2, 3485), dickkopf WNT signaling pathway inhibitor 1 (DKK1, 22943). The numbers in parentheses indicate the NCBI official symbol and gene ID, as well as alternative names. It was shown that a large proportion of secreted proteins involved in the inhibition of bone formation were secreted among the secreted proteins whose expression was increased by carnosine, and that carnosine increased the expression of many secreted proteins involved in the inhibition of bone formation. These results suggest that the bone differentiation inhibitory effect produced by umbilical cord MSCs cultured under carnosine-supplemented conditions is a paracrine effect caused by secreted proteins from MSCs.

[0138] Figures 20(a)–20(j) show the changes in the percent increase values ​​of the 10 genes listed above. Figures 20(a)–20(j) show the results for gremlin 1, DAN family BMP antagonist, KIT ligand, R-spondin 2, semaphorin 3B, fibroblast growth factor 11, TNF receptor superfamily member 11b, ADAM metallopeptidase domain 8, interleukin 34, insulin-like growth factor binding protein 2, and Dickkopf WNT signaling pathway inhibitor 1, respectively. Figures 20–22 show the results when DMEM / F12 (Sigma) was used as the supernatant collection medium and MSC medium B was used as the proliferation medium. The concentrations in the figures indicate the added carnosine concentration. Figure 21 is a graph showing the RPM values ​​of interleukin 34 expression in umbilical cord MSCs. When 10 mM or 30 mM carnosine was added, interleukin 34 expression was observed, but no expression was observed without its addition (0 mM). Figure 22 is a graph showing the RPM values ​​of ADAM metallopeptidase domain 8 expression levels in umbilical cord MSCs. When 10 mM, 20 mM, or 30 mM carnosine was added, ADAM metallopeptidase domain 8 expression was observed, but no expression was observed without its addition (0 mM).

[0139] Example 5: Analysis of culture supernatant and MSCs 5.1 Cultivation of MSCs using imidazole dipeptide-containing medium and collection of culture supernatant Using human umbilical cord-derived mesenchymal stem cells (CET03 line) established by the present inventors, culture supernatants were collected according to the experimental method up to the third production culture in Example 1. However, in this case, the MSC medium A used in Example 1 was replaced with αMEM (Sigma), DMEM (Sigma), or DMEM / F12 (Sigma). Furthermore, the concentration of carnosine added was varied to 0 or 30 mM (a 500 mM carnosine solution in distilled water was used for addition; in the case of 0 mM, the same amount of distilled water was used). As a result, the first, second, and third production culture supernatants were obtained and used for ELISA testing. RNA was extracted from the cells after that and used for RNAseq analysis, as described below.

[0140] 5.2 ELISA analysis The amount of cytokines contained in the culture supernatant obtained in 5.1 above was analyzed using an ELISA analysis kit (R&D Systems). The cytokines analyzed were osteoprotegerin and M-CSF (CSF1, 1435). These two cytokines have previously been reported to be involved in the inhibition of bone formation. In this experiment, the amount of cytokines was expressed as a value measured by ELISA using specific antibodies.

[0141] 5.3 Quantitative PCR (qPCR) analysis On day 27 after supernatant collection, cells were harvested, and RNA was extracted using the miRNeasy Mini Kit (Qiagen). Quantitative PCR (PowerUp SYBR Green Master Mix, Thermo Fisher Scientific) was used to analyze the expression levels of TNFRSF11B, the osteoprotegerin gene, and CSF1, the M-CSF gene, using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as an internal standard.

[0142] 5.4 Results Figures 23 to 25 show the results of ELISA analysis of osteoprotegerin, and Figures 26 to 28 show the results of ELISA analysis of M-CSF. The concentrations (mM) in the figures refer to carnosine concentrations. The supernatant collection medium used was αMEM in Figures 23 and 26, DMEM in Figures 24 and 27, and DMEM / F12 in Figures 25 and 28. Addition of carnosine tended to increase osteoprotegerin and M-CSF secretion levels in a concentration-dependent manner for the first, second, and third collection runs. Figure 29 shows the results of qPCR analysis of osteoprotegerin, and Figure 30 shows the results of qPCR analysis of M-CSF. The concentrations (mM) in the figures refer to carnosine concentrations, and the names of the media used refer to the supernatant collection medium used. Under each condition, addition of carnosine tended to increase osteoprotegerin and M-CSF gene expression levels in a concentration-dependent manner.

[0143] Example 6: Analysis of culture supernatant and MSCs 6.1 Cultivation of MSCs using imidazole dipeptide-containing medium and collection of culture supernatant Using human umbilical cord-derived mesenchymal stem cells (CET03 strain) or human bone marrow-derived mesenchymal stem cells (ATCC) established by the present inventors, culture supernatants were collected according to the experimental method described in Example 1 up to the third production culture. However, in this case, MSC medium B or MSC medium C (see Example 3 for composition) was used as the growth medium (MSC medium B in Example 1). Furthermore, DMEM / F12 (Sigma) was used as the supernatant collection medium (MSC medium A in Example 1). Carnosine was added at concentrations of 0, 10, 20, or 30 mM (a 500 mM carnosine solution in distilled water was used for the addition; an equal volume of distilled water was used for the 0 mM addition). Furthermore, the imidazole dipeptide anserine was added instead of carnosine to final concentrations of 0, 5, 10, or 20 mM. Consequently, production culture supernatants were obtained from the first, second, and third cultures and used for ELISA testing. RNA was then extracted from the cells and used for the quantitative PCR test described below. The procedures for ELISA analysis and quantitative PCR test were the same as in Example 5.

[0144] 6.2 Results Figure 31 shows the results of ELISA analysis of osteoprotegin. Figure 31(a) shows the results with carnosine added, and Figure 31(b) shows the results with anserine added. In both cases, umbilical cord MSCs were used, and MSC medium B was used as the growth medium. There was a tendency for the amount of osteoprotegin secreted to increase with the addition of carnosine or anserine, both in the first and second collections.

[0145] Figures 32 and 33 show the results of qPCR analysis of osteoprotegin. Figures 32(a) to (c) show the results under carnosine-supplemented conditions, and Figures 33(a) to (c) show the results under anserine-supplemented conditions. In each figure, UC_B_C indicates that umbilical cord MSCs were used as the cells, MSC medium B was used as the growth medium, and carnosine was used as an additive. In each figure, BM_B_C indicates that bone marrow MSCs were used as the cells, MSC medium B was used as the growth medium, and carnosine was used as an additive. In each figure, BM_C_C indicates that bone marrow MSCs were used as the cells, MSC medium C was used as the growth medium, and carnosine was used as an additive. In each figure, UC_B_A indicates that umbilical cord MSCs were used as the cells, MSC medium B was used as the growth medium, and anserine was used as an additive. In each figure, BM_B_A indicates that bone marrow MSCs were used as the cells, MSC medium B was used as the growth medium, and anserine was used as an additive. In each figure, BM_C_A indicates that bone marrow MSCs were used as the cells, MSC medium C was used as the growth medium, and anserine was used as the additive. Under each condition, the addition of carnosine or anserine tended to increase osteoprotegin gene expression levels.

[0146] Figure 34 shows the results of ELISA analysis of M-CSF. Figure 34(a) shows the results with the addition of carnosine, and Figure 34(b) shows the results with the addition of anserine. In both cases, umbilical cord MSCs were used, and MSC medium B was used as the growth medium. There was a tendency for the amount of M-CSF secreted to increase with the addition of carnosine or anserine, both in the first and second collections.

[0147] Figures 35 and 36 show the results of qPCR analysis of M-CSF. Figures 35(a) and (b) show the results under carnosine-supplemented conditions, and Figures 36(a) and (b) show the results under anserine-supplemented conditions. In each figure, UC_B_C indicates that umbilical cord MSCs were used as the cells, MSC medium B was used as the growth medium, and carnosine was used as the additive. In each figure, BM_B_C indicates that bone marrow MSCs were used as the cells, MSC medium B was used as the growth medium, and carnosine was used as the additive. In each figure, UC_B_A indicates that umbilical cord MSCs were used as the cells, MSC medium B was used as the growth medium, and anserine was used as the additive. In each figure, BM_B_A indicates that bone marrow MSCs were used as the cells, MSC medium B was used as the growth medium, and anserine was used as the additive. Under each condition, the addition of carnosine or anserine tended to increase M-CSF gene expression levels.

[0148] Example 7: Proliferation-promoting effect of MSCs 7.1 Experimental Method 7.1.1 MSC expansion culture Human umbilical cord-derived mesenchymal stem cells established by the present inventors were detached using TrypLE Select and seeded onto 24-well plates at 200 or 2,000 cells / cm. The culture media used were (1) MSC Expansion XSFM B (Fujifilm Wako Pure Chemical Corporation) (hereinafter also referred to as MSC medium B), (2) DMEM / F12 medium containing 10 ng / ml bFGF (Peprotech), 10 μg / ml insulin (Nacalai), 1,000 μg / ml albumin (Sigma), 10 μg / ml transferrin (Nacalai), and 1 ng / ml LIF (Peprotech) (hereinafter also referred to as MSC medium D), or (3) MSC medium D without LIF (hereinafter also referred to as MSC medium E). In addition, iMatrix-511 laminin fragment (Nippi Corporation) was added to each medium at a final concentration of 0 or 0.1%, and carnosine or anserine was added to final concentrations of 0, 0.2, 0.5, 1, 2, or 5 mM. Each sample was cultured for 4–7 days in the same medium used, with medium changes every 2–3 days. Subsequently, the cells were stained with DAPI, phalloidin, and CD44 antibody. Fluorescence images were taken using a CellVoyager CQ-1 confocal quantitative image cytometer (Yokogawa Electric Corporation). Cell counts were performed using DAPI, and the fluorescent areas of phalloidin (an actin filament marker) and CD44 (an MSC marker) were measured. A summary of each experimental condition is shown in Table 1. [Table 1]

[0149] Umbilical cord MSCs established by the present inventors were detached using TrypLE Select and seeded at 200 cells / cm2 in 24-well plates. The culture medium used was either (4) MSC medium D or (5) MSC medium D minus LIF (hereinafter also referred to as MSC medium E). Each medium was supplemented with iMatrix-511 laminin fragments (Nippi Corporation) at a final concentration of 0.1% and carnosine at final concentrations of 0, 0.2, 1, or 5 mM. Each sample was cultured for 7 days in the same medium used, with medium changes every 2–3 days. Subsequently, the cells were stained with DAPI and phalloidin. Fluorescence images were captured using a confocal quantitative image cytometer, CellVoyager CQ-1 (Yokogawa Electric Corporation). DAPI cell counts and phalloidin (actin filament marker) fluorescence area measurements were performed. Table 2 summarizes the experimental conditions. [Table 2]

[0150] 7.1.2 Measurement of MSC marker positivity rate Carnosine was added to MSC medium D, and umbilical cord MSCs were cultured at a density of 200 cells / cm. 2 The cells were seeded in a 5% CO2 solution and cultured in a T25 flask for 2 weeks (passage 2). The cells seeded in the T25 flask were detached using TrypLE Select, collected, and then single-celled. The collected cells were fixed in 4% formaldehyde and immunostained for CD31, 44, 45, 73, 90, 105, HLA-ABC, and HLA-DR. The percentage of positive cells for each marker was measured using a BD Accuri™ C6 Plus flow cytometer (BD Biosciences).

[0151] 7.2 Results Figures 37 and 38 show the results of cell counting for groups 1 to 4. In particular, in group 3, the addition of carnosine tended to increase the number of cells. Figure 39 shows the results of measuring the phalloidin fluorescence area for groups 1 to 4. In particular, in group 3, the addition of carnosine tended to increase the phalloidin area. Figure 40 shows the results of measuring the CD44 fluorescence area for groups 1 to 4. In particular, in group 3, the addition of carnosine tended to increase the CD44 area.

[0152] Figure 41 shows the results of taking fluorescent images (cell nucleus staining) of groups 1 and 3. A tendency for an increase in cell number was observed, particularly in group 3, where carnosine was 1 mM. Figure 42 shows the results of taking fluorescent images (phalloidin staining) of groups 1 and 3. A tendency for an increase in cell number and phalloidin area was observed, particularly in groups 1 and 3, where carnosine was 1 mM. No significant differences were observed in cell morphology. Figure 43 shows the results of taking fluorescent images (CD44 staining) of groups 1 and 3. A tendency for an increase in cell number and CD44 area was observed, particularly in group 3, where carnosine was 1 mM. No significant differences were observed in marker expression patterns.

[0153] Figures 44 and 45 show the results of cell counting for groups 5 to 8. In particular, in group 7, the addition of anserine tended to increase the cell count. Figures 46 and 47 show the results of measuring the phalloidin fluorescence area for groups 5 to 8. In particular, in group 7, the addition of anserine tended to increase the phalloidin area. Figures 48 and 49 show the results of measuring the CD44 fluorescence area for groups 5 to 8. In particular, in group 7, the addition of anserine tended to increase the CD44 area.

[0154] Figure 50 shows the results of photographing fluorescent images (phalloidin staining) for groups 5 and 7. In particular, under the anserine 1 mM condition in group 7, there was a tendency for the cell number and phalloidin area to increase. No significant differences were observed in cell morphology. Figure 51 shows the results of photographing fluorescent images (CD44 staining) for groups 5 and 7. In particular, under the anserine 1 mM condition in group 7, there was a tendency for the cell number and CD44 area to increase. No significant differences were observed in the marker expression pattern.

[0155] Figure 52 shows the results of measuring the positive rate of MSC markers after culturing umbilical cord MSCs in MSC medium D. While the cell number increased with the addition of carnosine, the levels of each MSC positive and negative marker remained unchanged with the addition of carnosine.

[0156] Figure 53 shows the results of cell counting for groups 9 and 10. In group 9, the addition of carnosine tended to increase the cell number, while no increase was observed in group 10. Figure 54 shows the results of measuring the phalloidin fluorescence area for groups 9 and 10. In group 9, the addition of carnosine tended to increase the phalloidin area, while no increase was observed in group 10.

[0157] Figure 55 shows the results of capturing fluorescent images (phalloidin staining) of Groups 9 and 10. In Group 9, where carnosine was added at 1 mM, a tendency for the phalloidin area to increase with the addition of carnosine was observed, while in Group 10, where carnosine was added at 1 mM, no tendency for an increase was observed.

[0158] These results demonstrate that the addition of carnosine to a medium containing LIF promotes the proliferation of MSCs.

[0159] The present invention has been described above based on the embodiments. However, these embodiments are merely illustrative, and it will be understood by those skilled in the art that various modifications are possible and that such modifications are also within the scope of the present invention.

Claims

1. A method for producing a culture supernatant, comprising the step of recovering a culture supernatant from a medium containing mesenchymal stem cells and an imidazole dipeptide.

2. The method according to claim 1, wherein the mesenchymal stem cells are cultured in a medium containing an imidazole dipeptide.

3. 3. The method according to claim 1, further comprising the step of culturing mesenchymal stem cells in a medium containing an imidazole dipeptide to produce cultured mesenchymal stem cells.

4. The method according to any one of claims 1 to 3, wherein the recovering step comprises centrifuging or filtering a medium containing mesenchymal stem cells and imidazole dipeptide.

5. The method according to any one of claims 1 to 4, further comprising a step of sterilizing the collected culture supernatant to produce a sterilized culture supernatant.

6. The method according to any one of claims 1 to 5, wherein the culture supernatant is a culture supernatant obtained by adhesion culture.

7. The method according to any one of claims 1 to 6, wherein the culture supernatant contains at least 500 pg / mL of G-CSF or at least 2700 pg / mL of IL-6.

8. The method according to any one of claims 1 to 7, wherein the culture supernatant contains G-CSF, MCP-1, VEGF-C, TGF-β1, IL-6, IL-7, IL-8, and exosomes.

9. The method according to any one of claims 1 to 8, wherein the imidazole dipeptide is a compound having the structure of the following formula (1) or (2): 【Chemistry 1】 【Chemistry 2】 Here, R 1 , R 2 , R 3 and R 4 are each independently H or C 1-6 is alkyl, R 5 and R 6 are each independently -NHR 7 or -CH 2 NHR 7 and R 7 H or -COR 8 and R 8 is H, C 1-6 Alkyl, optionally substituted phenyl, -OCH 2 R 9 or -CH=CHR 9 and R 9 , H, C 1-6 It is alkyl, or optionally substituted phenyl.

10. The method according to any one of claims 1 to 9, wherein the imidazole dipeptide is carnosine, anserine, balenine, or homocarnosine.

11. The method according to any one of claims 1 to 10, wherein the imidazole dipeptide is carnosine.

12. The method according to any one of claims 1 to 10, wherein the imidazole dipeptide is anserine.

13. A culture supernatant obtained by the production method according to any one of claims 1 to 12.

14. A pharmaceutical composition comprising a culture supernatant obtained by the production method according to any one of claims 1 to 12.

15. The culture supernatant according to claim 13 or the pharmaceutical composition according to claim 14, for use in inhibiting bone differentiation.

16. A syringe, a vial, or a medical bag comprising the culture supernatant according to claim 13 or the pharmaceutical composition according to claim 14 or 15.

17. A method for culturing cells, comprising the step of culturing mesenchymal stem cells in a serum-free medium containing an imidazole dipeptide to produce cultured cells.

18. The culture method according to claim 17, further comprising a step of recovering the cultured cells from the medium.

19. The culture method according to claim 17 or 18, wherein the culture comprises a step of culturing in the presence of an adhesion factor.

20. The culture method according to any one of claims 17 to 19, wherein the medium is insulin-free, IGF-free, or FGF-free.

21. The culture method according to any one of claims 17 to 19, wherein the culture medium is a growth factor-free medium.

22. The culture method according to any one of claims 17 to 19, wherein the medium is a protein-free medium.

23. A method for producing cells, comprising a step of carrying out the culture method according to any one of claims 17 to 22.

24. A cell obtained by the production method described in claim 23.

25. 25. A composition comprising a cell population of the cells of claim 24.

26. A pharmaceutical composition comprising cells obtained by the production method described in claim 23.

27. The pharmaceutical composition according to claim 26, for inhibiting bone differentiation.

28. A medium for culturing mesenchymal stem cells, the medium comprising an imidazole dipeptide and being serum-free.

29. 29. The medium of claim 28, which is an insulin-free, IGF-free, or FGF-free medium.

30. 30. The medium of claim 28 or 29, which is growth factor-free.

31. The medium according to any one of claims 28 to 30, which is protein-free.

32. The medium according to any one of claims 28 to 31, comprising mesenchymal stem cells.

33. A method for inhibiting osteoblast formation, comprising the step of culturing mesenchymal stem cells in a medium containing an imidazole dipeptide.

34. A method for inhibiting the production of osteoblasts, comprising the step of contacting mesenchymal stem cells with a culture supernatant obtained by culturing mesenchymal stem cells in a medium containing an imidazole dipeptide.

35. A composition for inhibiting osteoblast formation, comprising an imidazole dipeptide.

36. A composition for inhibiting osteoblast formation, comprising a culture supernatant obtained by culturing mesenchymal stem cells in a medium containing an imidazole dipeptide.

37. A method for promoting expression of G-CSF, MCP-1, VEGF-C, TGF-β1, IL-6, IL-7, IL-8, M-CSF, Osteoprotegrin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, semaphorin 3B, or an exosome marker, comprising the step of culturing mesenchymal stem cells in a medium containing an imidazole dipeptide.

38. A composition for promoting the expression of G-CSF, MCP-1, VEGF-C, TGF-β1, IL-6, IL-7, IL-8, M-CSF, Osteoprotegrin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, semaphorin 3B, or an exosome marker in mesenchymal stem cells, comprising an imidazole dipeptide.

39. A method for promoting exosome secretion, comprising a step of culturing mesenchymal stem cells in a medium containing an imidazole dipeptide.

40. A composition for promoting exosome secretion from mesenchymal stem cells, comprising an imidazole dipeptide.

41. A mesenchymal stem cell culture supernatant containing at least 500 pg / ml of G-CSF.

42. A mesenchymal stem cell culture supernatant containing at least 2700 pg / ml of IL-6.

43. Umbilical cord-derived mesenchymal stem cells that highly express or secrete G-CSF or IL-6.

44. 44. A composition comprising a cell population of the cells of claim 43.

45. IL-34 positive mesenchymal stem cells.

46. The mesenchymal stem cell of claim 45, which is purified.

47. ADAM8-positive mesenchymal stem cells.

48. The mesenchymal stem cell of claim 47, which is purified.

49. The mesenchymal stem cell according to any one of claims 45 to 48, which is positive for osteoprotegerin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, and semaphorin 3B.

50. The mesenchymal stem cell of claim 49, which is purified.

51. A pharmaceutical composition comprising the mesenchymal stem cells according to any one of claims 45 to 50.

52. The pharmaceutical composition according to claim 51, for inhibiting bone differentiation.

53. A pharmaceutical composition for inhibiting bone differentiation, comprising cells obtained by culturing mesenchymal stem cells in a medium containing an imidazole dipeptide.

54. 54. The pharmaceutical composition of claim 53, further comprising a pharma- ceutically acceptable carrier.

55. The pharmaceutical composition of claim 53 or 54, wherein the cells are positive for Osteoprotegrin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, and semaphorin 3B.

56. The pharmaceutical composition according to any one of claims 53 to 55, wherein the cells are obtained by culturing in a medium containing an imidazole dipeptide and LIF.

57. The pharmaceutical composition according to any one of claims 53 to 56, wherein the culture is an adherent culture.

58. A method for producing a pharmaceutical composition for inhibiting bone differentiation, comprising the step of culturing mesenchymal stem cells in a medium containing an imidazole dipeptide to produce cultured cells.

59. 59. The method of claim 58, further comprising the step of recovering the cultured cells from the medium.

60. 60. The method of claim 58 or 59, comprising the step of mixing the cultured cells with a pharma- ceutically acceptable carrier.

61. A medium containing an imidazole dipeptide and a LIF (leukemia inhibitory factor) component.

62. 62. The medium of claim 61, further comprising an attachment factor.

63. 63. The culture medium according to claim 61 or 62, for culturing mesenchymal stem cells.

64. The medium of any one of claims 61 to 63, comprising mesenchymal stem cells.

65. A method for producing cells, comprising the step of culturing mesenchymal stem cells in a medium according to any one of claims 61 to 64 to produce cultured cells.

66. The culture is an adherent culture.

66. The method of claim 65.

67. A cell obtained by the production method described in claim 65 or 66.

68. 68. A composition comprising a cell population of the cells of claim 67.

69. A pharmaceutical composition comprising cells obtained by the production method described in claim 65 or 66.

70. The pharmaceutical composition of claim 69, for inhibiting bone differentiation.

71. A kit comprising an imidazole dipeptide and a LIF component.

72. 72. The kit of claim 71 for use in preparing a medium for culturing mesenchymal stem cells.

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