Use of mesenchymal stem cells or their culture supernatant

Abstract

Provided are a culture supernatant of mesenchymal stem cells or mesenchymal stem cells that are useful for suppressing or improving a disease. A method for producing a culture supernatant that includes a step that recovers a culture supernatant from a medium containing mesenchymal stem cells and an imidazole dipeptide is utilized. A method for producing cells that includes a step that cultures mesenchymal stem cells in medium containing an imidazole dipeptide and generates cultured cells may be utilized.

Description

[Technical Field]

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

[0002] In the field of regenerative medicine, mesenchymal stem cells (MSCs) and MSC culture supernatants are used. MSC culture supernatants contain components secreted by cells, such as cytokines. MSC culture supernatants have cost advantages because they can be administered to patients without the need to purify the active ingredients individually.

[0003] Several studies have been reported on MSCs or the culture supernatant of MSCs. For example, Patent Document 1 and Non-Patent Document 1 describe the investigation of cytokine levels in the culture supernatant of MSCs (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 lower limb ischemia resulted in increased blood flow. Non-Patent Document 1 describes that the culture supernatant of MSCs showed effects of promoting the repair of skin photodamage and promoting 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. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] When considering its use for disease suppression or improvement, the composition of conventional MSC culture supernatants had room for improvement.

[0007] Meanwhile, the inventors conducted research on 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 improving diseases. Furthermore, the resulting cells were also useful for suppressing or improving diseases. Based on these findings, the present invention was completed. [Means for solving the problem]

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

[0009] Furthermore, according to another aspect of the present invention, a method for culturing cells is provided, comprising the step of culturing mesenchymal stem cells in a serum-free medium containing imidazole dipeptide to generate cultured cells. According to another aspect of the present invention, a medium for culturing mesenchymal stem cells is provided, which contains imidazole dipeptide and is serum-free. Furthermore, according to another aspect of the present invention, a method for suppressing osteoblast generation is provided, comprising the step of culturing mesenchymal stem cells in a medium containing imidazole dipeptide. According to another aspect of the present invention, a method for suppressing osteoblast generation is provided, comprising the step of contacting mesenchymal stem cells with a culture supernatant obtained by culturing mesenchymal stem cells in a medium containing imidazole dipeptide. According to another aspect of the present invention, a composition for suppressing osteoblast generation is provided, which contains imidazole dipeptide. According to another aspect of the present invention, a composition for suppressing osteoblast generation is provided, which contains a culture supernatant obtained by culturing mesenchymal stem cells in a medium containing imidazole dipeptide. Furthermore, according to another aspect of the present invention, a method is provided 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 an imidazole dipeptide. According to another aspect of the present invention, a composition is provided 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 from mesenchymal stem cells, comprising imidazole dipeptide. According to another aspect of the present invention, a method for promoting exosome secretion is provided, comprising the step of culturing mesenchymal stem cells in a medium containing imidazole dipeptide. According to another aspect of the present invention, a composition is provided for promoting exosome secretion from mesenchymal stem cells, comprising imidazole dipeptide.According to another aspect of the present invention, a culture supernatant of mesenchymal stem cells containing at least 500 pg / ml of G-CSF is provided. According to another aspect of the present invention, a culture supernatant of mesenchymal stem cells containing at least 2700 pg / ml of IL-6 is provided. According to another aspect of the present invention, umbilical cord-derived mesenchymal stem cells that highly express or secrete G-CSF or IL-6 are provided. According to another aspect of the present invention, IL-34-positive mesenchymal stem cells are provided. According to another aspect of the present invention, ADAM8-positive mesenchymal stem cells are provided. Furthermore, according to another aspect of the present invention, a pharmaceutical composition for inhibiting osteogenic differentiation is provided, comprising cells obtained by culturing mesenchymal stem cells in a medium containing imidazole dipeptide. According to another aspect of the present invention, a method for producing a pharmaceutical composition for inhibiting osteogenic differentiation is provided, comprising the step of culturing mesenchymal stem cells in a medium containing imidazole dipeptide to generate cultured cells. Furthermore, according to another aspect of the present invention, a culture medium comprising imidazole dipeptide and LIF (leukemia inhibitory factor) components is provided. According to another aspect of the present invention, a kit is provided comprising an imidazole dipeptide and a LIF component. [Brief explanation of the drawing]

[0010] [Figure 1] This is a micrograph of MSCs after being cultured in a carnosine-free medium. [Figure 2] This is a micrograph of MSCs after being cultured in a medium containing 1 mM carnosine. [Figure 3] This is a micrograph of MSCs after being cultured in a medium containing 10 mM carnosine. [Figure 4] This is a micrograph of MSCs after being cultured in a medium containing 30 mM carnosine. [Figure 5] This figure shows the count results for the number of MSCs. [Figure 6] This figure shows the results of examining the amount of exosome markers in the culture supernatant. The four bar graphs shown for each of the 1 to 4 collection cycles represent the results under conditions of carnosine addition at 0 mM, 1 mM, 10 mM, and 30 mM, respectively, from left to right. The same applies to Figures 7 to 14. [Figure 7] This figure shows the results of examining the amount of HGF in the culture supernatant. [Figure 8] This figure shows the results of examining the amount of G-CSF in the culture supernatant. [Figure 9] This figure shows the results of examining the amount of MCP-1 in the culture supernatant. [Figure 10] This figure shows the results of examining the amount of VEGF-C in the culture supernatant. [Figure 11] This figure 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 these were the detection limit. [Figure 13] This figure shows the results of examining the amount of IL-7 in the culture supernatant. [Figure 14] This figure shows the results of examining the amount of IL-8 in the culture supernatant. [Figure 15] This figure shows the quantitative results of calcium deposition in bone marrow MSCs. [Figure 16] This is an image of bone marrow MSCs stained with Alizarin Red S. [Figure 17] This figure shows the quantitative results of calcium deposition during co-culture of bone marrow MSCs and umbilical cord MSCs. [Figure 18] These are images of bone marrow MSCs and umbilical cord MSCs stained with Alizarin Red S during co-culture. [Figure 19] This figure shows the culture conditions for umbilical cord MSCs. In the % increase calculation formula described in the example, all RPM values ​​are increased by 1 before calculating the respective % increase. For example, even if the RPM value of M0 is 0, the % increase is calculated after adding 1, so the % increase value of M0 becomes (M0+1) / (M0+1)=(0+1) / (0+1), which is 1. [Figure 20]This figure shows the changes in the % increase values ​​of 10 different genes. Figures 20(a) to 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. [Figure 21] This figure shows the RPM values ​​of interleukin 34 expression levels in umbilical cord MSCs. [Figure 22] This figure shows the RPM values ​​of ADAM metallopeptidase domain 8 expression levels in umbilical cord MSCs. [Figure 23] This figure shows the results of the ELISA analysis of osteoprotegerin. [Figure 24] This figure shows the results of the ELISA analysis of osteoprotegerin. [Figure 25] This figure shows the results of the ELISA analysis of osteoprotegerin. [Figure 26] This figure shows the results of the ELISA analysis of M-CSF. [Figure 27] This figure shows the results of the ELISA analysis of M-CSF. [Figure 28] This figure shows the results of the ELISA analysis of M-CSF. [Figure 29] This figure shows the results of qPCR analysis of osteoprotegerin. [Figure 30] This figure shows the results of qPCR analysis of M-CSF. [Figure 31]This figure shows the results of ELISA analysis of Osteoprotegrin. Figure 31(a) shows the results under conditions with carnosine added, and Figure 31(b) shows the results under conditions with anserine added. [Figure 32] These are the results of qPCR analysis of osteoprotegrin. Figure 32(a) shows the results under the UC_B_C condition, Figure 32(b) shows the results under the BM_B_C condition, and Figure 32(c) shows the results under the BM_C_C condition. [Figure 33] These are the results of qPCR analysis of osteoprotegrin. Figure 33(a) shows the results for UC_B_A, Figure 33(b) for BM_B_A, and Figure 33(c) for BM_C_A. [Figure 34] This figure shows the results of ELISA analysis of M-CSF. Figure 34(a) shows the results under conditions with carnosine added, and Figure 34(b) shows the results under conditions with anserine added. [Figure 35] This figure shows the results of qPCR analysis of M-CSF. Figure 35(a) shows the results under the UC_B_C condition, and Figure 35(b) shows the results under the BM_B_C condition. [Figure 36] This figure shows the results of qPCR analysis of M-CSF. Figure 36(a) shows the results for UC_B_A, and Figure 36(b) shows the results for BM_B_A. [Figure 37] This figure shows the cell count results for groups 1 and 2. [Figure 38] This figure shows the cell count results for groups 3 and 4. [Figure 39] This figure shows the measurement results of the fluorescence area of ​​phalloidin in groups 1 to 4. [Figure 40] This figure shows the measurement results of the fluorescence area of ​​CD44 in groups 1 to 4. [Figure 41] This figure shows the fluorescence imaging results (nuclear staining) for groups 1 and 3. [Figure 42] This figure shows the fluorescence image acquisition results (phaloidin staining) for groups 1 and 3. [Figure 43] This figure shows the fluorescence image acquisition results (CD44 staining) for groups 1 and 3. [Figure 44] This figure shows the cell count results for groups 5 and 6. [Figure 45]This figure shows the cell count results for groups 7 and 8. [Figure 46] This figure shows the measurement results of the fluorescence area of ​​phalloidin in groups 5 and 6. [Figure 47] This figure shows the measurement results of the fluorescence area of ​​phalloidins in groups 7 and 8. [Figure 48] This figure shows the measurement results of the fluorescence area of ​​CD44 in groups 5 and 6. [Figure 49] This figure shows the measurement results of the fluorescence area of ​​CD44 in groups 7 and 8. [Figure 50] This figure shows the fluorescence image acquisition results (phaloidin staining) for groups 5 and 7. [Figure 51] This figure shows the fluorescence image acquisition results (CD44 staining) for groups 5 and 7. [Figure 52] This figure shows the results of measuring the positive rate of MSC markers after culturing umbilical cord MSCs in MSC medium D. [Figure 53] This figure shows the cell count results for groups 9 and 10. [Figure 54] This figure shows the measurement results of the fluorescence area of ​​phalloidins in groups 9 and 10. [Figure 55] This figure shows the fluorescence image acquisition results (phaloidin staining) for groups 9 and 10. [Modes for carrying out the invention]

[0011] The embodiments of the present invention will be described in detail below. To avoid repetition and unnecessary complexity, similar content will be omitted from explanation as appropriate.

[0012] (1) Method One embodiment of the present invention provides a method comprising the 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 improving a disease (for example, a culture supernatant containing at least one cytokine useful for suppressing or improving a disease in high concentration, or a culture supernatant useful for inhibiting osteogenic differentiation). This method may include, for example, the step of culturing MSCs in a medium containing an imidazole dipeptide to produce cultured MSCs, or the step of recovering a culture supernatant from a medium containing 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 the secretion of cytokines or exosomes, a method for inhibiting osteoblast generation, or a method for inhibiting osteogenic differentiation. Another embodiment of the present invention provides cells, a culture supernatant, a pharmaceutical composition, a cytokine-containing composition, or a container containing them obtained using this method.

[0013] (2) Method According to one embodiment of the present invention, a method for producing a culture supernatant is provided, comprising the step of recovering the culture supernatant from a medium containing MSCs and imidazole dipeptides. This method can be used to obtain a culture supernatant useful for suppressing or improving a disease (for example, a culture supernatant containing at least one cytokine useful for suppressing or improving a disease in high concentration, or a culture supernatant useful for suppressing osteogenic differentiation).

[0014] (3) Method One embodiment of the present invention provides a method for culturing or producing cells, comprising the step of culturing MSCs in a medium containing imidazole dipeptide to generate cultured cells. This method makes it possible to obtain MSCs useful for suppressing or improving diseases (for example, MSCs that secrete at least one cytokine useful for suppressing or improving diseases in high concentrations, or MSCs useful for inhibiting osteogenic differentiation). Furthermore, in this method, by culturing MSCs in a serum-free, serum albumin-free, insulin-free, IGF (insulin-like growth factor)-free (for example, IGF-1-free), FGF (fibroblast growth factor)-free (for example, FGF-1 or 2-free), growth factor-free, cytokine-free, or protein-free medium, it is possible to obtain MSCs with superior safety. By recovering the culture supernatant from the medium containing the cells obtained by this method, it is possible to obtain a culture supernatant useful for suppressing or improving diseases (for example, a culture supernatant containing at least one cytokine useful for suppressing or improving diseases in high concentrations, or a culture supernatant useful for inhibiting osteogenic differentiation).

[0015] (4) Culture supernatant According to one embodiment of the present invention, a culture supernatant obtained by culturing MSCs in a medium containing imidazole dipeptide is provided. By utilizing this culture supernatant, for example, the effects of the MSC culture supernatant, or effects useful for suppressing or improving diseases (for example, the effects brought about by at least one high concentration of cytokines useful for suppressing or improving diseases, or the effect of suppressing osteogenic differentiation) can be obtained.

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

[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 imidazole dipeptide. This composition can be used to suppress or improve osteogenic differentiation (for example, to suppress or improve diseases associated with osteogenic 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 imidazole dipeptide.

[0018] (7) Method One embodiment of the present invention provides a method for suppressing disease, comprising the step of administering cells obtained by culturing MSCs in a medium containing imidazole dipeptide to a target. This method can be used to suppress or improve osteogenic differentiation (for example, to suppress or improve diseases associated with osteogenic differentiation). Another aspect provides the use of a pharmaceutical composition for disease suppression, comprising cells obtained by culturing MSCs in a medium containing imidazole dipeptide.

[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 imidazole dipeptide. This composition can be used to suppress or improve osteogenic differentiation (for example, to suppress or improve diseases associated with osteogenic differentiation). In another aspect, a pharmaceutical composition for use in suppressing disease is provided, comprising cells obtained by culturing MSCs in a medium containing imidazole dipeptide.

[0020] (9) Method One embodiment of the present invention provides a method for inhibiting the osteogenic differentiation of target cells, comprising the step of administering to the target a culture supernatant obtained by culturing MSCs in a medium containing imidazole dipeptide. This method can be used to suppress the osteogenic differentiation of target cells. Another aspect provides the use of a pharmaceutical composition comprising a culture supernatant obtained by culturing MSCs in a medium containing imidazole dipeptide in the manufacture of a pharmaceutical composition for inhibiting the osteogenic differentiation of target cells. Another aspect provides a pharmaceutical composition comprising a culture supernatant obtained by culturing MSCs in a medium containing imidazole dipeptide for use in inhibiting the osteogenic differentiation of target cells.

[0021] (10) Method One embodiment of the present invention provides a method for inhibiting the osteogenic differentiation of target cells, comprising the step of administering cells obtained by culturing MSCs in a medium containing imidazole dipeptide to the target. This method can be used to suppress the osteogenic differentiation of cells in the target. Another aspect provides the 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 the osteogenic differentiation of target cells. Another aspect provides a pharmaceutical composition comprising cells obtained by culturing MSCs in a medium containing imidazole dipeptide for use in inhibiting the osteogenic differentiation of target cells.

[0022] (11) Culture medium According to one embodiment of the present invention, a culture medium for MSCs is provided, which contains an imidazole dipeptide and is serum-free, serum albumin-free, insulin-free, IGF-free, FGF-free, growth factor-free, cytokine-free, or protein-free. By culturing MSCs using this medium, cells useful for suppressing or improving diseases (for example, cells that secrete at least one cytokine useful for suppressing or improving diseases in high concentrations, or cells useful for inhibiting osteogenic differentiation) can be obtained. Alternatively, a culture supernatant useful for suppressing or improving diseases (for example, a culture supernatant containing at least one cytokine useful for suppressing or improving diseases in high concentrations, or a culture supernatant useful for inhibiting osteogenic differentiation) can be obtained. By culturing MSCs in this medium, MSCs or culture supernatants with excellent safety can be obtained.

[0023] (12) Method One embodiment of the present invention provides a method for inhibiting osteoblast generation, comprising the step of contacting MSCs with an imidazole dipeptide. This method can be used to inhibit osteoblast generation. Another aspect provides the use of a composition comprising an imidazole dipeptide in the manufacture of a composition for inhibiting osteoblast generation. Another aspect provides a method for inhibiting osteoblast generation, comprising the step of administering an imidazole dipeptide to a target.

[0024] (13) Method According to one embodiment of the present invention, a method for suppressing osteoblast generation is provided, which includes the step of culturing MSCs in a medium containing imidazole dipeptide. By using this method, osteoblast generation can be suppressed.

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

[0026] (15) Method One embodiment of the present invention provides a method for inhibiting osteoblast generation, comprising the step of contacting MSCs with a culture supernatant or MSC secretion obtained by culturing MSCs in a medium containing imidazole dipeptide. This method can be used to inhibit osteoblast generation. Another aspect provides the use of a composition comprising a culture supernatant or MSC secretion obtained by culturing MSCs in a medium containing imidazole dipeptide in the manufacture of a composition for inhibiting osteoblast generation. Another aspect provides a method for inhibiting osteoblast generation, comprising the step of administering a culture supernatant or MSC secretion obtained by culturing MSCs in a medium containing imidazole dipeptide to a target.

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

[0028] (17) Method According to one embodiment of the present invention, a method is provided for promoting the expression of G-CSF (granulocyte colony-stimulating factor), MCP-1 (monocyte chemotactic factor), 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, osteoprotoegrin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, semaphorin 3B, or an exosome marker, comprising the step of culturing MSCs in a medium containing an imidazole dipeptide. This method can be used to 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 According to one embodiment of the present invention, a method is provided 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 contacting MSCs with an imidazole dipeptide. This method can be used to 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. From another perspective, the use of compositions containing imidazole dipeptides in the preparation of compositions for promoting the expression of MSC 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. From another perspective, a method is provided for promoting the expression of MSCs 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, including the step of administering imidazole dipeptides to target MSCs.

[0030] (19) Composition According to one embodiment of the present invention, a composition is provided 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 from MSCs, comprising an imidazole dipeptide. 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, Osteoprotegrin, 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 the step of culturing MSCs in a medium containing an imidazole dipeptide. This method can be used to promote the secretion of exosomes from MSCs.

[0032] (21) Method One embodiment of the present invention provides a method for promoting exosome secretion, comprising the step of contacting MSCs with an imidazole dipeptide. This method can be used to promote the secretion of exosomes from MSCs. Another aspect provides the use of a composition comprising an imidazole dipeptide in the manufacture of a composition for promoting the secretion of exosomes from MSCs. Another aspect provides a method for promoting exosome secretion, comprising the step of administering an imidazole dipeptide to a target.

[0033] (22) Composition According to one embodiment of the present invention, a composition for promoting the secretion of exosomes from MSCs is provided, comprising an imidazole dipeptide. When this composition is brought into contact with MSCs, the secretion of exosomes from the MSCs can be promoted.

[0034] (23) Method According to one embodiment of the present invention, a method for producing a composition is provided, comprising the step of recovering a culture supernatant from a medium containing MSCs and imidazole dipeptides. This composition may be a culture supernatant or a cytokine-containing composition. By using this composition, the effects of MSC culture supernatant, or effects useful for suppressing or improving diseases (for example, the effects of at least one high concentration of cytokines useful for suppressing or improving diseases, or the effect of suppressing osteogenic differentiation) can be obtained.

[0035] (24) Method According to one embodiment of the present invention, a method for producing a composition containing MSC secretions is provided, comprising the step of contacting MSCs with an imidazole dipeptide. By using this composition, the effects of MSC secretions, or effects useful for suppressing or improving diseases (for example, the effects of at least one high concentration of cytokines useful for suppressing or improving diseases, or the effect of suppressing osteogenic differentiation) can be obtained.

[0036] (25) Culture supernatant According to one embodiment of the present invention, a culture supernatant of MSCs containing protein is provided. The culture supernatant may be, for example, a culture supernatant of MSCs containing at least 500 pg / ml of G-CSF. By using this culture supernatant, a disease suppression or improvement effect due to a high concentration of G-CSF can be obtained. The culture supernatant may also be, for example, a culture supernatant of MSCs containing at least 2700 pg / ml of IL-6. By using this culture supernatant, a disease suppression or improvement effect due to a high concentration of IL-6 can be obtained.

[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. Using this culture supernatant, a disease-suppressing or ameliorating effect due to a high concentration of IL-6 can be obtained. Using these cells, a disease-suppressing or ameliorating effect due to a high concentration of G-CSF or IL-6 can be obtained. Alternatively, recovering the culture supernatant from a medium containing these cells yields a culture supernatant containing a high concentration of G-CSF or IL-6. In another aspect, a composition is provided comprising a population of MSC cells, 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. Using this composition, a disease-suppressing or ameliorating effect due to a high concentration of G-CSF or IL-6 can be obtained. Recovering the culture supernatant from this composition yields a culture supernatant containing a high concentration of G-CSF or IL-6.

[0038] (27) Cell According to one embodiment of the present invention, MSCs positive for 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, or semaphorin 3B are provided. These MSCs may be purified or isolated MSCs. According to another aspect, a composition comprising purified or isolated MSCs is provided. According to yet another aspect, MSCs with elevated 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, or semaphorin 3B are provided. 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 containing these MSCs is provided. In yet another aspect, a method for treating a disease is provided, comprising the step of administering these MSCs to a target. Positivity can be evaluated, for example, by an immunological assay (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, for example, in suppressing osteogenic differentiation. These MSCs may be purified or isolated MSCs. In another aspect, a composition comprising the purified or isolated form of MSCs is provided. In another aspect, a pharmaceutical composition comprising the MSCs is provided. In another aspect, a method for treating a disease is provided, comprising the step of administering these MSCs to a target.

[0040] (29) Method According to one embodiment of the present invention, a method is provided to alter the properties of a culture medium, comprising the step of adding an imidazole dipeptide to the medium, so as to further promote the 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 during MSC culture. Using this method, a culture medium suitable for producing culture supernatant or MSCs useful for suppressing or improving disease can be prepared. In another aspect, according to one embodiment of the present invention, a method for preparing a culture 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, a culture medium is provided that contains an imidazole dipeptide and a LIF component. This culture medium can be used to promote the proliferation of MSCs.

[0042] (31) Method According to one embodiment of the present invention, a method for producing cells or culture supernatant is provided, comprising the step of culturing MSCs in a medium containing imidazole dipeptide and LIF components to generate cultured cells. The obtained cells can be used to obtain an effect that suppresses osteogenic differentiation. The obtained culture supernatant can be used to obtain the effects of the MSC culture supernatant, or effects useful for suppressing or improving diseases (for example, the effects brought about by at least one high concentration of cytokines useful for suppressing or improving diseases, or the effect of suppressing osteogenic differentiation).

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

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

[0045] (34) Method According to one embodiment of the present invention, a method for producing cells or culture supernatant is provided, comprising the step of culturing MSCs in a medium containing an imidazole dipeptide and a laminin fragment or a variant thereof to generate cultured cells. The obtained cells can be used to obtain an effect that suppresses osteogenic differentiation. The obtained culture supernatant can be used to obtain the effects of the MSC culture supernatant, or effects useful for suppressing or improving diseases (for example, the effects brought about by at least one high concentration of cytokines useful for suppressing or improving diseases, or the effect of suppressing osteogenic 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 culture medium (e.g., a medium for culturing MSCs). The components contained in this kit can be mixed to prepare a culture medium. The resulting medium can be used to promote the proliferation of MSCs. In another aspect, a method for preparing a culture medium is provided, comprising the step of mixing the imidazole dipeptide and LIF.

[0047] (36) Method According to one embodiment of the present invention, a method for producing a cytokine-containing composition is provided, comprising the step of processing a culture supernatant (for example, including (4) or (25) above) or a composition (for example, including (6), (8), (14), (16), (19) or (22) above) according to the embodiments of the present invention. By using the composition obtained by this method, the effects of MSC culture supernatant, the effects of at least one high concentration of cytokines useful for suppressing or improving disease, or the effect of suppressing osteogenic differentiation can be obtained. The processing may be, for example, formulation or filling into containers.

[0048] (37) Composition According to one embodiment of the present invention, a cytokine-containing composition is provided, obtained by carrying out a method of an embodiment of the present invention (for example, including (1) to (3), (5), (7), (9), (10), (12), (13), (15), (17), (18), (20), (21), (23), (24), (29), (31), (34), or (36) above). By using this composition, the effects of MSC culture supernatant, the effects of at least one high concentration of cytokines useful for suppressing or improving disease, or the effect of suppressing osteogenic differentiation can be obtained.

[0049] (38) Cell According to one embodiment of the present invention, cells are provided obtained by carrying out the methods of embodiments of the present invention (including, for example, (1) to (3), (31) or (34) above). These cells can secrete at high concentrations at least one cytokine useful for suppressing or improving a disease. These cells can be used to suppress or improve a disease. By collecting the culture supernatant from a medium containing these cells, a culture supernatant containing at least one cytokine useful for suppressing or improving a disease, or a culture supernatant useful for inhibiting osteogenic differentiation, can be obtained.

[0050] (39) Cell According to one embodiment of the present invention, a cell population comprising cells of an embodiment of the present invention (for example, including (26) to (28) or (38) above) is provided. By using these cells, a disease suppression or improvement effect due to a high concentration of G-CSF or IL-6, or an effect of suppressing osteogenic differentiation, can be obtained. Alternatively, by recovering the culture supernatant from a medium containing these cells, a culture supernatant containing a high concentration of G-CSF or IL-6, or a culture supernatant useful for suppressing or improving disease, can be obtained. According to another aspect, a composition comprising a cell population comprising cells of (25) or (28) above is provided. By using this composition, a disease suppression or improvement effect due to a high concentration of G-CSF or IL-6, or an effect of suppressing osteogenic differentiation, can be obtained. Alternatively, by recovering the culture supernatant from a medium containing this composition, a culture supernatant containing a high concentration of G-CSF or IL-6, or a culture supernatant useful for suppressing or improving disease, can be obtained.

[0051] (40) Method According to one embodiment of the present invention, a method for treating a disease is provided, comprising the step of administering a supernatant (including, for example, (4) or (25) above) or cells (including, for example, (26) to (28) or (38)) according to an embodiment of the present invention to a subject. The subject to be treated may be, for example, a patient requiring suppression of osteogenic differentiation. In another aspect, a pharmaceutical composition for use in treating a disease is provided, comprising the supernatant or cells of an embodiment of the present invention. In yet another aspect, the use of the supernatant or cells of an embodiment of the present invention for the production of a pharmaceutical composition for treating a disease is provided.

[0052] (41) Container According to one embodiment of the present invention, a container is provided that contains a culture supernatant (for example, including (4) or (25) above) or a composition (for example, including (6), (8), (14), (16), (19), (22), or (37) above) according to the embodiment of the present invention. By using this container, the effects of MSC culture supernatant, the effects of at least one high concentration of cytokines useful for suppressing or improving disease, or the effect of suppressing osteogenic differentiation can be obtained.

[0053] The methods of embodiments of the present invention (including, for example, (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 imidazole dipeptide to produce cultured MSCs, or (ii) recovering the culture supernatant from the cultured MSCs and the medium containing imidazole dipeptide. Furthermore, the methods of embodiments of the present invention (including, for example, (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 (iii) to (xvi).(iii) Suspending MSCs in a culture medium and seeding the resulting cell suspension into a culture vessel; (iv) Adding a culture medium containing adhesion factors to the culture medium after seeding and culturing the MSCs; (v) Mixing imidazole dipeptide and culture medium components; (vi) Replacing the culture medium in which the MSCs were cultured with a culture medium containing imidazole dipeptide; (vii) Culturing the MSCs in a culture medium containing imidazole dipeptide; (viii) Culturing the MSCs by adhesion culture to increase cell growth; (ix) Centrifuging or filtering the culture medium containing the MSCs and imidazole dipeptide after culturing; (x) Recovering the culture supernatant or the MSCs after culturing; (xi) 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 in the culture supernatant (xii) measuring the amount of IGFBP2, DKK1, semaphorin 3B, or an exosome marker; (xiii) sterilizing the recovered culture supernatant to produce a sterile culture supernatant; (xiv) formulating the culture supernatant or post-culture MSCs; (xv) administering a therapeutically effective amount of the culture supernatant or post-culture MSCs to the target; (xv) transferring the culture supernatant or post-culture MSCs to a medical container; or (xvi) transferring the culture supernatant or post-culture MSCs from the medical container to a syringe, vial, or medical bag. Employing 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 improving the disease, or a culture supernatant or MSCs useful for inhibiting osteogenic differentiation. If two or more steps are employed, the order is arbitrary and can be determined according to the desired operation.

[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 (mice, hamsters, etc.), rabbits, dogs, cats, horses, cattle, sheep, pigs, goats, marmosets, etc. MSCs may exist as a cell population. Unless otherwise specified herein, references to MSCs include MSCs that exist as a cell population. A cell population includes a plurality of cells produced 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, or 100% or more, and may be within the range of any two of these values. MSCs include MSCs before culturing by the method described above, and MSCs obtained by culturing by the method described above. 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 positive for CD44, CD73, CD90, or CD105, or negative for CD45, CD34, CD31, or HLA-DR. MSCs may be isolated or purified MSCs. Isolated or purified MSCs include, for example, a cell population that does not contain any cells other than MSCs, or MSCs in a formulation from which impurities have been removed. Impurities may include, for example, components of the culture medium, and removal may include, for example, partial removal.

[0055] In embodiments of the present invention (including, for example, (1) to (41) above), MSCs that highly express or secrete G-CSF or IL-6, for example, when cultured for 72 hours in a medium supplemented with carnosine (e.g., 10 mM), contain at least 5.56 × 10⁶ molecules. -3 pg / cells that secrete G-CSF or at least 3 × 10 -2It may be a cell that secretes IL-6 per pg / cell. The secretion amount of G-CSF is, 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, and may also be within the range of any two of these values. This value is, for example, 5.56×10 -3 ~3, 5.56×10 -3 ~3×10 -2 、5.56×10 -3 ~2×10 -2 、1.2×10 -2 ~3×10 -2 、or 1.2×10 -2 ~1.7×10 -2 pg / cell. The secretion amount of IL-6 is, for example, at least 3×10 -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, and may also be within the range of any two of these values. This value is, for example, 3×10 -2 ~20, 3×10 -2 ~2×10 -1 、3×10 -2 ~8×10 -2 、4×10 -2 ~8×10 -2 、or 4×10 -2 ~7×10 -2 pg / cell. The calculation of the secretion amount can be shown as the concentration of the secretion in the medium after culturing the cells (unit is, for example, pg / mL) × the amount of the medium used (unit is, for example, mL) ÷ the number of cells after culturing.

[0056] In embodiments of the present invention (including, for example, (1) to (41) above), 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 the structure of the following formula (1) or (2). [ka] [ka]

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

[0058] From the perspective of more efficiently promoting cytokine secretion by MSCs, R in equation (1) 1 and R 2 In this case, one is H and the other is C 1-6 It is preferable that it be alkyl, and more preferably that both are H. Here, R in formula (1) 1 and R 2 One of them is C 1-6In the case of alkyl, C 1-6 alkyl is preferably methyl. From the viewpoint of promoting cytokine secretion of MSC, R in formula (2) 3 and R 4 are preferably such that one is H and the other is C 1-6 alkyl, and more preferably both are H. Here, one of R 2 and R 3 in formula (2) is C 1-6 alkyl, in the case of C 1-6 alkyl is preferably methyl. From the viewpoint of promoting cytokine secretion of MSC, R in formula (1) or (2) 8 is preferably H, C 1-6 alkyl, or -OCH3, and more preferably methyl. From the viewpoint of promoting cytokine secretion of MSC, R 9 in formula (1) or (2) is preferably H or C 1-6 alkyl. R 7 in formula (1) or (2) may be formyl, acetyl, propionyl, benzoyl, or acryloyl. The optionally substituted phenyl in formula (1) or (2) may be, for example, unsubstituted or phenyl substituted with -OH, C 1-6 alkyl, or -OCH3. The substitution position may be the 2, 3, 4, 5, or 6 position of phenyl.

[0059] From the viewpoint of more efficiently promoting cytokine secretion of MSC, in formula (1) and formula (2), R 1 , R 2 , R 3 and R 4 may each independently be H or C 1-6 alkyl, either one of R 1 and R 2 may be H, either one of R 3 and R 4 may be H, R 5 and R 6 may each independently be -NHR 7 or -CH2NHR 7 , and R 7This can be H or -COCH3.

[0060] The alkyl group mentioned above includes a linear or branched hydrocarbon chain. 1-6 C is a hydrocarbon having 1, 2, 3, 4, 5, or 6 carbon atoms. 1-6 Alkyl is an alkyl group having 1, 2, 3, 4, 5, or 6 carbon atoms. 1-6 Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, and n-hexyl groups.

[0061] Regarding the methods for producing the compounds represented by formulas (1) and (2), the methods and principles for producing imidazole dipeptides described in Japanese Patent Publication Nos. 2020-022433, 2019-131532, 2010-31004, 2006-232686, or 2006-504701 can be utilized.

[0062] In embodiments of the present invention (including, for example, (1) to (41) above), the imidazole dipeptide includes carnosine, a methylated form of carnosine (e.g., anserine or balenine), or homocarnosine. The imidazole dipeptide includes a compound having the structure of formula (1) or (2) and possessing 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 the L-isomer or the D-isomer.

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

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

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

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

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

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

[0069] In embodiments of the present invention (including, for example, (1) to (41) above), inhibition of osteogenic differentiation is useful, for example, for inhibiting diseases associated with osteogenic differentiation. Diseases associated with osteogenic differentiation include, for example, ossification (e.g., heterotopic ossification). Inhibition of osteogenic differentiation includes, for example, inhibiting abnormal osteogenic differentiation. Furthermore, the use of inhibition of osteogenic differentiation includes the use of inhibiting or improving diseases associated with osteogenic differentiation. A pharmaceutical composition for inhibiting osteogenic differentiation includes a pharmaceutical composition for diseases associated with osteogenic differentiation such as heterotopic ossification. A method for inhibiting osteogenic differentiation includes a method for inhibiting or improving diseases associated with osteogenic differentiation such as heterotopic ossification.

[0070] In embodiments of the present invention (including, for example, (1) to (41) above), culturing means incubating cells under conditions suitable for growth or maintenance. Incubation may be carried out at approximately 37°C and in an atmosphere of approximately 5% CO2. Culturing may be carried out in serum-free, serum-free, serum albumin-free, insulin-free, IGF-free, FGF-free, growth factor-free, cytokine-free, or protein-free media. Free includes a state in which the target component is not added to the medium, a state in which the medium does not contain the target component at all, or a state in which the medium does not contain the component at a concentration above the detection limit. Free includes being substantially free or substantially ineligible. Culturing may be carried out by adherent culture in the presence of cell adhesion factors (e.g., mixed in the medium or in a pre-coated container). If culturing involves cell growth, culturing may include cell production. The culturing time may be, for example, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, or 50 days or more, and may be within the range of any two of these values. Culture may be carried out 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, it can be carried out until the cells cover 80-90% of the bottom area of ​​the culture vessel. Culturing of MSCs may be carried out by alternating between medium X, where medium X is a medium that does not contain imidazole dipeptides (e.g., a protein-containing medium), and medium Y is a medium that contains imidazole dipeptides (e.g., a protein-free medium). Alternating use includes starting culture in either X or Y, then replacing the medium with Y after culturing in X, and then replacing it with X after culturing in Y. For example, the following steps may be carried out in sequence: culture in X, replace with Y and culture, replace with X and culture, replace with Y and culture, replace with X and culture, replace with Y and culture, and replace with Y and culture. Under these conditions, where MSCs are cultured using X and Y alternately, the culture of Y may be performed, for example, at least 1, 2, 3, 4, 5, 6, or 7 times. The culture supernatant may be collected at any time after culturing with Y.Under the conditions described above, where MSCs are cultured using X and Y alternately, the culture supernatant may be collected, for example, after the 1st, 2nd, 3rd, 4th, 5th, 6th, or 7th culture cycle of Y. Alternatively, the supernatant may be collected at two or more of these timings, or at all of them, and the solution obtained by mixing all the resulting supernatants may be used as the target 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, (1) to (41) above), MSC culture may be carried out in a medium containing, for example, 0.1 to 100 mM of 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, and may be greater than or equal to these values, or within the range of any two of these values. From the viewpoint of more efficiently obtaining a culture supernatant containing at least one cytokine useful for suppressing or improving disease at a high concentration, 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. The culture may be adherent culture. A person skilled in the art can easily confirm whether cells are adhering to a container by, for example, tilting the container to confirm that no cell movement occurs, or by confirming that no cell movement occurs when the culture medium in the container is changed. Adherent culture may be carried out, for example, by culturing cells in the presence of cell adhesion factors (including, for example, bringing cells and adhesion factors into contact in a container, mixing a cell suspension containing cells and culture medium with adhesion factors, placing the resulting mixture in a container and culturing it, or coating the container with adhesion factors (e.g., pre-coating) and then seeding cells in the container). In this specification, cell adhesion factors and adhesion factors refer to the same thing. Adhesion factors include, for example, laminin, fibronectin, vitronectin, tenascin, cadherin, poly-L-lysine, poly-D-lysine, collagen, thrombospongin, galectin, or nidogen-1, or fragments thereof that have cell adhesion properties. Cell adhesion may include a state in which cells and a container are adhered via an extracellular matrix (e.g., adhesion factors). Adherent cells may include cells that can be cultured while attached, or cells that can proliferate while attached.

[0072] In the above-described adherent culture, the concentration of adhesion factors in the culture 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, and may be greater than or equal to 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 adhesion factors in the culture medium may be, for example, in a culture area of ​​1 cm² in a culture vessel. 2 The value may be 0.005, 0.01, 0.1, 0.25, 0.5, 1, 1.5, or 2 μg per unit area, and may be greater than or equal to any two of these values. This value is, for example, for a culture area of ​​1 cm² in a culture vessel. 2The amount may be 0.01 to 1.5, 0.1 to 1, or 0.1 to 0.5 μg per unit. The adhesion factor is a factor having adhesion activity, and may be, for example, laminin, fibronectin, vitronectin, tenascin, cadherin, poly-L-lysine, poly-D-lysine, collagen, thrombospongin, galectin, nidogen-1, fragments thereof, or modified versions thereof. The adhesion factor may be a laminin fragment or a modified version thereof. The laminin fragment may have integrin-binding activity and may be of human origin. The laminin fragment may be a laminin E8 fragment. The laminin fragment may be laminin 511 E8 fragment, laminin 521 E8 fragment, laminin 411 E8 fragment, laminin 421 E8 fragment, laminin 332 E8 fragment, laminin 311 E8 fragment, laminin 321 E8 fragment, laminin 211 E8 fragment, laminin 221 E8 fragment, laminin 213 E8 fragment, laminin 111 E8 fragment, or laminin 121 E8 fragment. Laminin 511 E8 fragment can be prepared, for example, by the method described in WO2011 / 043405A1. Laminin 511 contains laminin composed of α5, β1 and γ1 subunit chains. Modified laminin fragments may be known complexes consisting of a laminin fragment having integrin-binding activity and other functional molecules (for example, a complex of a laminin fragment having integrin-binding activity and a cell adhesion molecule, or a complex of 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). Modified laminin fragments can be produced as recombinant proteins using known genetic recombination techniques.The above laminin fragment or its variant may have integrin-binding activity. For example, it may include (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 with 90% or more homology to the amino acid sequence shown in SEQ ID NO: 1, a polypeptide having an amino acid sequence with 90% or more homology to the amino acid sequence shown in SEQ ID NO: 2, and a polypeptide having an amino acid sequence with 90% or more homology to the amino acid sequence shown in SEQ ID NO: 3; or (c) a trimer comprising a polypeptide having an amino acid sequence with a deletion, substitution, insertion, or addition of 1 to several amino acids relative to the amino acid sequence shown in SEQ ID NO: 1, a polypeptide having an amino acid sequence with a deletion, substitution, insertion, or addition of 1 to several amino acids relative to the amino acid sequence shown in SEQ ID NO: 2, and a polypeptide having an amino acid sequence with a deletion, substitution, insertion, or addition of 1 to several amino acids relative to the amino acid sequence shown in SEQ ID NO: 3. The above laminin fragment, variant, or subunit chain may optionally have a tag (see, for example, Mishra, Curr Protein Pept Sci. 2020;21(8):821-830) at the N or C terminus. The tag may be, for example, a purification tag. The purification tag may be, for example, a peptide tag (such as a His tag). The purification tag includes, for example, a biochemically inert tag. The seeding density of the cells may be, for example, 1×10e1, 1×10e2, 5x10e2, 1×10e3, 1.5×10e3, 1×10e4, 1×10e5, or 1x10e6 cells / mL, or may be greater than those values, or within the range of any two of those values. This value may be, for example, 1×10e1~1x10e5, 1x10e2~1x10e4, 5x10e2~5x10e4, or 5x10e2~1x10e4 cells / mL. The seeding density of the cells may be, for example, 0.002, 0.01, 0.1, 1, 50, 100, 300, 500, 1000, 1500, 2000 cells / cm². 2It may be any of these values, or it may be greater than or equal to any two of these values, or within the range of any two of these values. This value may be, for example, 0.01 to 2000, 0.1 to 1000, 1 to 1000, 1 to 500, or 1 to 100 cells / cm². 2 That's fine.

[0073] In embodiments of the present invention (including, for example, (1) to (41) above), the culture supernatant includes the culture supernatant obtained by culturing cells. The culture supernatant includes, for example, cytokine-containing culture supernatant. The culture supernatant may also 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 also contain imidazole dipeptide. The culture supernatant also includes supernatant obtained by subjecting the supernatant separated from cellular components to various treatments (e.g., centrifugation, filtration, freezing, lyophilization, storage, sterilization, etc.). The culture supernatant may also be the culture supernatant obtained using a medium containing only MSCs as cultured cells. From the viewpoint of reducing manufacturing costs or ensuring lot-to-lot uniformity, it is preferable that the cytokines contained in the culture supernatant are only cytokines derived from cultured cells. From the viewpoint of reducing manufacturing costs, it is preferable that the culture supernatant is unconcentrated. 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, (1) to (41) above), the culture supernatant may contain at least 500 pg / mL of G-CSF or at least 2700 pg / mL of IL-6. Using this culture supernatant, the effects of high concentrations of G-CSF or IL-6 can be obtained. This culture supernatant may contain G-CSF in concentrations of, for example, 500-2000, 700-1700, 800-1600, or 1000-1600 pg / mL. 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 within the range of any two of these values. The culture supernatant may contain IL-6 at concentrations of, for example, 2700-10000, 3000-10000, 4000-10000, or 3000-4000 pg / mL. This concentration may be, for example, at least 2700, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10000, 11000, or 12000 pg / mL, or within the range of any two of these values. In addition to the above concentrations of G-CSF and IL-6, this culture supernatant may also contain 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, or at least 6500 pg / mL of IL-8. Using this culture supernatant, the effects of high concentrations of G-CSF, IL-6, MCP-1, VEGF-C, TGF-β1, IL-7, or IL-8 can be obtained. This culture supernatant may contain MCP-1 at, for example, at least 6000, 7000, 8000, 9000, or 10000 pg / mL, or within the range of any two of these values. This culture supernatant may contain VEGF-C in amounts of, for example, at least 150, 200, 250, 300, 350, or 400 pg / mL, or within the range of any two of these values. This culture supernatant may also contain TGF-β1 in amounts of, for example, at least 670, 700, 750, 800, 850, 900, 950, or 1000 pg / mL, or within the range of 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 the range of any two of these values. This culture supernatant may also contain IL-8 at, for example, at least 6500, 7000, 8000, 9000, 10000, 11000, 12000, or 15000 pg / mL, or within the range of 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 within the above concentration ranges. Furthermore, the above culture supernatant may also contain M-CSF at, for example, at least 1350, 1370, 1390, 1400, 1420, 1440, 1450, 1470, 1490, or 1500 pg / mL, or within the range of any two of these values. Furthermore, the culture supernatant described above may contain osteoptegrin at, for example, at least 95, 100, 110, 120, 130, or 140 pg / mL, or within the range of 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 G-CSF in amounts 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 the range of any two of these values. The culture supernatant may also contain IL-6 in amounts of, 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, or within the range of any two of these values. This culture supernatant may contain MCP-1 in amounts of, for example, at least 6000, 7000, 8000, 9000, or 10000 pg / mL, or within the range of any two of these values. This culture supernatant may contain VEGF-C in amounts of, for example, at least 150, 200, 250, 300, 350, or 400 pg / mL, or within the range of any two of these values. This culture supernatant may contain TGF-β1 in amounts of, for example, at least 670, 700, 750, 800, 850, 900, 950, or 1000 pg / mL, or within the range of any two of these values. This culture supernatant may contain IL-7 in amounts of, for example, at least 3.5, 4, 5, 6, 7, 8, 9, or 10 pg / mL, or within the range of 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 the range of any two of these values.The culture supernatant may contain CD9 / CD63 fusion protein in amounts of, for example, at least 120, 140, 160, 180, 200, 250, or 300 pg / mL, or within the range of any two of these values. Furthermore, the culture supernatant may contain M-CSF in amounts of, for example, at least 1350, 1370, 1390, 1400, 1420, 1440, 1450, 1470, 1490, or 1500 pg / mL, or within the range of any two of these values. Furthermore, the culture supernatant may contain Osteoprotegrin in amounts of, for example, at least 95, 100, 110, 120, 130, or 140 pg / mL, or within the range of any two of these values.

[0076] In embodiments of the present invention (including, for example, (1) to (41) above), the culture supernatant may contain at least 1350 pg / mL of M-CSF or at least 95 pg / mL of Osteoprotegrin. Using this culture supernatant, the effects of high concentrations of M-CSF or Osteoprotegrin can be obtained. This culture supernatant may contain M-CSF at concentrations of, for example, 1350-1500, 1350-1450, 1350-1420, or 1350-1400 pg / mL. This concentration may be, for example, at least 1350, 1370, 1390, 1400, 1420, 1440, 1450, 1470, 1490, or 1500 pg / mL, and may be within the range of any two of these values. The culture supernatant may contain osteoprotogerin at concentrations of, for example, 95-140, 95-130, 95-120, or 95-110 pg / mL. This concentration may be, for example, at least 95, 100, 110, 120, 130, or 140 pg / mL, or within the range of any two of these values. In addition to the above concentrations of M-CSF and osteoprotogerin, the culture supernatant may also contain at least 500 pg / mL of G-CSF, at least 2700 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, or at least 6500 pg / mL of IL-8. Using this culture supernatant, the effects of high concentrations of M-CSF, osteoprotegrin, G-CSF, IL-6, MCP-1, VEGF-C, TGF-β1, IL-7, or IL-8 can be obtained. In another embodiment, this culture supernatant may contain G-CSF in amounts 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 the range of any two of these values.This culture supernatant may contain IL-6 in amounts of, 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, or within the range of any two of these values. This culture supernatant may contain MCP-1 in amounts of, for example, at least 6000, 7000, 8000, 9000, or 10000 pg / mL, or within the range of any two of these values. This culture supernatant may contain VEGF-C in amounts of, for example, at least 150, 200, 250, 300, 350, or 400 pg / mL, or within the range of any two of these values. This culture supernatant may contain TGF-β1 in amounts such as 670, 700, 750, 800, 850, 900, 950, or 1000 pg / mL, or within the range of any two of these values. This culture supernatant may contain IL-7 in amounts such as at least 3.5, 4, 5, 6, 7, 8, 9, or 10 pg / mL, or within the range of any two of these values. This culture supernatant may contain IL-8 in amounts such as at least 6500, 7000, 8000, 9000, 10000, 11000, 12000, or 15000 pg / mL, or within the range of any two of these values.

[0077] In embodiments of the present invention (including, for example, (1) to (41) above), the culture medium includes a culture medium used for culturing cells. The culture medium may be a liquid medium or a solid medium. The culture medium includes a culture solution. The culture medium may be any common cell culture medium, and its composition is not limited. The culture medium may include, for example, amino acids, inorganic salts, vitamins, minerals, or a carbon source (e.g., glucose). The culture medium may be, for example, serum medium (e.g., FBS), basal medium (e.g., MEM), compound medium, serum-free medium, etc. The culture medium may be one that is commercially available for the proliferation of human MSCs. Examples of basic cell culture media available from manufacturers include 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 RPMI1640 (e.g., Nacalai Tesque). For example, a medium prepared by adding amino acids to DMEM / F12 medium can be used. Commercially available amino acid solutions for culture media, such as MEM essential amino acid solution (Fujifilm Wako Pure Chemical Industries, Ltd.) or MEM non-essential amino acid solution (Fujifilm Wako Pure Chemical Industries, Ltd.), can be used. This DMEM / F12 medium-based medium does not contain any heterogeneous components, cytokines, insulin, proteins, or human serum. Considering that cultured cells and culture supernatants are used for regenerative medicine and therapeutic purposes, it is desirable that the culture medium be serum-free, serum albumin-free, insulin-free, IGF-free, FGF-free, growth factor-free, cytokine-free, protein-free, or xeno-free (free of heterogeneous components). In this case, there is the advantage of eliminating the risk of foreign active components other than those derived from cultured cells entering the patient's body. The culture medium may contain, for example, LIF components, adhesion molecules, FGF components, insulin components, albumin components, or transferrin components. From the viewpoint of particularly promoting the proliferation of MSCs, it is preferable that the culture medium contains LIF components.From the viewpoint of particularly promoting the proliferation of MSCs, it is preferable that the culture medium further contains adhesion molecules.

[0078] In embodiments of the present invention (including, for example, (1) to (41) above), LIF includes a component also referred to as, for example, leukemia inhibitory factor. Details such as the amino acid sequence of LIF can be found on websites such as NCBI or UniProt. The primary accession number for LIF listed on 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, (1) to (41) above), the LIF component includes a molecule that has LIF activity and is derived from wild-type human LIF. The LIF component may be, for example, (a) a protein or bioactive fragment thereof having the amino acid sequence shown in SEQ ID NO: 4 or 5, (b) a protein or bioactive fragment thereof having an amino acid sequence having 90% or more homology to the amino acid sequence shown in SEQ ID NO: 4 or 5, or (c) a protein or bioactive fragment thereof having an amino acid sequence having one to several amino acid deletions, substitutions, insertions, or additions to the amino acid sequence shown in SEQ ID NO: 4 or 5. LIF activity includes, for example, activity that promotes LIF signaling, activity that binds to LIF receptors, or activity that inhibits the proliferation of leukemia cells. The bioactive fragment includes, for example, a polypeptide having LIF activity in which a portion of LIF not involved in LIF activity is deleted. 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 LIF component concentration in the culture medium may be, for example, 0.01 ng / mL or higher. This concentration may be, for example, 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, and may be greater than or equal to any of these values, or within the range of any two of these values.This concentration may be, for example, 0.1-4, 0.1-3, 0.5-2, or 0.8-1.2 ng / ml. From the viewpoint of promoting the proliferation of MSCs, a concentration of 0.1 ng / ml or higher is preferred, 0.5 ng / ml or higher is more preferred, and 0.8 ng / ml or higher is even more preferred.

[0079] In embodiments of the present invention (including, for example, (1) to (41) above), FGF includes a component also known as fibroblast growth factor. FGF includes, for example, bFGF. bFGF includes a component also known as basic fibroblast growth factor. Details such as the amino acid sequence of bFGF can be found on websites such as NCBI or UniProt. The primary accession number for bFGF listed on UniProt is, for example, P09038. The amino acid sequence of bFGF may be, for example, the amino acid sequence shown in SEQ ID NOs: 6, 7, or 8. bFGF includes, for example, human-derived bFGF. In embodiments of the present invention (including, for example, (1) to (41) above), the bFGF component includes a molecule that has bFGF activity and is derived from wild-type human bFGF. The bFGF component may be, for example, (a) a protein or bioactive fragment thereof having the amino acid sequence shown in SEQ ID NO: 6, 7, or 8; (b) a protein or bioactive fragment thereof having an amino acid sequence having 90% or more homology to the amino acid sequence shown in SEQ ID NO: 6, 7, or 8; or (c) a protein or bioactive fragment thereof having an amino acid sequence having one to several amino acid deletions, substitutions, insertions, or additions to the amino acid sequence shown in SEQ ID NO: 6, 7, or 8. bFGF activity includes, for example, activity that promotes bFGF signaling, activity that binds to bFGF receptors, or activity that promotes fibroblast proliferation. The bioactive fragment includes, for example, a polypeptide having bFGF activity in which a portion of bFGF not involved in bFGF activity is 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). The purified tags include, for example, biochemically inactive tags. The concentration of bFGF components in the culture 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, and may be greater than or equal to any of these values, or within the range of any two of these values. This concentration may also be, for example, 1-40, 1-30, 5-20, or 8-12 ng / ml. From the viewpoint of promoting MSC growth, 1 ng / ml or more is preferred, 5 ng / ml or more is more preferred, and 8 ng / ml or more is even more preferred.

[0080] In embodiments of the present invention (including, for example, (1) to (41) above), insulin includes a type of hormone. Details such as the amino acid sequence of insulin can be found on websites such as NCBI or UniProt. The primary accession number for insulin listed on 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, human-derived insulin. In embodiments of the present invention (including, for example, (1) to (41) above), 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 or bioactive fragment thereof having the amino acid sequence shown in SEQ ID NO: 9, (b) a protein or bioactive fragment thereof having an amino acid sequence having 90% or more homology to the amino acid sequence shown in SEQ ID NO: 9, or (c) a protein or bioactive fragment thereof having an amino acid sequence having one to several amino acid deletions, substitutions, insertions or additions to the amino acid sequence shown in SEQ ID NO: 9. Insulin activity includes, for example, activity that promotes insulin signaling or activity that activates insulin receptor tyrosine kinase. The bioactive fragment includes, for example, an insulin-active polypeptide in which a portion of insulin not involved in insulin activity is deleted. The insulin component may optionally have a tag at its N or C terminus (see, e.g., Mishra, Curr Protein Pept Sci. 2020;21(8):821-830). The tag may be, for example, a purified tag. The purified tag may be, for example, a peptide tag (e.g., a His tag). The purified tag may include, for example, a biochemically inactive tag. The concentration of the insulin component in the culture 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, and may be greater than or equal to any of these values, or within the range of any two of these values. This concentration may be, for example, 1-40, 1-30, 5-20, or 8-12 μg / ml.From the viewpoint of promoting the proliferation of MSCs, a concentration of 1 μg / ml or higher is preferred, 5 μg / ml or higher is more preferred, and 8 μg / ml or higher is even more preferred.

[0081] In embodiments of the present invention (including, for example, (1) to (41) above), albumin includes a type of protein that is abundant in serum. Details such as the amino acid sequence of albumin can be found on websites such as NCBI or UniProt. The primary accession number for albumin listed on 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, human-derived albumin. In embodiments of the present invention (including, for example, (1) to (41) above), 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 or bioactive fragment thereof having the amino acid sequence shown in SEQ ID NO: 10, (b) a protein or bioactive fragment thereof having an amino acid sequence having 90% or more homology to the amino acid sequence shown in SEQ ID NO: 10, or (c) a protein or bioactive fragment thereof having an amino acid sequence having one to several amino acid deletions, substitutions, insertions or additions to the amino acid sequence shown in SEQ ID NO: 10. Albumin activity includes, for example, blood osmotic pressure regulating activity or binding activity to fatty acids or bilirubin. Bioactive fragments include, for example, polypeptides having albumin activity, in which a portion of albumin not involved in albumin activity is deleted. Albumin components 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 purified tag. The purified tag may be, for example, a peptide tag (e.g., a His tag). The purified tag may include, for example, a biochemically inactive tag. The concentration of the albumin component in the culture medium may be, for example, 10 μg / mL or higher. 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, and may be greater than or equal to any one of these values, or within the range of any two of these values.This concentration may be, for example, 100-4000, 10-3000, 500-2000, or 800-1200 ng / ml. From the viewpoint of promoting the proliferation of MSCs, a concentration of 100 μg / ml or higher is preferred, 500 μg / ml or higher is more preferred, and 800 μg / ml or higher is even more preferred.

[0082] In embodiments of the present invention (including, for example, (1) to (41) above), transferrin includes a type of protein present in serum. Details such as the amino acid sequence of transferrin can be found on websites such as NCBI or UniProt. The primary accession number for transferrin listed on UniProt is, for example, P02787. The amino acid sequence of human transferrin includes, for example, the amino acid sequence shown in SEQ ID NO: 11. Transferrin includes, for example, human-derived transferrin. In embodiments of the present invention (including, for example, (1) to (41) above), the transferrin component includes a molecule that has transferrin activity and is derived from wild-type human transferrin. The transferrin component may be, for example, (a) a protein or bioactive fragment thereof having the amino acid sequence shown in SEQ ID NO: 11, (b) a protein or bioactive fragment thereof having an amino acid sequence having 90% or more homology to the amino acid sequence shown in SEQ ID NO: 11, or (c) a protein or bioactive fragment thereof having an amino acid sequence having one to several amino acid deletions, substitutions, insertions, or additions to the amino acid sequence shown in SEQ ID NO: 11. Transferrin activity includes, for example, binding activity to transferrin receptors or binding activity to iron ions. The bioactive fragment includes, for example, a polypeptide having transferrin activity in which a portion of the 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 culture 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, and may be greater than or equal to any of these values, or within the range of any two of these values.This concentration may be, for example, 1-40, 1-30, 5-20, or 8-12 μg / ml. From the viewpoint of promoting the proliferation of MSCs, a concentration of 1 μg / ml or higher is preferred, 5 μg / ml or higher is more preferred, and 8 μg / ml or higher is even more preferred.

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

[0084] In embodiments of the present invention (including, for example, (1) to (41) above), the container for containing the culture supernatant may be a medical container. This container may include, for example, a syringe, vial, bottle, or bag. The medical container may be sterile (for example, the solution container is sterile), for infusion, or for cryopreservation. The medical bag may include a soft bag or a bag with a tube attached.

[0085] In embodiments of the present invention (including, for example, (1) to (41) above), the recovery step may include, for example, a step of separating the cell fraction from the culture supernatant, or a step of transferring the culture supernatant to a container. The separation step may include, for example, a step of separating the cell fraction from the culture supernatant by centrifugation or filtration of the culture medium containing MSCs and imidazole dipeptide. The recovery step may include a step of removing dead cells or impurities from the supernatant by centrifugation or filtration, a step of sterilizing the supernatant, or a step of transferring the supernatant to a medical container. The recovery step may include a step of removing culture medium components from the composition containing cells and culture medium. The recovery step may be carried out in a sterile environment.

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

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

[0088] In embodiments of the present invention (including, for example, (1) to (41) above), disease suppression includes treatment of the disease. Suppression includes, for example, exerting an inhibitory effect, a recurrence suppression effect, a symptom improvement effect, or a preventive effect on a patient's disease or one or more symptoms associated with the disease. Suppression also includes, for example, suppressing ossification in a patient, suppressing the generation of osteoblasts, or suppressing the ossification of cells (for example, suppressing the 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 include culture supernatant or cultured MSCs. If the pharmaceutical composition includes culture supernatant, the pharmaceutical composition includes a composition comprising the culture supernatant. The pharmaceutical composition may include, for example, cytokines or exosomes. In this case, the concentrations of cytokines and exosome markers may be within the concentration range described in the embodiments of the culture supernatant above. The pharmaceutical composition may include, for example, imidazole dipeptides. The concentration of imidazole dipeptides may be within the concentration range described in the embodiments of the culture medium above. The pharmaceutical composition includes a composition used for the suppression or improvement of the above-mentioned disease or a composition used for the prevention of the onset of the above-mentioned disease. The pharmaceutical composition may be manufactured, for example, by mixing an active ingredient with one or more pharmaceutically acceptable carriers by any method known in the art of pharmaceuticals. Furthermore, the form of use of the pharmaceutical composition is not limited as long as it is used for therapeutic purposes, and may be an active ingredient alone or a mixture of the active ingredient and any other component. Furthermore, the shape of the carrier is not particularly limited, and may be, for example, a solid or a liquid (e.g., a buffer solution). The amount of the carrier may be, for example, a pharmaceutically effective amount. The effective amount may be, for example, a sufficient amount for the pharmaceutical stability or delivery of the active ingredient. For example, a buffer solution is effective in stabilizing the active ingredient in the container. The pharmaceutical composition may also contain stabilizers, buffers, or pH adjusters. The dosage, administration interval, method of administration, and route of administration are not particularly limited and can be appropriately selected depending on the patient's age, weight, symptoms, target organ, etc. It is also preferable that the pharmaceutical composition contains a therapeutically effective amount or an effective amount of the active ingredient that exerts the desired effect. In one embodiment of the present invention, the therapeutically effective amount includes the amount necessary for clinically observed improvement or suppression of symptoms in the patient (for example, a sufficient amount for suppression of ossification of the target (e.g., suppression of heterotopic ossification)). In one embodiment of the present invention, pharmaceutically acceptable includes a state suitable for use in proportion to a reasonable benefit / risk ratio, within the range of reasonable medical judgment. There are no particular restrictions on components other than the culture supernatant or MSC in the pharmaceutical composition, as long as they do not impair the effects of the present invention, and can be appropriately selected according to the purpose.

[0090] In embodiments of the present invention (including, for example, (1) to (41) above), the subject (including the patient) includes one or more non-human mammals (for example, one or more species such as mice, guinea pigs, hamsters, rats, mice, rabbits, pigs, sheep, goats, cattle, horses, cats, dogs, marmosets, monkeys, or chimpanzees). The patient may also be a patient who requires suppression of ossification, a patient diagnosed with ossification, a patient who requires treatment for ossification, or a patient who requires 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, MSC, or pharmaceutical composition to the subject is preferably one that is effective for treatment, and may be, for example, intravenous, intra-arterial, subcutaneous, intra-lymph node, intramuscular, intraperitoneal, or oral. The form of administration is preferably one that is effective for treatment, and may be, for example, an injectable preparation (e.g., intravenous injection), a liquid preparation, or a solid preparation.

[0092] In embodiments of the present invention (including, for example, (1) to (41) above), the dosage, administration interval, and administration method of the culture supernatant, MSCs, or pharmaceutical composition administered to the subject can be appropriately selected depending on the patient's age, weight, symptoms, target organ, etc. For example, the dosage may be 0.01 to 1000 mL in the case of supernatant, and 1 × 10¹⁶ per administration in the case of cells. 3 ~1 × 10 11 This may also be the case. 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, (1) to (41) above), the suppression of osteoblast generation includes, for example, suppression caused by the suppression of differentiation of MSCs into osteoblasts, or suppression caused by the suppression of osteoblast proliferation. For example, the suppression of differentiation of MSCs into osteoblasts may occur by contact between MSCs and imidazole dipeptides.

[0094] In embodiments of the present invention (including, for example, (1) to (41) above), the method for suppressing osteoblast generation may include, for example, the steps of culturing MSCs in a medium containing imidazole dipeptide, collecting MSC secretions, and contacting the MSC secretions with other MSCs. In this case, the MSCs used for culturing and the MSCs used for contact are different MSCs.

[0095] In embodiments of the present invention (including, for example, (1) to (41) above), the MSC secretion includes, for example, 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. These may be of human origin.

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

[0097] In one embodiment of the present invention, the homology (%) may be calculated, for example, by determining the percentage of homologous amino acids among 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 in parts of the sequences if necessary to maximize the percentage of homologous / identical amino acids. Alignment methods, percentage calculation methods, and related computer programs are conventionally well known in the art. Homology is calculated, for example, using global or local alignment. The former may be obtained using the Needleman-Wunsch algorithm (Needleman et al., J Mol Biol. 1970 Mar;48(3):443-53.) or calculated using 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 can be obtained 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 being compared) × 100. Homology is preferably calculated using global alignment. Unless otherwise specified, a homology of the sequence being examined being greater than or equal to a certain value includes at least one of the above algorithms yielding a value greater than or equal to that value. Any of the above algorithms can be used with default settings.

[0098] In one embodiment of the present invention, the above-mentioned 90% or more, which is stated as the homology value, may be, for example, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, and may be greater than or equal to any of these values, or within the 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 above-mentioned number of items may be, for example, 15, 10, 8, 6, 5, 4, 3, 2, 1, or 0, and may be less than or equal to any of these values, or within the 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 substitution occurs in relation 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, a conservative amino acid substitution includes substituting an amino acid residue with an amino acid residue having a side chain of 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), non-charged 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 acids are 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 contains a "specific amino acid sequence," any of the amino acids in that amino acid sequence may be chemically modified. Also, any of the amino acids in that amino acid sequence may form a salt or a solvate. Also, any of the amino acids in that amino acid sequence may be L-type or D-type. Even in such cases, the protein according to an embodiment of the present invention can be said to contain the above-mentioned "specific amino acid sequence." Examples of chemical modifications that amino acids contained in proteins 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 assessed, for example, by using Student's t-test (one-sided or two-sided) to determine statistical significance when p < 0.05 or p < 0.01. Alternatively, significance may be assessed when a substantial difference exists.

[0102] All publications cited herein are incorporated by reference in their entirety. In this specification, “or” is used when “at least one” of the items listed in the text can be adopted. The same applies to “or.” In this specification, when “within the range of two values” is specified, that range includes the two values ​​themselves. In this specification, “A to B” includes A and B. In this specification, “(1) to (41) above” includes references to 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] One 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 the culture supernatant from a medium containing mesenchymal stem cells and imidazole dipeptide. 2. The production method according to paragraph 1, wherein the mesenchymal stem cells are mesenchymal stem cells cultured in a medium containing imidazole dipeptide. 3. The production method according to 1 or 2 above, comprising the step of culturing mesenchymal stem cells in a medium containing imidazole dipeptide to produce cultured mesenchymal stem cells. 4. The production method according to any one of 1 to 3 above, wherein the recovery step includes a step of centrifugation or filtration of a culture 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 recovered 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 the culture supernatant obtained by adherent culture. 7. The production method according to 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 according to 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 according to any one of items 1 to 8 above, wherein the imidazole dipeptide is a compound having the structure of formula (1) or (2) described above. 10. The production method according to any one of items 1 to 9 above, wherein the imidazole dipeptide is carnosine, anserine, balenine, or homocarnosine. 11. The production method according to any one of 1 to 10 above, wherein the imidazole dipeptide is carnosine. 12. The production method according to any one of claims 1 to 10 above, wherein the imidazole dipeptide is anserine. 13. Culture supernatant obtained by any of the production methods described in 1 to 12 above. 14. A pharmaceutical composition comprising a culture supernatant obtained by any of the production methods described in 1 to 12 above. 15. The culture supernatant described in 13 above or the pharmaceutical composition described in 14 above, for the purpose of inhibiting osteogenic differentiation. 16. A syringe, vial, or medical bag containing the culture supernatant described in item 13 above or the pharmaceutical composition described in item 14 or 15 above. 17. A cell culture method comprising the step of culturing mesenchymal stem cells in a serum-free medium containing imidazole dipeptide to generate cultured cells. 18. The culture method according to 17, further comprising the step of recovering the cultured cells from the culture medium. 19. The culture method according to 17 or 18 above, wherein the culture includes the step of culturing in the presence of adhesion factors. 20. The culture method according to any one of paragraphs 17 to 19 above, wherein the culture medium is insulin-free, IGF-free, or FGF-free. 21. The culture method according to any one of paragraphs 17 to 19 above, wherein the culture medium is a growth factor-free culture medium. 22. The culture method according to any one of paragraphs 17 to 19 above, wherein the culture medium is a protein-free culture medium. 23. A method for producing cells, comprising the step of carrying out the culture method described in any of items 17 to 22 above. 24. Cells obtained by the production method described in item 23 above. 25. A composition comprising a population of cells described in item 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 osteogenic differentiation. 28. A culture medium for mesenchymal stem cell culture, comprising an imidazole dipeptide and being serum-free. 29. The culture medium described in 28 above, which is insulin-free, IGF-free, or FGF-free. 30. The culture medium described in 28 or 29 above, which is growth factor-free. 31. A protein-free culture medium as described in any of items 28-30 above. 32. A culture medium containing mesenchymal stem cells, as described in any of items 28 to 31 above. 33. A method for suppressing osteoblast generation, comprising the step of culturing mesenchymal stem cells in a medium containing imidazole dipeptide. 34. A method for inhibiting osteoblast generation, comprising the step of contacting mesenchymal stem cells with a culture supernatant obtained by culturing mesenchymal stem cells in a medium containing imidazole dipeptide. 35. A composition containing an imidazole dipeptide for inhibiting osteoblast formation. 36. A composition for inhibiting osteoblast generation, comprising the 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 containing imidazole dipeptide for promoting the expression of mesenchymal stem cell markers such as 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. 39. A method for promoting exosome secretion, comprising the step of culturing mesenchymal stem cells in a medium containing imidazole dipeptide. 40. A composition containing an imidazole dipeptide for promoting the secretion of exosomes from mesenchymal stem cells. 41. Culture supernatant of mesenchymal stem cells containing at least 500 pg / ml of G-CSF. 42. Culture supernatant of mesenchymal stem cells 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 population of cells described in 43 above. 45. IL-34-positive mesenchymal stem cells. 46. ​​Purified mesenchymal stem cells as described in 45 above. 47. ADAM8-positive mesenchymal stem cells. 48. Purified mesenchymal stem cells as described in 47 above. 49. Mesenchymal stem cells as described in any of 45-48 above, which are positive for Osteoprotegrin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, and semaphorin 3B. 50. Purified mesenchymal stem cells as described in 49 above. 51. A pharmaceutical composition comprising mesenchymal stem cells as described in any of items 45 to 50 above. 52. The pharmaceutical composition described in 51 above for inhibiting osteogenic differentiation. 53. A pharmaceutical composition for inhibiting osteogenic differentiation, comprising cells obtained by culturing mesenchymal stem cells in a medium containing imidazole dipeptide. 54. The pharmaceutical composition according to 53, further comprising a pharmaceutically acceptable carrier. 55. The pharmaceutical composition according to 53 or 54, wherein the cells are positive for osteoporotegrin, 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 cells obtained by culturing them in a medium containing imidazole dipeptide and LIF. 57. The pharmaceutical composition according to any one of the above 53 to 56, wherein the culture is an adherent culture. 58. A method for producing a pharmaceutical composition for inhibiting osteogenic differentiation, comprising the step of culturing mesenchymal stem cells in a medium containing imidazole dipeptide to generate cultured cells. 59. The production method according to 58 above, further comprising the step of recovering the cultured cells from the culture medium. 60. The production method according to 58 or 59, comprising the step of mixing the cultured cells with a pharmaceutically acceptable carrier. 61. A culture medium containing imidazole dipeptide and LIF (leukemia inhibitory factor) components. 62. The culture medium according to 61 above, further comprising an adhesion factor. 63. The culture medium described in 61 or 62 above for culturing mesenchymal stem cells. 64. A culture medium containing mesenchymal stem cells, as described in any of the above 61-63. 65. A method for producing cells, comprising the step of culturing mesenchymal stem cells in a culture medium described in any of the above 61 to 64 to generate cultured cells. 66. The culture is an adherent culture. The production method described in 65 above. 67. Cells obtained by the production method described in 65 or 66 above. 68. A composition comprising a 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. The pharmaceutical composition described in 69 above for inhibiting osteogenic differentiation. 71. A kit containing imidazole dipeptide and LIF components. 72. The kit described in 71 above for use in preparing culture media for mesenchymal stem cells.

[0104] Although 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 other configurations can be adopted. Furthermore, the present invention can be adopted by combining or independently the configurations or features described in the above embodiments. [Examples]

[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: Culturing MSCs in 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 10 e5 in 20 ml of MSC Expansion XSFM B (Fujifilm Wako Pure Chemical Corporation) (hereinafter also referred to as MSC medium B). 10 μl of iMatrix-511 laminin fragment (0.5 μg / μl) (Nippi Corporation) was added to 20 ml of the cell suspension, and the resulting cell suspension was seeded in a T150 flask (Sumitomo Bakelite Co., Ltd.).

[0107] Seven days after sowing, 10 ml of MSC medium B containing 5 μl of iMatrix-511 laminin fragment (0.5 μg / μl) (Nippi Corporation) was added. Fourteen days after sowing (day 14), the medium was replaced twice with PBS to wash away any remaining medium. Then, 30 ml of protein-free medium (hereinafter also referred to as MSC medium A), based on DMEM / F12 medium with added 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). At this time, L-carnosine (Fujifilm Wako Pure Chemical Corporation), an imidazole dipeptide, was added to MSC medium A either at no concentration (0 mM), or at concentrations of 1 mM, 10 mM, or 30 mM. These carnosine-supplemented MSC medium A was added to the cells, and they were 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 in the culture supernatant was analyzed using an ELISA kit (R&D Systems) or a Milliplex kit (Merck Millipore). The cytokines analyzed were HGF (Hepatocyte growth factor), G-CSF (Granulocyte colony stimulating factor), MCP-1 (Monocyte chemotactic protein 1), VEGF-C (Vascular endothelial growth factor-C), TGF-β1 (Transforming growth factor-β1), IL-6 (Interleukin-6), IL-7 (Interleukin-7), and IL-8 (Interleukin-8). In addition, the amount of exosomes in the culture supernatant was analyzed using a CD9 / CD63 ELISA kit (CosmoBio) with exosome marker protein (CD9 / CD63 fusion protein) as an indicator. In this example, the amount of cytokines and the amount of exosome marker proteins refer to values ​​measured by ELISA using specific antibodies.

[0109] After the first production culture, the cells were returned to MSC medium B and cultured for 2 days (recovery culture). After 2 days of recovery culture, the cells were again replaced with MSC medium A supplemented with carnosine, as described above, and cultured for 3 days (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 cells were returned to MSC medium B and cultured for 2 days (recovery culture). After 2 days of recovery culture, the cells were again replaced with MSC medium A supplemented with carnosine, as described above, and cultured for 3 days (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 cycle, the culture supernatant of MSC medium A supplemented with carnosine was collected. Cytokines in the culture supernatant were analyzed using an ELISA kit (R&D Systems).

[0113] After the third production culture, the cells were returned to MSC medium B and cultured for 2 days (recovery culture). After 2 days of recovery culture, the cells were again replaced with MSC medium A supplemented with carnosine, as described above, and cultured for 3 days (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 cycle, the culture supernatant of MSC medium A supplemented with carnosine was collected. Cytokines in the culture supernatant were analyzed using an ELISA kit (R&D Systems).

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

[0116] 1.2 Morphological observation of cells Figures 1-4 are micrographs of MSCs after culturing in carnosine-containing or carnosine-free medium. No significant cell death was observed, and no clear differences in cell morphology were seen at each carnosine concentration.

[0117] 1.3 Counting the number of cells Figure 5 shows the cell count results for MSCs after culturing in carnosine-containing medium and carnosine-free medium. No significant change in cell count was observed when carnosine was added to the medium.

[0118] 1.4 Exosome marker levels Figure 6 shows the results of examining the amount of exosome markers in the culture supernatant. Adding carnosine to the culture medium resulted in an increasing trend in exosome marker levels.

[0119] 1.5 Cytokine levels Figures 7-14 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. Adding carnosine to the culture medium hardly changed the amount of HGF. On the other hand, G-CSF, MCP-1, VEGF-C, TGF-β1, IL-6, IL-7, and IL-8 showed an increasing trend (the values ​​in parentheses represent the NCBI Official Symbol and Gene ID). In particular, the increase rates of G-CSF and IL-6 were significant. When comparing the results at the fourth culture supernatant collection, the G-CSF concentration increased 14.7 times (approximately 1621 pg / mL) and the IL-6 concentration increased more than 22 times (more than 7000 pg / mL) when 10 mM carnosine was added.

[0120] Furthermore, in the experimental method described above, umbilical cord-derived MSCs were replaced with adipose-derived MSCs for culturing. Subsequently, the amounts of G-CSF and MCP-1 in the culture supernatant were measured. As a result, the amount of both increased when carnosine was added to the culture medium.

[0121] Example 2: Inhibitory effect of culture supernatant on osteogenic differentiation 2.1 Experimental Method 2.1.1 Culture of MSCs and recovery of culture supernatant using imidazole dipeptide-containing medium Using human umbilical cord-derived mesenchymal stem cells (CET03 strain) established by the present inventor, the culture supernatant was collected according to the experimental method up to the third production culture described in Example 1 above. However, this was carried out under conditions in which the MSC medium A used in Example 1 was replaced with DMEM(Sigma), DMEM / F12(Sigma), or IMDM(Sigma). In addition, the concentration of carnosine added was changed to 0 or 10 mM. As a result, the first, second, and third production culture supernatants were obtained. Furthermore, equal volumes 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 denoted as DMEM_supernatant, DMEM_C_supernatant, DMEM / F12_supernatant, DMEM / F12_C_supernatant, IMDM_supernatant, and IMDM_C_supernatant (the name of the culture medium used is indicated on the left side of these supernatant names. Supernatants obtained under carnosine supplementation conditions are indicated with a "C" to the right of the culture medium name).

[0122] 2.1.2 MSC Osteogenesis Induction and Calcium Deposition Analysis Procedure 1. Human bone marrow MSCs (primary cultured human bone marrow MSCs) were cultured in αMEM medium containing 10% FBS (Hyclone), treated with trypsin-EDTA solution, and then separated into single cells. The resulting single cells were seeded in basal medium containing 10% FBS (DMEM (Sigma) with 0.6 mM CaCl2) at a concentration of 8 × 10 e4 cells / well in fibronectin-coated 24-well plates. The day after seeding, the medium was changed with basal medium (DMEM (Sigma) with 0.6 mM CaCl2) (hereinafter also referred to as osteogenic medium) supplemented with osteogenic differentiation-inducing 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 also referred to as osteogenic differentiation-inducing medium). The medium used for this medium change was changed every 2-3 days, and the cells were cultured for 10-14 days until calcium deposition was observed in the cells. The amount of intracellular calcium deposition in the cultured cells was quantified by a calcium assay. The obtained cells are denoted as DMEM_medium_cells and DMEM_C_medium_cells (the cell names are indicated with the name of the medium used on the left. Cells obtained under carnosine supplementation conditions are indicated with a "C" to the right of the medium name).

[0123] Step 2. Step 1 was carried out under the same conditions, but with the basal medium changed to DMEM / F12(Sigma) with 0.6 mM CaCl2 added. The resulting cells are referred to as DMEM / F12_medium_cells and DMEM / F12_C_medium_cells (the cell names are indicated with the name of the medium used on the left. Cells obtained under carnosine-added conditions are indicated with a "C" to the right of the medium name).

[0124] Step 3. Step 1 was performed under the same conditions as above, but with the basal medium changed to IMDM (Sigma) with 2 mM L-glutamin added. The resulting cells are denoted as IMDM_medium_cells and IMDM_C_medium_cells (the cell names indicate the medium used on the left. Cells obtained under carnosine-added conditions are indicated with a "C" to the right of the medium name). In addition, the same procedure as Step 3 was performed separately, and calcium deposition images were taken of cells after osteogenic differentiation induction culture using an Alizarin Red S staining kit (CosmoBio).

[0125] Step 4. Step 1 was carried out under the same conditions as above, but with the osteogenicity induction medium replaced with the umbilical cord MSC culture supernatant (DMEM_supernatant or DMEM_C_supernatant) obtained in 2.1.1, to which the components for osteogenicity induction (10% FBS, 0.1 μM dexamethasone, 50 μM ascorbic acid, and 10 mM β-glycerophosphate) were added. The resulting cells are referred to as DMEM_supernatant_cells and DMEM_C_supernatant_cells (the cell names are indicated with the name of the medium used on the left. Cells obtained under carnosine-added conditions are indicated with a "C" to the right of the medium name).

[0126] Step 5. Step 2 was carried out under the condition that the osteogenic differentiation induction medium was replaced with the umbilical cord MSC culture supernatant (DMEM / F12_supernatant or DMEM / F12_C_supernatant) obtained in 2.1.1 above, to which the components for osteogenic differentiation induction (10% FBS, 0.1 μM dexamethasone, 50 μM ascorbic acid, and 10 mM β-glycerophosphate) were added. The resulting cells are referred to as DMEM / F12_supernatant_cells and DMEM / F12_C_supernatant_cells (the cell names are indicated with the name of the medium used on the left. Cells obtained under carnosine-added conditions are indicated with a "C" to the right of the medium name).

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

[0128] 2.2 Results Figure 15 shows the results of quantifying calcium deposition in bone marrow MSCs. The culture medium names in the figure are the names of the basal media used. When bone marrow MSC osteogenic differentiation was quantified by calcium content, cells obtained using carnosine-free medium (C_0mM in the figure; corresponding samples are DMEM_C_cells, DMEM / F12_C_cells, and IMDM_C_cells) and cells obtained using the supernatant of MSCs cultured in carnosine-free medium (Supernatant_C_10mM in the figure; corresponding samples are DMEM_C_supernatant_cells, DMEM / F12_C_supernatant_cells, and IMDM_C_supernatant_cells) showed significantly lower calcium content compared to cells obtained using carnosine-free medium (Supernatant_C_0mM in the figure; corresponding samples are DMEM_C_supernatant_cells, DMEM / F12_C_supernatant_cells, and IMDM_C_supernatant_cells). Furthermore, the calcium content was significantly lower compared to cells obtained using carnosine-supplemented medium (C medium 10mM in the figure; corresponding samples are DMEM C medium cells, DMEM / F12 C medium cells, and IMDM C medium cells). The supernatant of MSCs cultured in carnosine-supplemented medium showed a significant inhibitory effect on osteogenic differentiation.

[0129] Figure 16 shows images of bone marrow MSCs stained with Alizarin Red S. When bone marrow MSCs were stained with Alizarin Red S after induction of osteogenic differentiation, cells obtained using a carnosine-free medium (DMEM_medium_cells), cells obtained using the supernatant of MSCs cultured in a carnosine-free medium (DMEM_supernatant_cells), and cells obtained using a carnosine-supplemented medium (DMEM_C_medium_cells) all showed staining with Alizarin Red. On the other hand, cells obtained using the supernatant of MSCs cultured in a carnosine-supplemented medium (DMEM_C_supernatant_cells) did not show staining with Alizarin Red. The supernatant of MSCs cultured in a carnosine-supplemented medium showed a significant inhibitory effect on osteogenic differentiation.

[0130] Example 3: Inhibitory effect of MSC on osteogenic differentiation 3.1 Experimental Method 3.1.1 Culture of umbilical cord MSCs using imidazole dipeptide-containing medium Human umbilical cord MSCs established by the inventors of this application were cultured in DMEM / F12 medium (hereinafter also referred to as MSC medium C) containing 10 ng / ml bFGF (Peprotec, AF-100-18C), 10 μg / ml insulin (Nacalai, 12878-44), 1000 μg / ml albumin (Sigma, A9511), 10 ug / ml transferrin (Nacalai, 12879-34), and 1 ng / ml LIF (Peprotec, AF-300-05), and then treated with trypsin EDTA solution to form single cells. Carnosine was added to the same medium at a final concentration of 0 or 5 mM, and iMatrix-511 laminin fragment (Nippi Corporation) was added at a final concentration of 0.25 μg / ml, and the cells were seeded in T25 flasks (Corning). After culturing for two weeks, the cells were washed with PBS to remove carnosine, then treated with trypsin-EDTA solution to separate 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), treated with trypsin-EDTA solution, and then separated into single cells. These bone marrow MSCs were mixed with umbilical cord MSCs obtained by culturing in the carnosine 0 or 5 mM supplemented medium described in 3.1.1 above, in ratios of 1:0, 0:1, 1:1, and 3:1, respectively. Furthermore, the combined total number of MSCs was seeded into 24-well plates in αMEM medium with 10% FBS to a total of 8.0 x 10 e4 / well. On the day after seeding, the wells of each co-cultured cell condition were changed with either (1) 10% FBS αMEM medium supplemented with osteogenic components (0.1 μM dexamethasone, 50 μM ascorbic acid, and 10 mM β-glycerophosphate), or (2) 10% FBS αMEM medium without osteogenic components. The medium was changed with the same medium every 2-3 days, and the cells were cultured for 9 days. Osteogenic differentiation was evaluated by calcium assay and alizarin red S staining.

[0132] 3.2 Results Figure 17 shows the quantitative results of calcium deposition during co-culture of bone marrow MSCs and umbilical cord MSCs. While significant calcium deposition was observed under 1:1 co-culture conditions for bone marrow MSCs and umbilical cord MSCs (BM:UC(1:1)), calcium deposition was significantly suppressed under 1:1 co-culture conditions for bone marrow MSCs and umbilical cord MSCs pre-cultured with carnosine (BM:UC_preC(1:1)). This indicates that pre-cultured umbilical cord MSCs with carnosine have an inhibitory effect on MSC osteogenic differentiation. Since carnosine was removed by washing the umbilical cord MSCs with PBS after pre-culture, and no carnosine was added to the culture medium during osteogenic differentiation induction, the effect observed is purely from the cells (umbilical cord MSCs pre-cultured with carnosine). The explanations of the terms in the figure are as follows. BM: Non-co-culture conditions using bone marrow MSCs (8 x 10^4 cells / well). Conditions in αMEM medium with osteogenic differentiation-inducing components. BM_NC: Non-co-culture conditions using bone marrow MSCs (8 x 10^4 cells / well). Conditions in αMEM medium without osteogenic differentiation-inducing components. UC: Non-co-culture conditions using umbilical cord MSCs (8 x 10^4 cells / well) pre-cultured for 2 weeks in MSC medium C without carnosine. Conditions using αMEM medium with osteogenic components. UC_preC: Non-co-culture conditions using 8 x 10^4 cells / well umbilical cord MSCs pre-cultured for 2 weeks in MSC medium C containing 5 mM carnosine. αMEM medium conditions with induced osteogenic differentiation. 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 are mixed and seeded in 4 x 10^4 cells / wells. αMEM medium conditions with bone differentiation-inducing components. BM:UC_preC(1:1): Co-culture conditions in which bone marrow MSCs and umbilical cord MSCs pre-cultured for 2 weeks in CET original medium containing 5 mM carnosine are mixed and seeded in 4 x 10^4 cells / wells. Conditions in αMEM medium with bone differentiation-inducing components. BM:UC(3:1): Co-culture conditions in which bone marrow MSCs (6 x 10^4 cells / well) and umbilical cord MSCs (2 x 10^4 cells / well) pre-cultured for 2 weeks in MSC medium C without carnosine are mixed and seeded. αMEM medium conditions with osteogenic differentiation-inducing components. BM:UC_preC(3:1): Co-culture conditions in which bone marrow MSCs (6 x 10^4 cells / well) and umbilical cord MSCs (2 x 10^4 cells / well) pre-cultured for 2 weeks in CET original medium containing 5 mM carnosine are mixed and seeded. αMEM medium conditions with osteogenic differentiation-inducing components.

[0133] Figure 18 shows images stained with Alizarin Red S during co-culture of bone marrow MSCs and umbilical cord MSCs. While significant calcium deposition is observed under the osteogenic differentiation conditions (BM:UC(1:1)) for co-culture of bone marrow MSCs and umbilical cord MSCs, calcium deposition is significantly suppressed under the osteogenic differentiation conditions (BM:UC_preC(1:1)) for co-culture of bone marrow MSCs and umbilical cord MSCs pre-cultured with carnosine. This demonstrates that pre-cultured umbilical cord MSCs with carnosine have an inhibitory effect on MSC osteogenic differentiation.

[0134] Example 4: Analysis of MSCs 4.1 Experimental Method 4.1.1 RNA recovery from MSCs cultured using imidazole dipeptide-containing medium Using human umbilical cord-derived mesenchymal stem cells (CET03 strain) established by the present inventor, the culture supernatant was collected according to the experimental method up to the third production culture described in Example 1 above. However, at this time, the growth 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. For the supernatant collection medium, αMEM (Sigma), DMEM (Sigma), or DMEM / F12 (Sigma) was used. For the growth medium, MSC medium B or MSC medium C (composition is shown in Example 3) was used. The carnosine addition concentration was 0 mM, 10, 20, or 30 mM. The resulting first, second, and third production culture supernatants were used for ELISA testing. RNA was extracted from the cells thereafter and used for RNA-seq analysis described later.

[0135] 4.1.2 RNA-seq analysis Cells were collected on day 27 after supernatant collection, and RNA was recovered using the miRNeasy Mini Kit (Qiagen) and used for RNA-seq analysis. A NextSeq500 (Illumina) was used as the sequencing instrument. The sequencing read length was 75 bp per read, and the analysis was performed using the equivalent of 1 million reads. The analysis program used was StrandNGS ver4.0 (Strand Life Sciences Pvt Ltd). Fastq reads after removal of unreliable bases were mapped to the human reference genome hg38, and RPM expression level correction was performed. RPM correction values ​​were calculated for each gene for a total of 19 samples.

[0136] 4.1.3 Listing of genes with increased expression levels by RNA-seq analysis For a total of 13 cell samples obtained under carnosine supplementation conditions, the average percentage increase in gene expression (comparison between conditions with 0 mM carnosine and conditions with 10-30 mM carnosine) was calculated. Genes with high average percentage increases in cells obtained under carnosine supplementation 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-seq analysis identified the top 21 secreted protein-coding genes whose expression levels increased with carnosine. Of these, the following 10 are factors previously reported to be involved in inhibiting 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 terms in parentheses represent the NCBI's Official Symbol and Gene ID, as well as alternative names. It was shown that a large proportion of the secreted proteins whose expression was enhanced by carnosine were those involved in inhibiting bone formation, and that carnosine enhanced the expression of various secreted proteins involved in inhibiting bone formation. These results suggest that the inhibitory effect on bone differentiation produced by umbilical cord MSCs cultured under carnosine supplementation is due to a paracrine effect by proteins secreted from the MSCs.

[0138] Figures 20(a) to 20(j) show the results of the change in the % increase values ​​of the 10 genes mentioned above. Figures 20(a) to 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 to 22 all show the results when DMEM / F12 (Sigma) was used as the supernatant collection medium and MSC medium B was used as the growth medium. The concentrations in the figures are the concentrations of added carnosine. Figure 21 is a graph showing the RPM values ​​of interleukin 34 expression in umbilical cord MSCs. Interleukin 34 expression was observed when carnosine was added at 10 mM or 30 mM, but not when no carnosine was added (0 mM). Figure 22 is a graph showing the RPM values ​​of ADAM metallopeptidase domain 8 expression in umbilical cord MSCs. ADAM metallopeptidase domain 8 expression was observed when carnosine was added at 10 mM, 20 mM, or 30 mM, but not when no carnosine was added (0 mM).

[0139] Example 5: Analysis of culture supernatant and MSCs 5.1 Culture of MSCs and recovery of culture supernatant using imidazole dipeptide-containing medium Using human umbilical cord-derived mesenchymal stem cells (CET03 strain) established by the present inventor, the culture supernatant was collected according to the experimental method up to the third production culture described in Example 1 above. However, this was carried out under conditions in which the MSC medium A used in Example 1 was replaced with αMEM (Sigma), DMEM (Sigma), or DMEM / F12 (Sigma). In addition, the concentration of carnosine added was changed to 0 or 30 mM (a 500 mM carnosine solution in distilled water was used for addition; the same amount of distilled water was used for 0 mM). As a result, the first, second, and third production culture supernatants were obtained and used for ELISA testing. RNA was extracted from the cells thereafter and used for RNA-seq analysis described later.

[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). Osteoprotegerin and M-CSF (CSF1, 1435) were analyzed as cytokines. These two cytokines are factors that have been previously reported to be involved in the inhibition of bone formation. In this experiment, the amount of cytokines is shown as a value measured by ELISA using a specific antibody.

[0141] 5.3 Quantitative PCR (qPCR) analysis Cells were collected on day 27 after supernatant collection, RNA was recovered using the miRNeasy Mini Kit (Qiagen), and the expression levels of the osteoprotegerin gene TNFRSF11B and the M-CSF gene CSF1 were analyzed using quantitative PCR (PowerUp SYBR Green Master Mix, Thermo Fisher) with the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as the internal standard.

[0142] 5.4 Results Figures 23-25 ​​show the results of ELISA analysis of Osteoprotegerin, and Figures 26-28 show the results of ELISA analysis of M-CSF. The concentration (mM) in the figures refers to the carnosine concentration. For supernatant collection, αMEM was used in Figures 23 and 26, DMEM in Figures 24 and 27, and DMEM / F12 in Figures 25 and 28. A concentration-dependent increase in Osteoprotegrin and M-CSF secretion was observed with the addition of carnosine for each of the 1st, 2nd, and 3rd collection cycles. Figure 29 shows the results of qPCR analysis of Osteoprotegerin, and Figure 30 shows the results of qPCR analysis of M-CSF. The concentration (mM) in the figures refers to the carnosine concentration, and the medium name refers to the supernatant collection medium used. Under each condition, a concentration-dependent increase in Osteoprotegrin and M-CSF gene expression levels was observed with the addition of carnosine.

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

[0144] 6.2 Results Figure 31 shows the results of ELISA analysis of osteoprotegrin. Figure 31(a) shows the results under conditions with carnosine added, and Figure 31(b) shows the results under conditions with anserine added. In both cases, umbilical cord MSCs were used, and MSC medium B was used as the growth medium. In both the first and second collection cycles, there was a tendency for osteoprotegrin secretion to increase with the addition of carnosine or anserine, respectively.

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

[0146] Figure 34 shows the results of ELISA analysis of M-CSF. Figure 34(a) shows the results under conditions with carnosine added, and Figure 34(b) shows the results under conditions with anserine added. In both cases, umbilical cord MSCs were used, and MSC medium B was used as the growth medium. In both the first and second collection cycles, there was a tendency for M-CSF secretion to increase with the addition of carnosine or anserine.

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

[0148] Example 7: Effect of promoting the proliferation of MSCs 7.1 Experimental Method 7.1.1 MSC Growth Culture Human umbilical cord-derived mesenchymal stem cells established by the inventors of the present invention were detached using TrypLE Select and seeded in 24-well plates at a cell count of 200 or 2000 cells / cm2. The culture medium used was (1) MSC Expansion XSFM B (Fujifilm Wako Pure Chemical Industries, Ltd.) (hereinafter also referred to as MSC medium B), (2) DMEM / F12 medium containing 10 ng / ml bFGF (Peprotec), 10 μg / ml insulin (Nacalai), 1000 μg / ml albumin (Sigma), 10 ug / ml transferrin (Nacalai), and 1 ng / ml LIF (Peprotec) (hereinafter also referred to as MSC medium D), or (3) medium obtained by removing LIF from MSC medium D (hereinafter also referred to as MSC medium E). Furthermore, iMatrix-511 laminin fragment (Nippi Corporation) was added to each culture medium to a final concentration of 0 or 0.1%, and carnosine or anserine was added to a final concentration of 0, 0.2, 0.5, 1, 2, or 5 mM, and cells were seeded. Each sample was cultured for 4 to 7 days in the same culture medium used, changing the medium every 2 to 3 days. After that, the cells were stained with DAPI, phalloidin, and CD44 antibody, and fluorescence images were taken using a confocal quantitative image cytometer CellVoyager CQ-1 (Yokogawa Electric Co., Ltd.). Cell counts using DAPI and fluorescence area measurements for phalloidin (actin filament marker) and CD44 (MSC marker) were performed. A summary of each experimental condition is shown in Table 1. [Table 1]

[0149] Furthermore, umbilical cord MSCs established by the inventors of this application were detached using TrypLE Select and seeded in a 24-well plate at a cell count of 200 cells / cm2. The culture medium used was (4) MSC medium D, or (5) medium obtained by removing LIF from MSC medium D (hereinafter also referred to as MSC medium E). In addition, iMatrix-511 laminin fragment (Nippi Corporation) was added to each medium to a final concentration of 0.1%, and carnosine was added to a final concentration of 0, 0.2, 1, or 5 mM before seeding. Each sample was cultured for 7 days in the same medium used, changing the medium every 2-3 days. After that, staining with DAPI and phalloidin was performed, and fluorescence images were taken using a confocal quantitative image cytometer CellVoyager CQ-1 (Yokogawa Electric Co., Ltd.), and cell counting by DAPI and fluorescence area of ​​phalloidin (actin filament marker) were measured. The outline of each experimental condition is shown in Table 2. [Table 2]

[0150] 7.1.2 Measurement of the positive rate of MSC markers Carnosine was added to MSC medium D, and umbilical cord MSCs were added at a rate of 200 cells / cm². 2 The cells were seeded and cultured in T25 flasks for two weeks (two passages). The seeded cells in the T25 flasks were detached and collected using TrypLE Select and separated into single cells. The collected cells were fixed with 4% formaldehyde solution, and then immunostained with CD31, 44, 45, 73, 90, 105, HLA-ABC, and HLA-DR. The percentage of cells positive for each marker was measured using a BD Accuri™ C6 Plus flow cytometer (BD Bioscience).

[0151] 7.2 Results Figures 37-38 show the cell count results for groups 1-4. In particular, in group 3, an increasing trend in cell number was observed with the addition of carnosine. Figure 39 shows the results of phalloidin fluorescence area measurements for groups 1-4. In particular, in group 3, an increasing trend in phalloidin area was observed with the addition of carnosine. Figure 40 shows the results of CD44 fluorescence area measurements for groups 1-4. In particular, in group 3, an increasing trend in CD44 area was observed with the addition of carnosine.

[0152] Figure 41 shows the fluorescence imaging results (nuclear staining) for groups 1 and 3. In particular, a trend of increasing cell number was observed in group 3 under the condition of 1 mM carnosine. Figure 42 shows the fluorescence imaging results (phalloidin staining) for groups 1 and 3. In particular, a trend of increasing cell number and phalloidin area was observed in groups 1 and 3 under the condition of 1 mM carnosine. No significant differences were observed in cell morphology. Figure 43 shows the fluorescence imaging results (CD44 staining) for groups 1 and 3. In particular, a trend of increasing cell number and CD44 area was observed in group 3 under the condition of 1 mM carnosine. No significant differences were observed in marker expression patterns.

[0153] Figures 44-45 show the cell count results for groups 5-8. In particular, in group 7, an increasing trend in cell number was observed with the addition of anserine. Figures 46-47 show the results of phalloidin fluorescence area measurements for groups 5-8. In particular, in group 7, an increasing trend in phalloidin area was observed with the addition of anserine. Figures 48-49 show the results of CD44 fluorescence area measurements for groups 5-8. In particular, in group 7, an increasing trend in CD44 area was observed with the addition of anserine.

[0154] Figure 50 shows the fluorescence imaging results (phalloidin staining) for groups 5 and 7. In particular, group 7 showed an increasing trend in cell number and phalloidin area under the condition of 1 mM anserine. No significant differences were observed in cell morphology. Figure 51 shows the fluorescence imaging results (CD44 staining) for groups 5 and 7. In particular, group 7 showed an increasing trend in cell number and CD44 area under the condition of 1 mM anserine. No significant differences were observed in marker expression patterns.

[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 number of cells increased with the addition of carnosine, the levels of each MSC-positive and negative marker did not change with the addition of carnosine.

[0156] Figure 53 shows the cell count results for groups 9 and 10. In group 9, the addition of carnosine showed a tendency for the cell number to increase, while in group 10, no such trend was observed. Figure 54 shows the results of phalloidin fluorescence area measurements for groups 9 and 10. In group 9, the addition of carnosine showed a tendency for the phalloidin area to increase, while in group 10, no such trend was observed.

[0157] Figure 55 shows the fluorescence image acquisition results (phalloidin staining) for groups 9 and 10. In group 9, under the condition of 1 mM carnosine, an increasing trend in phalloidin area was observed with the addition of carnosine, whereas no increasing trend was observed in group 10 under the condition of 1 mM carnosine.

[0158] These results indicate that adding carnosine to a culture medium containing LIF promotes the growth of MSCs.

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

Claims

1. A method for producing a culture supernatant, comprising the step of recovering the culture supernatant from a medium containing mesenchymal stem cells and an imidazole dipeptide, wherein the imidazole dipeptide is carnosine, anserine, balenine, or homocarnosine.

2. The production method according to claim 1, wherein the mesenchymal stem cells are mesenchymal stem cells cultured in a medium containing carnosine, anserine, balenine, or homocarnosine.

3. The production method according to claim 1, comprising the step of culturing mesenchymal stem cells in a medium containing carnosine, anserine, balenine, or homocarnosine to produce cultured mesenchymal stem cells.

4. The production method according to claim 1, wherein the recovery step includes a step of centrifugation or filtering of a culture medium containing mesenchymal stem cells and an imidazole dipeptide, the imidazole dipeptide being carnosine, anserine, balenine, or homocarnosine.

5. The production method according to claim 1, further comprising the step of sterilizing the recovered culture supernatant to produce a sterilized culture supernatant.

6. The production method according to claim 1, wherein the culture supernatant is the culture supernatant obtained by adherent culture.

7. The production method according to claim 1, 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 according to claim 1, wherein the culture supernatant comprises G-CSF, MCP-1, VEGF-C, TGF-β1, IL-6, IL-7, IL-8, and exosomes.

9. The production method according to claim 1, wherein the imidazole dipeptide is carnosine.

10. The production method according to claim 1, wherein the imidazole dipeptide is anserine.

11. Culture supernatant obtained by the production method according to any one of claims 1 to 10.

12. A pharmaceutical composition comprising a culture supernatant obtained by the production method described in any one of claims 1 to 10.

13. A pharmaceutical composition according to claim 12 for inhibiting osteogenic differentiation.

14. A syringe, vial, or medical bag containing the culture supernatant described in claim 11.

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