Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
  • C12N5/0662—Stem cells
  • C12N5/0668—Mesenchymal stem cells from other natural sources
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/999—Small molecules not provided for elsewhere

Definitions

• the technical field of the present invention relates to mesenchymal stem cells or mesenchymal stem cell culture supernatants, etc.
• MSCs Mesenchymal stem cells
• MSC culture supernatants contain components secreted by cells, such as cytokines.
• MSC culture supernatants have the advantage in terms of cost, as they can be administered to patients without the need to purify the active ingredients into a single component.
• Patent Document 1 and Non-Patent Document 1 describe an investigation into the amount of cytokines in MSC culture supernatants (see the Examples in Patent Document 1 and the Abstract in Non-Patent Document 1).
• Patent Document 1 also describes that administration of an MSC cell suspension to a rat model of lower limb ischemia resulted in an increase in blood flow.
• Non-Patent Document 1 describes that MSC culture supernatants were found to promote the repair of skin photodamage and promote vascular regeneration.
• the inventors conducted research into methods for culturing MSCs. Surprisingly, they found that by adding imidazole dipeptide to the medium used for culturing MSCs, a culture supernatant useful for suppressing or ameliorating diseases was obtained. Furthermore, the cells obtained were also useful for suppressing or ameliorating diseases. Based on these findings, the present invention was completed.
• a method for producing a culture supernatant comprising the step of recovering a culture supernatant from a medium containing mesenchymal stem cells and an imidazole dipeptide.
• a culture supernatant useful for suppressing or ameliorating a disease can be obtained.
• a method for producing cells comprising the step of culturing mesenchymal stem cells in a medium containing an imidazole dipeptide to produce cultured cells.
• a method for culturing cells comprising the step of culturing mesenchymal stem cells in a serum-free medium containing an imidazole dipeptide to produce cultured cells.
• a medium for culturing mesenchymal stem cells the medium containing an imidazole dipeptide and being serum-free.
• a method for inhibiting the production of osteoblasts comprising the step of culturing mesenchymal stem cells in a medium containing an imidazole dipeptide.
• a method for inhibiting the production of osteoblasts comprising the step of contacting mesenchymal stem cells with a culture supernatant obtained by culturing mesenchymal stem cells in a medium containing an imidazole dipeptide.
• a composition for inhibiting the production of osteoblasts comprising an imidazole dipeptide.
• a composition for inhibiting the production of osteoblasts comprising a culture supernatant obtained by culturing mesenchymal stem cells in a medium containing an imidazole dipeptide.
• a method for promoting expression of G-CSF, MCP-1, VEGF-C, TGF- ⁇ 1, 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.
• a composition for promoting expression of G-CSF, MCP-1, VEGF-C, TGF- ⁇ 1, IL-6, IL-7, IL-8, M-CSF, Osteoprotegrin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, semaphorin 3B, or an exosome marker in mesenchymal stem cells the composition comprising an imidazole dipeptide.
• a method for promoting exosome secretion the method comprising culturing mesenchymal stem cells in a medium comprising an imidazole dipeptide.
• a composition for promoting exosome secretion in mesenchymal stem cells comprising an imidazole dipeptide.
• a culture supernatant of mesenchymal stem cells comprising at least 500 pg/ml of G-CSF.
• an umbilical cord-derived mesenchymal stem cell that highly expresses or highly secretes G-CSF or IL-6.
• an IL-34-positive mesenchymal stem cell According to another aspect of the present invention, there is provided an ADAM8-positive mesenchymal stem cell. According to another aspect of the present invention, there is provided a pharmaceutical composition for inhibiting bone differentiation, comprising cells obtained by culturing mesenchymal stem cells in a medium containing an imidazole dipeptide. According to another aspect of the present invention, there is provided a method for producing a pharmaceutical composition for inhibiting bone differentiation, comprising a step of culturing mesenchymal stem cells in a medium containing an imidazole dipeptide to generate cultured cells.
• a medium containing an imidazole dipeptide and a LIF (leukemia inhibitory factor) component.
• a kit containing an imidazole dipeptide and a LIF component is provided.
• FIG. 1 is a micrograph of MSCs after culture in carnosine-free medium.
• 1 shows micrographs of MSCs after culturing in medium containing 1 mM carnosine.
• 1 shows micrographs of MSCs after culturing in medium containing 10 mM carnosine.
• 1 shows micrographs of MSCs after culture in medium containing 30 mM carnosine.
• FIG. 1 shows the results of counting the number of MSC cells. This figure shows the results of examining the amount of exosome markers in the culture supernatant.
• the four bar graphs shown for collection times 1 to 4 respectively represent the results under conditions with 0 mM, 1 mM, 10 mM, and 30 mM carnosine added, from the left. The same applies to Figures 7 to 14.
• FIG. 1 shows the results of examining the amount of HGF in the culture supernatant.
• FIG. 1 shows the results of examining the amount of G-CSF in the culture supernatant.
• FIG. 1 shows the results of examining the amount of MCP-1 in the culture supernatant.
• FIG. 1 shows the results of examining the amount of VEGF-C in the culture supernatant.
• FIG. 1 shows the results of examining the amount of TGF- ⁇ 1 in the culture supernatant.
• FIG. 1 shows the results of examining the amount of IL-7 in the culture supernatant.
• FIG. 1 shows the results of examining the amount of IL-8 in the culture supernatant.
• FIG. 1 shows the quantification of calcium deposition in bone marrow MSCs. Alizarin Red S stained images of bone marrow MSCs.
• FIG. 1 shows the quantification results of calcium deposition during co-culture of bone marrow MSCs and umbilical cord MSCs.
• 13 shows Alizarin Red S staining images of bone marrow MSCs and umbilical cord MSCs in co-culture. This is a diagram showing the culture conditions of umbilical cord MSC. In the % increase calculation formula described in the examples, all RPM values are incremented by +1 before calculating each % increase.
• 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.
• FIG. 1 shows the results of ELISA analysis of osteoprotegerin.
• FIG. 1 shows the results of ELISA analysis of osteoprotegerin.
• FIG. 1 shows the results of ELISA analysis of osteoprotegerin.
• FIG. 1 shows the results of ELISA analysis of osteoprotegerin.
• FIG. 1 shows the results of ELISA analysis of M-CSF.
• FIG. 1 shows the results of ELISA analysis of M-CSF.
• FIG. 1 shows the results of ELISA analysis of M-CSF.
• FIG. 1 shows the results of qPCR analysis of osteoprotegerin.
• FIG. 1 shows the results of qPCR analysis of M-CSF.
• FIG. 31 shows the results of ELISA analysis of osteoprotegerin, with (a) showing the condition with the addition of carnosine and (b) showing the condition with the addition of anserine. These are the results of qPCR analysis of osteoprotegerin.
• Fig. 32(a) shows the conditions of UC_B_C
• Fig. 32(b) shows the conditions of BM_B_C
• Fig. 32(c) shows the conditions of BM_C_C.
• Fig. 33(a) shows the conditions of UC_B_A
• Fig. 33(b) shows the conditions of BM_B_A
• Fig. 33(c) shows the conditions of BM_C_A.
• FIG. 34 shows the results of ELISA analysis of M-CSF, where (a) shows the condition with the addition of carnosine, and (b) shows the condition with the addition of anserine.
• Fig. 35 shows the results of qPCR analysis of M-CSF
• Fig. 35(a) shows the results under UC_B_C conditions
• Fig. 35(b) shows the results under BM_B_C conditions.
• Fig. 36 shows the results of qPCR analysis of M-CSF
• Fig. 36(a) shows the results under UC_B_A conditions
• Fig. 36(b) shows the results under BM_B_A conditions.
• FIG. 1 shows the results of cell counting in groups 1 and 2.
• FIG. 1 shows the results of cell counting in groups 3 and 4.
• FIG. 1 shows the results of measuring the fluorescent area of phalloidin in groups 1 to 4.
• FIG. 1 shows the results of measuring the fluorescent area of CD44 in groups 1 to 4.
• FIG. 13 shows the results of capturing fluorescent images (cell nucleus staining) of groups 1 and 3.
• FIG. 1 shows the results of capturing fluorescent images (phalloidin staining) of groups 1 and 3.
• FIG. 13 shows the results of capturing fluorescent images (CD44 staining) of groups 1 and 3.
• FIG. 1 shows the results of cell counting in groups 5 and 6.
• FIG. 1 shows the results of cell counting in groups 7 and 8.
• FIG. 1 shows the results of measuring the fluorescence area of phalloidin in groups 5 and 6.
• FIG. 1 shows the results of measuring the fluorescence area of phalloidin in groups 7 and 8.
• FIG. 13 shows the results of measuring the CD44 fluorescence area in groups 5 and 6.
• FIG. 13 shows the results of measuring the CD44 fluorescence area in groups 7 and 8.
• FIG. 13 shows the results of capturing fluorescent images (phalloidin staining) of groups 5 and 7.
• FIG. 13 shows the results of capturing fluorescent images (CD44 staining) of groups 5 and 7.
• FIG. 13 shows the results of measuring the positive rate of MSC markers after culturing umbilical cord MSCs in MSC medium D.
• FIG. 13 shows the results of cell counting for groups 9 and 10.
• FIG. 1 shows the results of measuring the fluorescence area of phalloidin in groups 9 and 10.
• FIG. 13 shows the results of capturing fluorescent images (phalloidin staining) of groups 9 and 10.
• a method which includes a step of contacting mesenchymal stem cells (MSCs) with an imidazole dipeptide.
• MSCs mesenchymal stem cells
• a culture supernatant useful for suppressing or ameliorating a disease for example, a culture supernatant containing at least one or more cytokines useful for suppressing or ameliorating a disease at a high concentration, or a culture supernatant useful for suppressing bone differentiation
• This method may include, for example, a step of culturing MSCs in a medium containing an imidazole dipeptide to generate cultured MSCs, or a 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 secretion of cytokines or exosomes, a method for suppressing the production of osteoblasts, or a method for suppressing bone differentiation.
• a cell obtained by using this method a culture supernatant, a pharmaceutical composition, a cytokine-containing composition, or a container containing them is provided.
• a method for producing a culture supernatant comprising a step of recovering a culture supernatant from a medium containing MSCs and an imidazole dipeptide.
• a culture supernatant useful for suppressing or ameliorating a disease for example, a culture supernatant containing at least one or more cytokines useful for suppressing or ameliorating a disease at a high concentration, or a culture supernatant useful for suppressing bone differentiation
• a culture supernatant useful for suppressing or ameliorating a disease for example, a culture supernatant containing at least one or more cytokines useful for suppressing or ameliorating a disease at a high concentration, or a culture supernatant useful for suppressing bone differentiation
• a method for culturing or producing cells which includes a step of culturing MSCs in a medium containing an imidazole dipeptide to generate cultured cells.
• MSCs useful for suppressing or ameliorating a disease e.g., MSCs secreting at high concentrations of at least one or more cytokines useful for suppressing or ameliorating a disease, or MSCs useful for inhibiting bone differentiation
• MSCs useful for inhibiting or ameliorating a disease e.g., MSCs secreting at high concentrations of at least one or more cytokines useful for suppressing or ameliorating a disease, or MSCs useful for inhibiting bone differentiation
• MSCs can be cultured in a serum-free, serum albumin-free, insulin-free, IGF (insulin-like growth factor)-free (e.g., IGF-1-free), FGF (fibroblast growth factor)-free (e.g., FGF-1 or 2-free), growth factor-free, cytokine-free, or protein-free medium to obtain MSCs with excellent safety.
• IGF insulin-free
• IGF-1-free insulin-free
• FGF fibroblast growth factor-free
• a culture supernatant useful for suppressing or ameliorating a disease e.g., a culture supernatant containing at least one or more cytokines useful for suppressing or ameliorating a disease at high concentrations, or a culture supernatant useful for inhibiting bone differentiation
• a culture supernatant useful for suppressing or ameliorating a disease e.g., a culture supernatant containing at least one or more cytokines useful for suppressing or ameliorating a disease at high concentrations, or a culture supernatant useful for inhibiting bone differentiation
• a culture supernatant obtained by culturing MSCs in a medium containing an imidazole dipeptide.
• the effect of the MSC culture supernatant or an effect useful for suppressing or ameliorating a disease for example, the effect of at least one or more high concentrations of cytokines useful for suppressing or ameliorating a disease, or the effect of suppressing bone differentiation
• a disease for example, the effect of at least one or more high concentrations of cytokines useful for suppressing or ameliorating a disease, or the effect of suppressing bone differentiation
• a method for suppressing a disease comprising a step of administering to a subject a culture supernatant obtained by culturing MSCs in a medium containing an imidazole dipeptide.
• bone differentiation can be suppressed or ameliorated (e.g., a disease associated with bone differentiation can be suppressed or ameliorated).
• a pharmaceutical composition comprising a culture supernatant obtained by culturing MSCs in a medium containing an imidazole dipeptide in the manufacture of a pharmaceutical composition for suppressing a disease.
• a pharmaceutical composition comprising a culture supernatant obtained by culturing MSCs in a medium containing an imidazole dipeptide.
• the use of this composition makes it possible to suppress or ameliorate bone differentiation (e.g., suppress or ameliorate a disease associated with bone differentiation).
• a pharmaceutical composition for use in suppressing a disease comprising a culture supernatant obtained by culturing MSCs in a medium containing an imidazole dipeptide.
• a method for suppressing a disease comprising administering to a subject cells obtained by culturing MSCs in a medium containing an imidazole dipeptide.
• bone differentiation can be suppressed or ameliorated (e.g., a disease associated with bone differentiation can be suppressed or ameliorated).
• a pharmaceutical composition comprising cells obtained by culturing MSCs in a medium containing an imidazole dipeptide in the manufacture of a pharmaceutical composition for suppressing a disease.
• a pharmaceutical composition comprising cells obtained by culturing MSCs in a medium containing an imidazole dipeptide.
• the use of this composition makes it possible to inhibit or ameliorate bone differentiation (e.g., inhibit or ameliorate a disease associated with bone differentiation).
• a pharmaceutical composition for use in inhibiting a disease comprising cells obtained by culturing MSCs in a medium containing an imidazole dipeptide.
• a method for inhibiting bone differentiation of cells of a subject comprising a step of administering to the subject a culture supernatant obtained by culturing MSCs in a medium containing an imidazole dipeptide.
• a pharmaceutical composition comprising a culture supernatant obtained by culturing MSCs in a medium containing an imidazole dipeptide in the manufacture of a pharmaceutical composition for inhibiting bone differentiation of cells of a subject.
• a pharmaceutical composition comprising a culture supernatant obtained by culturing MSCs in a medium containing an imidazole dipeptide, for use in inhibiting bone differentiation of cells of a subject.
• a method for inhibiting bone differentiation of cells of a subject comprising a step of administering to the subject cells obtained by culturing MSCs in a medium containing an imidazole dipeptide.
• a pharmaceutical composition comprising cells obtained by culturing MSCs in a medium containing an imidazole dipeptide in the manufacture of a pharmaceutical composition for inhibiting bone differentiation of cells of a subject.
• a pharmaceutical composition comprising cells obtained by culturing MSCs in a medium containing an imidazole dipeptide for use in inhibiting bone differentiation of cells of a subject.
• a medium for culturing MSCs 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.
• cells useful for suppressing or improving a disease e.g., cells secreting at high concentrations of at least one or more cytokines useful for suppressing or improving a disease, or cells useful for inhibiting bone differentiation
• a disease e.g., cells secreting at high concentrations of at least one or more cytokines useful for suppressing or improving a disease, or cells useful for inhibiting bone differentiation
• a culture supernatant useful for suppressing or improving a disease e.g., a culture supernatant containing at high concentrations of at least one or more cytokines useful for suppressing or improving a disease, or a culture supernatant useful for inhibiting bone differentiation
• a culture supernatant useful for suppressing or improving a disease e.g., a culture supernatant containing at high concentrations of at least one or more cytokines useful for suppressing or improving a disease, or a culture supernatant useful for inhibiting bone differentiation
• MSCs or culture supernatants with excellent safety can be obtained.
• a method for inhibiting osteoblast formation comprising the step of contacting MSCs with an imidazole dipeptide.
• osteoblast formation can be inhibited.
• a use of a composition comprising an imidazole dipeptide in the manufacture of a composition for inhibiting osteoblast formation comprising the step of administering an imidazole dipeptide to a subject.
• a method for inhibiting osteoblast formation comprising the step of culturing MSCs in a medium containing an imidazole dipeptide. By using this method, osteoblast formation can be inhibited.
• compositions for inhibiting osteoblast formation comprising an imidazole dipeptide.
• a composition for use in inhibiting osteoblast formation comprising an imidazole dipeptide.
• a method for inhibiting osteoblast production comprising the step of contacting MSCs with a culture supernatant or an MSC secretion product obtained by culturing MSCs in a medium containing an imidazole dipeptide.
• a composition comprising a culture supernatant or an MSC secretion product obtained by culturing MSCs in a medium containing an imidazole dipeptide in the manufacture of a composition for inhibiting osteoblast production.
• a method for inhibiting osteoblast production comprising the step of administering to a subject a culture supernatant or an MSC secretion product obtained by culturing MSCs in a medium containing an imidazole dipeptide.
• compositions for inhibiting osteoblast formation comprising a culture supernatant or an MSC secretion product obtained by culturing MSCs in a medium containing an imidazole dipeptide.
• a composition for use in inhibiting osteoblast formation comprising a culture supernatant or an MSC secretion product obtained by culturing MSCs in a medium containing an imidazole dipeptide.
• a method for promoting expression of G-CSF granulocyte-colony stimulating factor
• MCP-1 monocyte chemotactic protein
• VEGF-C vascular endothelial growth factor-C
• TGF- ⁇ 1 transforming growth factor beta 1
• IL-6 interleukin 6
• IL-7 interleukin 7
• IL-8 interleukin 8
• M-CSF osteoprotegrin
• Gremlin 1 SCF
• ADAM8 IL-34 IGFBP2, DKK1, semaphorin 3B
• an exosome marker comprising the step of culturing MSCs in a medium containing an imidazole dipeptide.
• a method for promoting expression of G-CSF, MCP-1, VEGF-C, TGF- ⁇ 1, 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.
• a composition comprising an imidazole dipeptide in the manufacture of a composition for promoting expression of G-CSF, MCP-1, VEGF-C, TGF- ⁇ 1, IL-6, IL-7, IL-8, M-CSF, Osteoprotegrin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, semaphorin 3B, or an exosome marker in MSCs.
• a method for promoting expression of G-CSF, MCP-1, VEGF-C, TGF- ⁇ 1, IL-6, IL-7, IL-8, M-CSF, Osteoprotegrin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, semaphorin 3B, or an exosome marker in MSCs comprising the step of administering an imidazole dipeptide to a subject.
• Contacting this composition with MSCs can promote the expression or secretion of G-CSF, MCP-1, VEGF-C, TGF- ⁇ 1, 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 the MSCs.
• a method for promoting exosome secretion comprising the step of contacting MSCs with an imidazole dipeptide. Using this method, it is possible to promote exosome secretion from MSCs. According to another aspect, there is provided a use of a composition comprising an imidazole dipeptide in the manufacture of a composition for promoting exosome secretion from MSCs. According to another aspect, there is provided a method for promoting exosome secretion, comprising the step of administering an imidazole dipeptide to a subject.
• composition for promoting exosome secretion from MSCs, comprising an imidazole dipeptide. By contacting the composition with MSCs, exosome secretion from MSCs can be promoted.
• a method for producing a composition comprising a step of recovering a culture supernatant from a medium containing MSCs and an imidazole dipeptide.
• the composition may be a culture supernatant or a cytokine-containing composition.
• a method for producing a composition containing a secretion product of MSCs comprising a step of contacting MSCs with an imidazole dipeptide.
• the composition can be used to obtain the effect of the secretion product of MSCs or an effect useful for suppressing or ameliorating a disease (e.g., the effect of at least one or more high concentrations of cytokines useful for suppressing or ameliorating a disease, or the effect of suppressing bone differentiation).
• a culture supernatant of MSC containing a protein is provided.
• the culture supernatant may be, for example, a culture supernatant of MSC containing at least 500 pg/ml of G-CSF. By using this culture supernatant, a disease suppressing or ameliorating effect due to a high concentration of G-CSF can be obtained.
• the culture supernatant may be, for example, a culture supernatant of MSC containing at least 2700 pg/ml of IL-6. By using this culture supernatant, a disease suppressing or ameliorating effect due to a high concentration of IL-6 can be obtained.
• MSCs preferably umbilical cord-derived MSCs
• G-CSF or IL-6 preferably umbilical cord-derived MSCs
• a disease suppressing or ameliorating effect due to high concentrations of IL-6 can be obtained.
• a disease suppressing or ameliorating effect due to high concentrations of G-CSF or IL-6 can be obtained.
• a culture supernatant containing a high concentration of G-CSF or IL-6 can be obtained.
• a composition that contains a cell population of MSCs, in which 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.
• MSCs preferably umbilical cord-derived MSCs
• IL-6 IL-6-derived MSCs
• a disease suppressing or ameliorating effect due to high concentrations of G-CSF or IL-6 can be obtained.
• a culture supernatant containing a high concentration of G-CSF or IL-6 can be obtained.
• MSCs positive for G-CSF, MCP-1, VEGF-C, TGF- ⁇ 1, IL-6, IL-7, IL-8, M-CSF, Osteoprotegrin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, or semaphorin 3B.
• the MSCs may be purified or isolated MSCs.
• a composition comprising MSCs in a purified or isolated form.
• MSCs in which expression of G-CSF, MCP-1, VEGF-C, TGF- ⁇ 1, IL-6, IL-7, IL-8, M-CSF, Osteoprotegrin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, or semaphorin 3B is increased.
• the increased expression may be, for example, an increase compared to MSCs cultured in the absence of imidazole dipeptide.
• a pharmaceutical composition comprising the MSCs.
• a method for treating a disease comprising the step of administering the MSCs to a subject. Whether or not the result is positive can be evaluated by, for example, an immunological measurement method (e.g., ELISA) or PCR (e.g., quantitative PCR), etc.
• IL-34-positive or ADAM8-positive MSCs are provided.
• the MSCs are particularly suitable for use in, for example, inhibiting bone differentiation.
• the MSCs may be purified or isolated MSCs.
• a composition comprising the MSCs in a purified or isolated form is provided.
• a pharmaceutical composition comprising the MSCs is provided.
• a method for treating a disease comprising the step of administering the MSCs to a subject.
• a medium suitable for producing a culture supernatant or MSCs useful for suppressing or improving a disease can be prepared.
• a method for preparing a medium comprising the step of adding an imidazole dipeptide to the medium.
• a culture medium comprising an imidazole dipeptide and an LIF component.
• the medium can be used to promote cell proliferation of MSCs.
• a method for producing cells or culture supernatant comprising a step of culturing MSCs in a medium containing an imidazole dipeptide and an LIF component to generate cultured cells.
• the effect of suppressing bone differentiation can be obtained by using the obtained cells.
• the effect of MSC culture supernatant or an effect useful for suppressing or ameliorating a disease e.g., an effect brought about by at least one or more high concentrations of cytokines useful for suppressing or ameliorating a disease, or an effect of suppressing bone differentiation
• kits that includes an imidazole dipeptide and an LIF component.
• This kit may be, for example, a kit for preparing a medium (e.g., a medium for culturing MSCs). The components included in this kit can be mixed to prepare a medium. The obtained medium can be used to promote cell proliferation of MSCs.
• a method for preparing a medium is provided, which includes a step of mixing an imidazole dipeptide and an LIF component.
• a culture medium comprising an imidazole dipeptide and a laminin fragment or a modified form thereof.
• the use of this culture medium can promote cell proliferation of MSCs.
• a method for producing cells or culture supernatant comprising the steps of culturing MSCs in a medium containing an imidazole dipeptide and a laminin fragment or a modified form thereof to produce cultured cells.
• the use of the resulting cells provides an effect of suppressing bone differentiation.
• the use of the resulting culture supernatant provides an effect possessed by MSC culture supernatant, or an effect useful for suppressing or ameliorating a disease (e.g., an effect provided by at least one or more high concentrations of cytokines useful for suppressing or ameliorating a disease, or an effect of suppressing bone differentiation).
• kits that includes an imidazole dipeptide and a laminin fragment or a variant thereof.
• This kit may be, for example, a kit for preparing a medium (e.g., a medium for culturing MSCs). The components included in this kit can be mixed to prepare a medium. The obtained medium can be used to promote cell proliferation of MSCs.
• a method for preparing a medium is provided, which includes a step of mixing an imidazole dipeptide and LIF.
• a method for producing a cytokine-containing composition comprising a step of processing a culture supernatant (e.g., containing the above (4) or (25)) or a composition (e.g., containing the above (6), (8), (14), (16), (19) or (22)) according to an embodiment of the present invention.
• a composition obtained by this method it is possible to obtain the effect of an MSC culture supernatant, the effect of at least one or more high concentrations of cytokines useful for suppressing or ameliorating a disease, or the effect of suppressing bone differentiation.
• the processing may be, for example, formulation or filling into a container.
• composition obtained by carrying out a method according to any of the embodiments of the present invention (including, for example, the above (1) to (3), (5), (7), (9), (10), (12), (13), (15), (17), (18), (20), (21), (23), (24), (29), (31), (34), or (36)).
• a cytokine-containing composition obtained by carrying out a method according to any of the embodiments of the present invention (including, for example, the above (1) to (3), (5), (7), (9), (10), (12), (13), (15), (17), (18), (20), (21), (23), (24), (29), (31), (34), or (36)).
• a cell obtained by carrying out a method according to an embodiment of the present invention (including, for example, the above (1) to (3), (31) or (34)).
• the cell is capable of secreting at high concentrations of at least one or more cytokines useful for suppressing or ameliorating a disease.
• the cell can be used for suppressing or ameliorating a disease.
• a culture supernatant containing at high concentrations of at least one or more cytokines useful for suppressing or ameliorating a disease, or a culture supernatant useful for suppressing bone differentiation can be obtained.
• a cell population is provided that includes the cells of the embodiments of the present invention (e.g., including the above (26) to (28) or (38)).
• a disease suppressing or ameliorating effect by high concentrations of G-CSF or IL-6, or an effect of suppressing bone differentiation can be obtained.
• a culture supernatant containing a high concentration of G-CSF or IL-6, or a culture supernatant useful for suppressing or ameliorating a disease can be obtained.
• a composition is provided that includes a cell population that includes the cells of the above (25) or (28).
• a disease suppressing or ameliorating effect by high concentrations of G-CSF or IL-6, or an effect of suppressing bone differentiation can be obtained.
• a culture supernatant containing a high concentration of G-CSF or IL-6, or a culture supernatant useful for suppressing or ameliorating a disease can be obtained.
• a method for treating a disease comprising the step of administering to a subject a supernatant (e.g., comprising (4) or (25) above) or cells (e.g., comprising (26) to (28) or (38)) according to an embodiment of the present invention.
• the subject to be treated may be, for example, a patient in need of inhibition of bone differentiation.
• a pharmaceutical composition for use in treating a disease comprising the supernatant or cells according to an embodiment of the present invention.
• use of the supernatant or cells according to an embodiment of the present invention for the production of a pharmaceutical composition for treating a disease.
• a container containing a culture supernatant e.g., containing the above-mentioned (4) or (25)
• a composition e.g., containing the above-mentioned (6), (8), (14), (16), (19), (22) or (37)
• a container containing a culture supernatant (e.g., containing the above-mentioned (4) or (25)) or a composition (e.g., containing the above-mentioned (6), (8), (14), (16), (19), (22) or (37)) according to an embodiment of the present invention.
• Methods according to embodiments of the present invention may include one or more of the following steps (i) to (ii): (i) culturing MSCs in a medium containing an imidazole dipeptide to produce cultured MSCs, or (ii) recovering a culture supernatant from the cultured MSCs and the medium containing an imidazole dipeptide.
• the method of the embodiment of the present invention may include any one or more of the following steps (iii) to (xvi).
• Employing one or more of these steps is useful for obtaining a culture supernatant containing at least one or more cytokines useful for suppressing or ameliorating a disease at a high concentration, or a culture supernatant or MSCs useful for suppressing bone differentiation. If two or more are used, the order is arbitrary and can be determined according to the desired operation.
• the MSCs include, for example, umbilical cord-derived MSCs, adipose-derived MSCs, bone marrow-derived MSCs, placenta-derived MSCs, or umbilical cord blood-derived MSCs.
• the MSCs may be mammalian MSCs, and are preferably human MSCs. Mammals include, for example, humans, monkeys, rodents (e.g., mice, hamsters, etc.), rabbits, dogs, cats, horses, cows, sheep, pigs, goats, marmosets, etc.
• the MSCs may exist as a cell population.
• references to MSCs include MSCs that exist as a cell population.
• a cell population includes a plurality of cells generated by cell division.
• the proportion of the MSCs of the embodiments of the present invention (including, for example, (25) or (28) above) in the cell population may be, for example, 30, 40, 50, 60, 70, 80, 90, or 100% or more, or may be within a range of any two of these values.
• MSCs include MSCs before being cultured by the above-mentioned method, and MSCs obtained by culturing by the above-mentioned method.
• MSCs include MSCs cultured in a medium containing an 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 is substantially free of cells other than MSCs, or MSCs in a preparation from which impurities have been removed. Impurities may include, for example, medium components, and removal may include, for example, partial removal.
• MSCs that highly express or secrete G-CSF or IL-6 may be, for example, cells that secrete at least 5.56 ⁇ 10 ⁇ 3 pg/cell of G-CSF or at least 3 ⁇ 10 ⁇ 2 pg/cell of IL-6 when cultured for 72 hours in a medium supplemented with carnosine (e.g., 10 mM).
• the amount of G-CSF secreted may be, for example, at least 5.56 ⁇ 10 ⁇ 3 , 6 ⁇ 10 ⁇ 3 , 8 ⁇ 10 ⁇ 3 , 1 ⁇ 10 ⁇ 2 , 1.2 ⁇ 10 ⁇ 2 , 1.7 ⁇ 10 ⁇ 2 , 2 ⁇ 10 ⁇ 2 , 3 ⁇ 10 ⁇ 2 , 1 ⁇ 10 ⁇ 1 , 1, or 3 pg/cell, or may be within a range between any two of these values.
• This value may be, for example, 5.56 ⁇ 10 -3 to 3, 5.56 ⁇ 10 -3 to 3 ⁇ 10 -2 , 5.56 ⁇ 10 -3 to 2 ⁇ 10 -2 , 1.2 ⁇ 10 -2 to 3 ⁇ 10 -2 , or 1.2 ⁇ 10 -2 to 1.7 ⁇ 10 -2 pg/cell.
• the amount of IL-6 secreted may be, 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, or may be within a range of any two of these values.
• This value may be, for example, 3 ⁇ 10 -2 to 20, 3 ⁇ 10 -2 to 2 ⁇ 10 -1 , 3 ⁇ 10 -2 to 8 ⁇ 10 -2 , 4 ⁇ 10 -2 to 8 ⁇ 10 -2 , or 4 ⁇ 10 -2 to 7 ⁇ 10 -2 pg/cell.
• the amount of secretion can be calculated by the concentration of the secretion product in the medium after culturing the cells (unit: pg/mL, for example) ⁇ the amount of medium used (unit: mL, for example) ⁇ the number of cells after culturing.
• the imidazole dipeptide includes a compound having a structure in which an amino acid having an imidazole group is bonded to another amino acid.
• the imidazole dipeptide includes, for example, a compound having a structure of the following formula (1) or (2).
• R 1 to R 6 are as follows.
• R 1 , R 2 , R 3 and R 4 are each independently H or C 1-6 alkyl.
• R 5 and R 6 are each independently -NHR 7 or -CH 2 NHR 7.
• R 7 is H or -COR 8.
• R 8 is H, C 1-6 alkyl, optionally substituted phenyl, -OCH 2 R 9 or -CH ⁇ CHR 9.
• R 9 is H, C 1-6 alkyl or optionally substituted phenyl.
• the compound having the structure of formula (1) or (2) may have an effect of promoting cytokine secretion from MSCs.
• one of R 1 and R 2 in formula (1) is H and the other is C 1-6 alkyl, and more preferably both are H.
• one of R 1 and R 2 in formula (1) is C 1-6 alkyl
• it is preferable that the C 1-6 alkyl is methyl.
• one of R 3 and R 4 in formula (2) is H and the other is C 1-6 alkyl, and more preferably both are H.
• one of R 2 and R 3 in formula (2) is C 1-6 alkyl, it is preferable that the C 1-6 alkyl is methyl.
• R 8 in formula (1) or (2) is H, C 1-6 alkyl, or -OCH 3 , and more preferably is methyl.
• R 9 in formula (1) or (2) is H or C 1-6 alkyl.
• R7 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 substituted with -OH, C1-6 alkyl, or -OCH3 .
• the substitution position may be the 2-, 3-, 4-, 5-, or 6-position of the phenyl.
• 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 -CH 2 NHR 7 , and R 7 may be H or -COCH 3 .
• the alkyl group includes straight or branched hydrocarbon chains.
• the C1-6 group includes hydrocarbons having 1, 2, 3, 4, 5, or 6 carbon atoms. That is, the C1-6 alkyl group includes alkyls having 1, 2, 3, 4, 5, or 6 carbon atoms. Examples of the C1-6 alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, and n-hexyl groups.
• the imidazole dipeptide includes carnosine, a methylated form of carnosine (e.g., anserine or balenine, etc.), or homocarnosine.
• the imidazole dipeptide includes a compound having a structure of formula (1) or (2) and having antioxidant activity.
• carnosine, anserine, balenine, and homocarnosine are known to have antioxidant activity (see, for example, Boldyrev et al., Physiol Rev. 2013 Oct;93(4):1803-45.).
• the imidazole dipeptide may be in the L-form or the D-form.
• carnosine includes L-carnosine or D-carnosine unless otherwise specified.
• L-carnosine can also be referred to as beta-alanyl-L-histidine.
• L-carnosine can be represented by the CAS Registry Number 305-84-0.
• anserine includes L-anserine or D-anserine unless otherwise specified.
• L-anserine can also be represented by the name beta-alanyl-3-methyl-L-histidine.
• L-anserine can be represented by the CAS Registry Number 584-85-0.
• balenine includes L-balenine or D-balenine.
• L-balenine can also be represented by the name beta-alanyl-1-methyl-L-histidine.
• L-balenine can be represented by the CAS registry number 331-38-4.
• homocarnosine includes L-homocarnosine or D-homocarnosine, unless otherwise specified.
• L-homocarnosine can also be referred to as gamma-aminobutyryl-L-histidine.
• L-homocarnosine can be represented by the CAS Registry Number 3650-73-5.
• 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- ⁇ 1, 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 inhibits bone formation, it is believed that high concentrations of G-CSF can inhibit bone formation.
• IL-6 is a cytokine that inhibits bone formation
• high concentrations of IL-6 can inhibit bone formation.
• IL-6, VEGF, MCP-1, VEGF-C, TGF- ⁇ 1, and IL-8 are angiogenic factors, it is believed that high concentrations of these cytokines can promote angiogenesis.
• G-CSF, MCP-1, TGF- ⁇ 1, and IL-7 are immune regulatory factors, and it is believed that these cytokines can 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 the inhibition of bone formation, and it is believed that a combination of these cytokines can suppress bone formation.
• the disease or symptom to be inhibited or improved includes, for example, bone differentiation, disease associated with bone differentiation (e.g., heterotopic ossification), ischemic disease (e.g., lower limb ischemia, myocardial infarction, cerebral infarction, spinal cord infarction, or chronic arterial occlusion), wound (e.g., epithelial wound or burn), sarcopenia associated with aging, arthritis (e.g., rheumatism, herniated disc, osteoarthritis, etc.), inflammatory disease (e.g., nephritis, keratitis, cytokine storm, etc.), psychiatric disease (e.g., autism or insomnia thought to be caused in part by neuroinflammation), immune disease (e.g., GVHD (graft versus host disease), Sjogren's syndrome, atopic dermatitis, collagen disease, multiple myocardial infarction, cerebral infarction, spinal cord infarction, or chronic
• the disease or symptom may be inhibited or improved, for example, by administering to a subject the culture supernatant, cells, or composition in the embodiments of the present invention (including, for example, the above (1) to (41)).
• the disease or condition may be suppressed or improved, for example, by the effect of a high concentration of cytokines.
• the above-mentioned ectopic ossification includes a phenomenon in which bone formation occurs abnormally in a site where bone formation does not normally occur.
• Ectopic ossification includes, for example, ossification occurring in soft tissues (e.g., tendons, membranes, ligaments, muscles, joint capsules, etc.).
• Ectopic ossification may be a state having a trabecular structure.
• Ectopic ossification includes, for example, ossification of the posterior longitudinal ligament, ossification of the ligamentum flavum, fibrodysplasia ossificans progressiva, myositis ossificans progressiva, myositis ossificans traumatic, or diffuse idiopathic osteophytosis.
• the inhibition of bone differentiation is useful, for example, for the inhibition of diseases associated with bone differentiation.
• Diseases associated with bone differentiation include, for example, ossification (e.g., ectopic ossification).
• the inhibition of bone differentiation includes, for example, the inhibition of abnormal bone differentiation.
• the use of the inhibition of bone differentiation includes the use of inhibiting or ameliorating diseases associated with bone differentiation.
• Pharmaceutical compositions for inhibiting bone differentiation include pharmaceutical compositions for diseases associated with bone differentiation, such as ectopic ossification.
• Methods for inhibiting bone differentiation include methods for inhibiting or ameliorating diseases associated with bone differentiation, such as ectopic ossification.
• culturing includes incubating cells under conditions suitable for growth or maintenance. Incubation may be performed at about 37° C. and in an atmosphere of about 5% CO 2. Culturing may be performed in a medium that is serum-free, serum-free, serum albumin-free, insulin-free, IGF-free, FGF-free, growth factor-free, cytokine-free, or protein-free. Free includes a state in which the target component is not added to the medium, a state in which the medium is completely free of the target component, or a state in which the medium does not contain the component at a concentration above the detection limit. Free includes being substantially free or being substantially free.
• Culturing may be performed by adhesion culture in the presence of a cell adhesion factor (e.g., mixed in the medium or in a pre-coated container).
• culturing may include cell production.
• the time of culturing may be, for example, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, or 50 days or more, or may be within a range of any two of these values.
• the culture may be performed until the cells reach a confluent state.
• the confluent state includes a state in which the cells cover 80% or more of the bottom area of the culture vessel. More preferably, the culture may be performed until the cells cover 80-90% of the bottom area of the culture vessel.
• the culture of MSCs may be performed by alternately using X and Y, where X is a medium that does not contain imidazole dipeptide (e.g., a protein-containing medium) and Y is a medium that contains imidazole dipeptide (e.g., a protein-free medium).
• the alternate use includes starting the culture with X or Y, and then replacing the medium with Y after the culture with X, or with X after the culture with Y. For example, the following may be performed in order: culture with X, replacement with Y and culture, replacement with X and culture, replacement with Y and culture, replacement with X and culture, replacement with Y and culture, replacement with X and culture, and replacement with Y and culture.
• 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 timing after the culture of Y.
• the culture supernatant may be collected, for example, after the 1st, 2nd, 3rd, 4th, 5th, 6th, or 7th culture of Y.
• the supernatant may be collected at any two or more times or at all times, and the solution obtained by mixing all the supernatants may be used as the target supernatant.
• the time from the start of culture to the replacement can be the same as the above-mentioned culture time.
• MSCs may be cultured in a medium containing, for example, 0.1 to 100 mM imidazole dipeptide.
• This concentration may be, for example, 0.1, 0.2, 0.5, 1, 2, 5, 7, 10, 15, 20, 25, 30, 35, 40, 50, 60, 80, or 100 mM, or may be equal to or greater than these values, or may be within a range of any two of these values.
• 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 an adherent culture. Whether or not cells are attached to a container can be easily confirmed by a person skilled in the art, for example, by tilting the container to confirm that no cell movement occurs, or by confirming that no cell movement occurs when changing the medium in the container.
• Adhesion culture may be performed, for example, by culturing cells in the presence of a cell adhesion factor (for example, by contacting cells and an adhesion factor in a container, mixing a cell suspension containing cells and a medium with an adhesion factor, placing the resulting mixture in a container and culturing it, or coating (for example, pre-coating) the container with an adhesion factor and then seeding the cells in the container).
• a cell adhesion factor for example, by contacting cells and an adhesion factor in a container, mixing a cell suspension containing cells and a medium with an adhesion factor, placing the resulting mixture in a container and culturing it, or coating (for example, pre-coating) the container with an adhesion factor and then seeding the cells in the container.
• a cell adhesion factor for example, by contacting cells and an adhesion factor in a container, mixing a cell suspension containing cells and a medium with an adhesion factor, placing the resulting mixture in
• Adhesion factors include, for example, laminin, fibronectin, vitronectin, tenascin, cadherin, poly-L-lysine, poly-D-lysine, collagen, thrombospondin, galectin, or nidogen-1, or fragments thereof having a cell adhesion effect.
• Cell adhesion may include a state in which cells are attached to a container via an extracellular matrix (for example, an adhesion factor).
• Adherent cells may include cells that can be cultured as adherent cells or cells that can grow while attached.
• the concentration of the adhesion factor in the medium may be, for example, 0.01, 0.1, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 2, or 3 ⁇ g/ml, or may be equal to or greater than these values, or may be within a range of any two of these values. This value may be, for example, 0.01 to 2, 0.1 to 1.5, or 0.5 to 1 ⁇ g/ml.
• the concentration of the adhesion factor in the medium may be, for example, 0.005, 0.01, 0.1, 0.25, 0.5, 1, 1.5, or 2 ⁇ g per cm 2 of culture area of the culture vessel, or may be equal to or greater than these values, or may be within a range of any two of these values.
• the adhesion factor may be a factor having adhesive activity, such as laminin, fibronectin, vitronectin, tenascin, cadherin, poly-L-lysine, poly-D-lysine, collagen, thrombospondin, galectin, nidogen-1, a fragment thereof, or a variant thereof.
• the adhesion factor may be a laminin fragment or a variant 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 a laminin 511 E8 fragment, a laminin 521 E8 fragment, a laminin 411 E8 fragment, a laminin 421 E8 fragment, a laminin 332 E8 fragment, a laminin 311 E8 fragment, a laminin 321 E8 fragment, a laminin 211 E8 fragment, a laminin 221 E8 fragment, a laminin 213 E8 fragment, a laminin 111 E8 fragment, or a laminin 121 E8 fragment.
• the laminin 511 E8 fragment can be prepared, for example, by the method described in WO2011/043405A1.
• Laminin 511 includes laminins composed of ⁇ 5, ⁇ 1, and ⁇ 1 subunit chains.
• the modified laminin fragment may be a known complex composed of a laminin fragment having integrin-binding activity and another functional molecule (e.g., a complex between a laminin fragment having integrin-binding activity and a cell adhesion molecule, or a complex between a laminin fragment having integrin-binding activity and a growth factor-binding molecule (e.g., heparan sulfate)) (see, for example, WO2012/137970, WO2014/103534, and WO2016/010082).
• another functional molecule e.g., a complex between a laminin fragment having integrin-binding activity and a cell adhesion molecule, or a complex between a laminin fragment having integrin-binding activity and a growth factor-binding molecule (e.g., heparan sulf
• the modified laminin fragment can be produced as a recombinant protein by using known gene recombination techniques.
• the laminin fragment or variant thereof may have integrin-binding activity, and may include, for example, (a) a trimer comprising a polypeptide having an amino acid sequence shown in SEQ ID NO:1, a polypeptide having an amino acid sequence shown in SEQ ID NO:2, and a polypeptide having an amino acid sequence shown in SEQ ID NO:3; (b) a trimer comprising a polypeptide having an amino acid sequence having 90% or more homology to the amino acid sequence shown in SEQ ID NO:1, a polypeptide having an amino acid sequence having 90% or more homology to the amino acid sequence shown in SEQ ID NO:2, and a polypeptide having an amino acid sequence having 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 having one to several amino acid deletions, substitutions, insertions
• the laminin fragments, variants, or subunit chains may optionally have a tag (see, e.g., 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 (e.g., a His tag).
• the purification tag may include, for example, a biochemically inactive tag.
• the seeding density of the cells may be, for example, 1x10e1, 1x10e2, 5x10e2, 1x10e3, 1.5x10e3, 1x10e4, 1x10e5, or 1x10e6 cells/mL, or may be greater than or equal to any two of these values.
• This value may be, for example, 1x10e1 to 1x10e5, 1x10e2 to 1x10e4, 5x10e2 to 5x10e4, or 5x10e2 to 1x10e4 cells/mL.
• the cell seeding density may be, for example, 0.002, 0.01, 0.1, 1, 50, 100, 300, 500, 1000, 1500, 2000 cells/ cm2 , or may be greater than or equal to 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 .
• the culture supernatant includes a culture supernatant obtained by culturing cells.
• the culture supernatant includes, for example, a cytokine-containing culture supernatant.
• the culture supernatant may include, for example, cell metabolites (e.g., amino acids, lipids, sugars, etc.), secreted proteins (e.g., hormones, peptides, cytokines, extracellular matrix, etc.), or exosomes.
• the culture supernatant may include imidazole dipeptide.
• the culture supernatant also includes a supernatant separated from a cellular component and subjected to various treatments (e.g., centrifugation, filtration, freezing, lyophilization, storage, sterilization, etc.).
• the culture supernatant may be a culture supernatant obtained using a medium containing only MSCs as cultured cells. From the viewpoint of reducing production costs or uniformity between lots, it is preferable that the cytokines contained in the culture supernatant are only cytokines derived from the cultured cells. From the viewpoint of reducing production costs, it is preferable that the culture supernatant is non-concentrated.
• the culture supernatant may contain, for example, HGF, G-CSF, MCP-1, VEGF-C, TGF- ⁇ 1, 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.
• the culture supernatant may contain at least 500 pg/mL of G-CSF or at least 2700 pg/mL of IL-6. Use of this culture supernatant can provide the effects of high concentrations of G-CSF or IL-6.
• This culture supernatant may contain, for example, 500 to 2000, 700 to 1700, 800 to 1600, or 1000 to 1600 pg/mL of G-CSF.
• This concentration may be, for example, at least 500, 600, 700, 800, 900, 1000, 1250, 1500, 1600, 1700, 1800, 1900, or 2000 pg/mL, or may be within the range of any two of these values.
• the culture supernatant may contain IL-6 at, for example, 2700 to 10000, 3000 to 10000, 4000 to 10000, or 3000 to 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 may be within a range of any two of these values.
• the culture supernatant may contain at least 6000 pg/mL of MCP-1, at least 150 pg/mL of VEGF-C, at least 670 pg/mL of TGF- ⁇ 1, at least 3.5 pg/mL of IL-7, or at least 6500 pg/mL of IL-8.
• the effects of high concentrations of G-CSF, IL-6, MCP-1, VEGF-C, TGF- ⁇ 1, IL-7, or IL-8 can be obtained.
• the culture supernatant may contain, for example, at least 6000, 7000, 8000, 9000, or 10000 pg/mL of MCP-1, or may contain MCP-1 within the range of any two of these values.
• the culture supernatant may contain, for example, at least 150, 200, 250, 300, 350, or 400 pg/mL of VEGF-C, or within a range between any two of these values.
• the culture supernatant may contain, for example, at least 670, 700, 750, 800, 850, 900, 950, or 1000 pg/mL of TGF- ⁇ 1, or within a range between any two of these values.
• the culture supernatant may contain, for example, at least 3.5, 4, 5, 6, 7, 8, 9, or 10 pg/mL of IL-7, or within a range between any two of these values.
• the culture supernatant may contain IL-8 at, for example, at least 6500, 7000, 8000, 9000, 10000, 11000, 12000, or 15000 pg/mL, or within any two of these values. From the viewpoint of enhancing the effects of these cytokines, the culture supernatant preferably contains the above seven cytokines in the above concentration ranges.
• the culture supernatant may contain M-CSF at, for example, at least 1350, 1370, 1390, 1400, 1420, 1440, 1450, 1470, 1490, or 1500 pg/mL, or within any two of these values.
• the above culture supernatant may contain osteoprotegerin, for example, at least 95, 100, 110, 120, 130, or 140 pg/mL, or may contain osteoprotegerin within a range between any two of these values.
• the culture supernatant may contain at least 100 pg/mL G-CSF, at least 550 pg/mL IL-6, at least 6000 pg/mL MCP-1, at least 150 pg/mL VEGF-C, at least 670 pg/mL TGF- ⁇ 1, at least 3.5 pg/mL IL-7, at least 6500 pg/mL IL-8, or at least 120 pg/mL CD9/CD63 fusion protein.
• the culture supernatant may contain, for example, at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1600, 1700, 1800, 1900, or 2000 pg/mL of G-CSF, or within a range between any two of these values.
• the culture supernatant may contain, for example, at least 550, 800, 1000, 2000, 2500, 2700, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10000, 11000, or 12000 pg/mL of IL-6, or within a range between any two of these values.
• the culture supernatant may contain, for example, at least 6000, 7000, 8000, 9000, or 10000 pg/mL of MCP-1, or may contain within any two of these values.
• the culture supernatant may contain, for example, at least 150, 200, 250, 300, 350, or 400 pg/mL of VEGF-C, or may contain within any two of these values.
• the culture supernatant may contain, for example, at least 670, 700, 750, 800, 850, 900, 950, or 1000 pg/mL of TGF- ⁇ 1, or may contain within any two of these values.
• the culture supernatant may contain, for example, at least 3.5, 4, 5, 6, 7, 8, 9, or 10 pg/mL of IL-7, or may contain within any two of these values.
• the culture supernatant may contain, for example, at least 6500, 7000, 8000, 9000, 10000, 11000, 12000, or 15000 pg/mL of IL-8, or within a range between any two of these values.
• the culture supernatant may contain, for example, at least 120, 140, 160, 180, 200, 250, or 300 pg/mL of CD9/CD63 fusion protein, or within a range between any two of these values.
• the culture supernatant may contain, for example, at least 1350, 1370, 1390, 1400, 1420, 1440, 1450, 1470, 1490, or 1500 pg/mL of M-CSF, or within a range between any two of these values.
• the above culture supernatant may contain osteoprotegerin, for example, at least 95, 100, 110, 120, 130, or 140 pg/mL, or may contain osteoprotegerin within a range between any two of these values.
• the culture supernatant may contain at least 1350 pg/mL M-CSF or at least 95 pg/mL Osteoprotegrin. Use of this culture supernatant can provide the effects of high concentrations of M-CSF or Osteoprotegrin.
• This culture supernatant may contain, for example, 1350 to 1500, 1350 to 1450, 1350 to 1420, or 1350 to 1400 pg/mL M-CSF.
• This concentration may be, for example, at least 1350, 1370, 1390, 1400, 1420, 1440, 1450, 1470, 1490, or 1500 pg/mL, or may be within the range of any two of these values.
• the culture supernatant may contain osteoprotegerin at, for example, 95 to 140, 95 to 130, 95 to 120, or 95 to 110 pg/mL.
• This concentration may be, for example, at least 95, 100, 110, 120, 130, or 140 pg/mL, or may be within a range of any two of these values.
• the culture supernatant may 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- ⁇ 1, at least 3.5 pg/mL of IL-7, or at least 6500 pg/mL of IL-8.
• the culture supernatant allows for the effects of high concentrations of M-CSF, Osteoprotegrin, G-CSF, IL-6, MCP-1, VEGF-C, TGF- ⁇ 1, IL-7, or IL-8 to be obtained.
• the culture supernatant may contain G-CSF at, for example, at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1600, 1700, 1800, 1900, or 2000 pg/mL, or within a range between any two of these values.
• the culture supernatant may contain, for example, at least 550, 800, 1000, 2000, 2500, 2700, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10000, 11000, or 12000 pg/mL of IL-6, or within any two of these values.
• the culture supernatant may contain, for example, at least 6000, 7000, 8000, 9000, or 10000 pg/mL of MCP-1, or within any two of these values.
• the culture supernatant may contain, for example, at least 150, 200, 250, 300, 350, or 400 pg/mL of VEGF-C, or within any two of these values.
• the culture supernatant may contain, for example, 670, 700, 750, 800, 850, 900, 950, or 1000 pg/mL of TGF- ⁇ 1, or within a range between any two of these values.
• the culture supernatant may contain, for example, at least 3.5, 4, 5, 6, 7, 8, 9, or 10 pg/mL of IL-7, or within a range between any two of these values.
• the culture supernatant may contain, for example, at least 6500, 7000, 8000, 9000, 10000, 11000, 12000, or 15000 pg/mL of IL-8, or within a range between any two of these values.
• the medium comprises a medium used for culturing cells.
• the medium may be a liquid medium or a solid medium.
• the medium comprises a culture solution.
• the medium may be any common cell culture medium, and the composition is not limited.
• the medium may comprise, for example, amino acids, inorganic salts, vitamins, minerals, or a carbon source (e.g., glucose).
• the medium may be, for example, a serum medium (e.g., FBS, etc.), a basal medium (MEM, etc.), a complex medium, a serum-free medium, etc.
• the medium may be a commercially available medium for the growth 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), RPMI1640 (e.g., Nacalai Tesque), etc.
• the medium to be used may be, for example, DMEM/F12 medium to which amino acids have been added.
• the amino acids to be added may be commercially available amino acid solutions for medium additives, such as MEM essential amino acid solution (Fujifilm Wako Pure Chemical Industries, Ltd.) and MEM non-essential amino acid solution (Fujifilm Wako Pure Chemical Industries, Ltd.).
• MEM essential amino acid solution Flujifilm Wako Pure Chemical Industries, Ltd.
• MEM non-essential amino acid solution Flujifilm Wako Pure Chemical Industries, Ltd.
• This DMEM/F12 medium-based medium does not contain any xenogeneic components, cytokines, insulin, proteins, or human serum.
• the medium be a serum-free, serum albumin-free, insulin-free, IGF-free, FGF-free, growth factor-free, cytokine-free, protein-free, or xeno-free medium.
• the medium may contain, for example, an LIF component, an adhesion molecule, an FGF component, an insulin component, an albumin component, or a transferrin component. From the viewpoint of particularly promoting cell proliferation of MSCs, it is preferable that the medium contains an LIF component. From the viewpoint of particularly promoting cell proliferation of MSCs, it is preferable that the medium further contains an adhesion molecule.
• LIF includes, for example, a component also referred to as leukemia inhibitory factor. Details of the amino acid sequence of LIF can be found on websites such as NCBI or UniProt. The primary accession number of LIF listed in UniProt is, for example, P15018. The amino acid sequence of LIF may be, for example, the amino acid sequence shown in SEQ ID NO: 4 or 5. LIF includes, for example, LIF derived from human. In embodiments of the present invention (including, for example, (1) to (41) above), the LIF component includes a molecule having LIF activity and derived from wild-type human LIF.
• the LIF component may be, for example, (a) a protein having an amino acid sequence shown in SEQ ID NO: 4 or 5, or a biologically active fragment thereof; (b) a protein having an amino acid sequence having 90% or more homology to the amino acid sequence shown in SEQ ID NO: 4 or 5, or a biologically active fragment thereof; or (c) a protein 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, or a biologically active fragment thereof.
• LIF activity includes, for example, an activity of promoting LIF signaling, an activity of binding to a LIF receptor, or an activity of inhibiting the proliferation of leukemic cells.
• a biologically active fragment includes, for example, a polypeptide having LIF activity, in which a portion of LIF that is not involved in LIF activity is deleted.
• the LIF component 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 (e.g., a His tag).
• the purification tag includes, for example, a biochemically inactive tag.
• the concentration of the LIF component in the medium may be, for example, 0.01 ng/mL or more.
• 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, or may be any value or more, or may be within a range of any two values.
• This concentration may be, for example, 0.1 to 4, 0.1 to 3, 0.5 to 2, or 0.8 to 1.2 ng/ml. From the viewpoint of promoting the proliferation of MSCs, 0.1 ng/ml or more is preferable, 0.5 ng/ml or more is more preferable, and 0.8 ng/ml or more is even more preferable.
• the FGF includes, for example, a component also referred to as fibroblast growth factor.
• the FGF includes, for example, bFGF.
• the bFGF includes, for example, a component also referred to as basic fibroblast growth factor. Details of the amino acid sequence of bFGF, etc., can be confirmed on websites such as NCBI or UniProt. The primary accession number of bFGF listed in UniProt is, for example, P09038.
• the amino acid sequence of bFGF may be, for example, the amino acid sequence shown in SEQ ID NO: 6, 7 or 8.
• the bFGF includes, for example, bFGF derived from a human.
• the bFGF component includes a molecule having bFGF activity and derived from wild-type human bFGF.
• the bFGF component may be, for example, (a) a protein having an amino acid sequence shown in SEQ ID NO: 6, 7, or 8, or a biologically active fragment thereof; (b) a protein 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 a biologically active fragment thereof; or (c) a protein 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, or a biologically active fragment thereof.
• the bFGF activity includes, for example, an activity of promoting bFGF signaling, an activity of binding to a bFGF receptor, or an activity of promoting the proliferation of fibroblasts.
• the biologically active fragment includes, for example, a polypeptide having bFGF activity, in which a portion of bFGF that is not involved in bFGF activity is deleted.
• the FGF component 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 (e.g., a His tag).
• the purification tag may include, for example, a biochemically inactive tag.
• the concentration of the bFGF component in the medium may be, for example, 0.1 ng/mL or more. This concentration may be, for example, 0.1, 0.5, 1, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 80, or 100 ng/mL, or may be any value or more thereof, or may be within a range of any two values thereof. This concentration may be, for example, 1 to 40, 1 to 30, 5 to 20, or 8 to 12 ng/ml. From the viewpoint of promoting the proliferation of MSCs, 1 ng/ml or more is preferable, 5 ng/ml or more is more preferable, and 8 ng/ml or more is even more preferable.
• insulin includes a type of hormone. Details of the amino acid sequence of insulin, etc., can be confirmed on websites such as NCBI or UniProt. The primary accession number of insulin listed in UniProt is, for example, P01308.
• the amino acid sequence of human insulin includes, for example, the amino acid sequence shown in SEQ ID NO: 9.
• Insulin includes, for example, insulin derived from humans.
• the insulin component includes a molecule that has insulin activity and is derived from wild-type human insulin.
• the insulin component may be, for example, (a) a protein having the amino acid sequence shown in SEQ ID NO: 9 or a biologically active fragment thereof, (b) a protein having an amino acid sequence having 90% or more homology to the amino acid sequence shown in SEQ ID NO: 9 or a biologically active fragment thereof, or (c) a protein having an amino acid sequence having one to several amino acid deletions, substitutions, insertions or additions to the amino acid sequence shown in SEQ ID NO: 9 or a biologically active fragment thereof.
• Insulin activity includes, for example, activity to promote insulin signaling or activity to activate insulin receptor tyrosine kinase.
• Bioactive fragments include, for example, polypeptides having insulin activity, in which a portion of insulin not involved in insulin activity is deleted.
• the insulin component 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 (e.g., a His tag).
• the purification tag includes, for example, a biochemically inactive tag.
• the concentration of the insulin component in the medium may be, for example, 0.1 ⁇ g/mL or more.
• 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 ⁇ g/mL, or may be any of these values or more, or may be within a range of any two of these values.
• This concentration may be, for example, 1 to 40, 1 to 30, 5 to 20, or 8 to 12 ⁇ g/ml. From the viewpoint of promoting the proliferation of MSCs, 1 ⁇ g/ml or more is preferable, 5 ⁇ g/ml or more is more preferable, and 8 ⁇ g/ml or more is even more preferable.
• albumin includes a type of protein that is abundant in serum. Details of the amino acid sequence of albumin can be found on websites such as NCBI or UniProt. The primary accession number of albumin listed in UniProt is, for example, Q56G89.
• the amino acid sequence of human albumin includes, for example, the amino acid sequence shown in SEQ ID NO: 10.
• Albumin includes, for example, albumin derived from humans.
• the albumin component has albumin activity and includes a molecule derived from wild-type human albumin.
• the albumin component may be, for example, (a) a protein having an amino acid sequence as set forth in SEQ ID NO: 10 or a biologically active fragment thereof, (b) a protein having an amino acid sequence having 90% or more homology to the amino acid sequence as set forth in SEQ ID NO: 10 or a biologically active fragment thereof, or (c) a protein having an amino acid sequence having one to several amino acid deletions, substitutions, insertions or additions to the amino acid sequence as set forth in SEQ ID NO: 10 or a biologically active fragment thereof.
• Albumin activity includes, for example, blood osmoregulation activity, or binding activity to fatty acids or bilirubin.
• Biologically active fragments include, for example, polypeptides having albumin activity, in which a portion of albumin not involved in albumin activity is deleted.
• the albumin component 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 (e.g., a His tag).
• the purification tag includes, for example, a biochemically inactive tag.
• the albumin component concentration in the medium may be, for example, 10 ⁇ g/mL or more.
• This concentration may be, for example, 10, 50, 100, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000, 3000, 4000, 5000, 8000, or 10000 ⁇ g/mL, or may be any value or more thereof, or may be within a range of any two values thereof.
• This concentration may be, for example, 100 to 4000, 10 to 3000, 500 to 2000, or 800 to 1200 ng/ml. From the viewpoint of promoting the proliferation of MSCs, 100 ⁇ g/ml or more is preferable, 500 ⁇ g/ml or more is more preferable, and 800 ⁇ g/ml or more is even more preferable.
• transferrin comprises a type of protein present in serum. Details of the amino acid sequence of transferrin can be found on websites such as NCBI or UniProt. The primary accession number of transferrin listed in UniProt is, for example, P02787.
• the amino acid sequence of human transferrin comprises, for example, the amino acid sequence shown in SEQ ID NO: 11. Transferrin includes, for example, transferrin derived from humans.
• the transferrin component has transferrin activity and comprises a molecule derived from wild-type human transferrin.
• the transferrin component may be, for example, (a) a protein having the amino acid sequence shown in SEQ ID NO: 11 or a biologically active fragment thereof, (b) a protein having an amino acid sequence having 90% or more homology to the amino acid sequence shown in SEQ ID NO: 11 or a biologically active fragment thereof, or (c) a protein 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 or a biologically active fragment thereof.
• Transferrin activity includes, for example, binding activity to a transferrin receptor or binding activity to iron ions.
• Bioactive fragments include, for example, polypeptides having transferrin activity from which a portion of transferrin not involved in transferrin activity has been deleted.
• the transferrin component 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 (e.g., a His tag).
• the purification tag includes, for example, a biochemically inactive tag.
• the concentration of the transferrin component in the medium may be, for example, 0.1 ⁇ g/mL or more.
• 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 ⁇ g/mL, or may be any value or more, or may be within a range of any two values.
• This concentration may be, for example, 1 to 40, 1 to 30, 5 to 20, or 8 to 12 ⁇ g/ml. From the viewpoint of promoting the proliferation of MSCs, 1 ⁇ g/ml or more is preferable, 5 ⁇ g/ml or more is more preferable, and 8 ⁇ g/ml or more is even more preferable.
• the container used for culture may be, for example, a flat-bottomed container.
• the flat-bottomed container may be, for example, a plate-type, petri dish-type, or flask-type.
• the shape of the bottom or top of the container may be square or round.
• the container that holds the culture supernatant may be a medical container.
• This container includes, for example, a syringe, a vial, a bottle, or a bag.
• the medical container may be a sterile container (for example, the solution-holding portion is sterile), a container for intravenous drip, or a container for cryopreservation.
• Medical bags include soft bags and bags connected to tubes.
• the recovering step may include, for example, a step of separating a cell fraction from a culture supernatant, or a step of transferring the culture supernatant to a container.
• the separating step may include, for example, a step of centrifuging or filtering a medium containing MSCs and an imidazole dipeptide to separate a cell fraction from a culture supernatant.
• the recovering step may include a step of centrifuging or filtering the supernatant to remove dead cells or impurities from the supernatant, a step of sterilizing the supernatant, or a step of transferring the supernatant to a medical container.
• the recovering step may include a step of removing medium components from a composition containing cells and a medium. The recovering step may be performed in a sterile environment.
• the method of the embodiment of the present invention may include a step of sterilizing the collected culture supernatant to produce a sterilized culture supernatant.
• the sterilization may include, for example, a step of passing the culture supernatant through a sterilizing filter.
• the contacting step may be carried out by culturing the MSCs in a medium containing imidazole dipeptide.
• the contacting step may be carried out in vitro or in vivo.
• suppressing a disease includes treating the disease.
• Suppression includes, for example, exerting an effect of suppressing, suppressing recurrence, improving symptoms, or preventing a patient's disease or one or more symptoms associated with the disease.
• Suppression also includes, for example, suppressing ossification in a patient, suppressing osteoblast production, or suppressing bone differentiation of cells (for example, suppressing differentiation of MSCs into osteoblasts or chondrocytes).
• the pharmaceutical composition may contain a culture supernatant or cultured MSCs.
• the pharmaceutical composition includes a composition consisting of the culture supernatant.
• the pharmaceutical composition may contain, for example, cytokines or exosomes. In this case, the concentrations of the cytokines and exosome markers may be in the concentration ranges described in the above embodiment of the culture supernatant.
• the pharmaceutical composition may contain, for example, imidazole dipeptide. The concentration of the imidazole dipeptide may be in the concentration ranges described in the above embodiment of the medium.
• the pharmaceutical composition includes a composition used for suppressing or ameliorating the above disease or a composition used for preventing the onset of the above disease.
• the pharmaceutical composition may be produced, for example, by mixing an active ingredient with one or more pharma- ceutical acceptable carriers and by any method known in the technical field of formulation science.
• the pharmaceutical composition may be used in any form as long as it is used for treatment, and may be the active ingredient alone or a mixture of the active ingredient and any ingredient.
• the shape of the carrier is not particularly limited, and may be, for example, a solid or liquid (e.g., a buffer solution).
• the content of the carrier may be, for example, a pharmaceutical effective amount.
• the effective amount may be, for example, an amount sufficient for pharmaceutical stability or delivery of the active ingredient.
• a buffer solution is effective for stabilizing the active ingredient in a container.
• the pharmaceutical composition may also contain a stabilizer, a buffering agent, or a pH adjuster.
• the dosage, administration interval, administration method, and administration route are not particularly limited and can be appropriately selected depending on the age and weight of the patient, symptoms, target organ, and the like.
• the pharmaceutical composition preferably contains a therapeutically effective amount, or an effective amount of the active ingredient that exerts a desired effect.
• the therapeutically effective amount includes an amount necessary for clinically observing improvement or suppression of symptoms in the patient (for example, an amount sufficient to suppress bone differentiation (e.g., suppression of heterotopic ossification) in the subject).
• being pharmaceutical acceptable includes a state suitable for use in accordance with a reasonable benefit/risk ratio within the scope of reasonable medical judgment.
• 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 they can be appropriately selected according to the purpose.
• the subject includes a human or a non-human mammal (e.g., one or more of mouse, guinea pig, hamster, rat, mouse, rabbit, pig, sheep, goat, cow, horse, cat, dog, marmoset, monkey, or chimpanzee).
• a human or a non-human mammal e.g., one or more of mouse, guinea pig, hamster, rat, mouse, rabbit, pig, sheep, goat, cow, horse, cat, dog, marmoset, monkey, or chimpanzee.
• the patient may also be a patient in need of inhibition of bone differentiation, a patient diagnosed with developing ossification, a patient in need of treatment for ossification, or a patient in need of increased expression of G-CSF, MCP-1, VEGF-C, TGF- ⁇ 1, IL-6, IL-7, IL-8, M-CSF, Osteoprotegrin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, semaphorin 3B, or an exosome marker.
• the route of administration of the culture supernatant, MSCs, or pharmaceutical composition to the subject is preferably one that is effective for the treatment, and may be, for example, intravenous, intraarterial, subcutaneous, intralymphatic, intramuscular, intraperitoneal, or oral.
• the form of administration is preferably one that is effective for the treatment, and may be, for example, an injection (e.g., intravenous injection), a liquid, or a solid preparation.
• the amount, administration interval, and administration method of the culture supernatant, MSCs, or pharmaceutical composition administered to a subject can be appropriately selected depending on the age, weight, symptoms, target organ, and the like of the patient.
• the administration amount may be, for example, 0.01 to 1000 mL in the case of a supernatant, and 1 x 10 3 to 1 x 10 11 per administration in the case of cells.
• the administration interval may be, for example, once or twice every 1 to 28 days or 1 to 4 weeks.
• inhibition of osteoblast production includes, for example, inhibition caused by inhibition of differentiation of MSCs into osteoblasts, or inhibition caused by inhibition of osteoblast proliferation.
• inhibition of differentiation of MSCs into osteoblasts may occur by contacting MSCs with an imidazole dipeptide.
• the method for inhibiting osteoblast formation may include, for example, a step of culturing MSCs in a medium containing an imidazole dipeptide, recovering an MSC secretion product, and a step of contacting the MSC secretion product with another MSC.
• the MSCs during culture and the MSCs during contact are different MSCs.
• the MSC secretions include, for example, G-CSF, MCP-1, VEGF-C, TGF- ⁇ 1, 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.
• the percentage of the composition or cell population containing cells of (26) to (28) or (38) above may be, for example, 30, 40, 50, 60, 70, 80, 90, 95, 98, 99, or 100%, or may be equal to or greater than these values, or within a range of any two of these values. This percentage may be, for example, 30-100%, 60-100%, 80-100%, 60-90%, 80-90%, or 90-100%.
• the homology (%) may be calculated, for example, by calculating the percentage of amino acids that are homologous between a plurality of amino acid sequences according to methods known in the art. Before calculating the percentage, the sequences to be compared are aligned and gaps are introduced into some of the sequences if necessary to maximize the percentage of homologous/identical amino acids. Methods for alignment, methods for calculating percentages, and related computer programs are conventionally well known in the art. The homology may be calculated, for example, using global or local alignments. The former may be determined using the Needleman-Wunsch algorithm (Needleman et al., J Mol Biol.
• EMBOSS Needle (Rice et al., EMBOSS User's Guide: Practical Bioinformatics, 25 July 2011.).
• Default settings for EMBOSS Needle may include, for example, MATRIX: BLOSUM62, Gap Open Penalty: 10, Gap Extend Penalty: 0.5, Output formats: pair, End Gap Penalty: false, End Gap Open Penalty: 10, End Gap Extend Penalty: 0.5.
• the latter may be determined with 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.
• 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 source amino acid sequence) x 100. Homology is preferably calculated using a global alignment. Unless otherwise specified, a test homology equal to or greater than a particular value includes a value equal to or greater than a particular value calculated by at least one of the above algorithms. Any of the above algorithms may be used with default settings.
• the above-mentioned 90% or more described as the homology value may be, for example, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or may be any of these values or more, or within a range of any two of these values. From the viewpoint of maintaining functional equivalence, this ratio 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 several or less may be, for example, 15, 10, 8, 6, 5, 4, 3, 2, 1, or 0, or may be any of these values or less, or within a range of any two of these values. From the viewpoint of maintaining functional equivalence, this number is preferably 15 or less, more preferably 10 or less, and particularly preferably 5 or less.
• conservative amino acid substitutions are preferred when substitutions are made to the original sequence.
• conservative amino acid substitutions involve replacing 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), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-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.
• the amino acid is a general term for an organic compound having an amino group and a carboxyl group.
• a protein according to an embodiment of the present invention contains a "specific amino acid sequence”
• any of the amino acids in the amino acid sequence may be chemically modified.
• Any of the amino acids in the amino acid sequence may form a salt or a solvate.
• Any of the amino acids in the amino acid sequence may be 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 a protein undergo in vivo include N-terminal modifications (e.g., acetylation, myristoylation, etc.), C-terminal modifications (e.g., amidation, glycosylphosphatidylinositol addition, etc.), and side chain modifications (e.g., phosphorylation, sugar chain addition, etc.).
• N-terminal modifications e.g., acetylation, myristoylation, etc.
• C-terminal modifications e.g., amidation, glycosylphosphatidylinositol addition, etc.
• side chain modifications e.g., phosphorylation, sugar chain addition, etc.
• significance may be evaluated as being significant when, for example, p ⁇ 0.05 or p ⁇ 0.01 using a Student's t-test (one-tailed or two-tailed) to determine statistical significance.
• significance may be evaluated as being significant when a substantial difference is present.
• the above (1) to (41) includes reference 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).
• An embodiment of the present invention may include, for example, the following embodiments.
• a method for producing a culture supernatant comprising the step of recovering a culture supernatant from a medium containing mesenchymal stem cells and an imidazole dipeptide.
• the mesenchymal stem cells are mesenchymal stem cells cultured in a medium containing an imidazole dipeptide.
• the imidazole dipeptide is a compound having the structure of the aforementioned formula (1) or (2).
• a pharmaceutical composition comprising a culture supernatant obtained by the production method described in any one of 1 to 12 above.
• a method for culturing cells comprising the step of culturing mesenchymal stem cells in a serum-free medium containing an imidazole dipeptide to produce cultured cells. 18.
• the culture method described in 17 above further comprising a step of recovering the cultured cells from the medium.
• the culture method described in 17 or 18 above wherein the culture comprises a step of culturing in the presence of an adhesion factor.
• 20. The culture method described in any one of 17 to 19 above, wherein the medium is insulin-free, IGF-free, or FGF-free.
• 24. A cell obtained by the production method described in 23 above.
• 25. A composition comprising a cell population of cells as described in 24 above.
• 26. A pharmaceutical composition comprising cells obtained by the production method described in 23 above.
• 28. A medium for culturing mesenchymal stem cells, the medium containing an imidazole dipeptide and being serum-free. 29.
• a composition for inhibiting osteoblast formation comprising a culture supernatant obtained by culturing mesenchymal stem cells in a medium containing an imidazole dipeptide.
• a method for promoting exosome secretion comprising a step of culturing mesenchymal stem cells in a medium containing imidazole dipeptide. 40.
• a composition for promoting exosome secretion from mesenchymal stem cells comprising an imidazole dipeptide. 41. A mesenchymal stem cell culture supernatant containing at least 500 pg/ml of G-CSF. 42. A mesenchymal stem cell culture supernatant containing at least 2700 pg/ml of IL-6. 43. Umbilical cord-derived mesenchymal stem cells that highly express or secrete G-CSF or IL-6. 44. A composition comprising a cell population of cells according to 43 above. 45. IL-34 positive mesenchymal stem cells. 46. A purified mesenchymal stem cell according to 45 above. 47. ADAM8 positive mesenchymal stem cells. 48.
• the pharmaceutical composition described in 53 above further comprising a pharma- ceutically acceptable carrier.
• 55. The pharmaceutical composition described in 53 or 54 above, wherein the cells are positive for Osteoprotegrin, Gremlin 1, SCF, FGF11, R-spondin 2, ADAM8, IL-34, IGFBP2, DKK1, and semaphorin 3B.
• 56. The pharmaceutical composition according to any one of claims 53 to 55, wherein the cells are obtained by culturing in a medium containing an imidazole dipeptide and LIF.
• 57. A pharmaceutical composition described in any one of 53 to 56 above, wherein the culture is an adherent culture. 58.
• a method for producing a pharmaceutical composition for inhibiting bone differentiation comprising the step of culturing mesenchymal stem cells in a medium containing an imidazole dipeptide to produce cultured cells. 59. The production method described in 58 above, further comprising a step of recovering the cultured cells from the medium. 60. A production method described in 58 or 59 above, comprising a step of mixing the cultured cells with a pharma- ceutically acceptable carrier. 61. A medium containing an imidazole dipeptide and a LIF (leukemia inhibitory factor) component. 62. The medium described in 61 above, further comprising an adhesion factor. 63.
• 64. A medium described in any one of 61 to 63 above, containing mesenchymal stem cells.
• 65. A method for producing cells, comprising the step of culturing mesenchymal stem cells in a medium described in any one of 61 to 64 above to generate cultured cells.
• 66. The method for producing the cell according to claim 65, wherein the culture is an adherent culture.
• 67. A cell obtained by the production method described in 65 or 66 above.
• 68. A composition comprising a cell population of cells as described in 67 above.
• 69. A pharmaceutical composition comprising cells obtained by the production method described in 65 or 66 above. 70.
• a kit comprising an imidazole dipeptide and a LIF component.
• Example 1 Analysis of culture supernatant 1.1
• Experimental method Cultivation of MSCs using a medium containing imidazole dipeptide and collection of culture supernatant In this experiment, carnosine was used as the imidazole dipeptide.
• the experimental procedure is as follows. Human umbilical cord-derived mesenchymal stem cells were suspended in 20 ml of MSC Expansion XSFM B (FUJIFILM Wako Pure Chemical Industries, Ltd.) (hereinafter also referred to as MSC medium B) at a cell number of 0.3x10e5.
• iMatrix-511 laminin fragment (0.5 ⁇ g/ ⁇ l) (Nippi Corporation) was added to 20 ml of the cell suspension, and the obtained cell suspension was seeded in a T150 flask (Sumitomo Bakelite Co., Ltd.).
• MSC medium B containing 5 ⁇ l of iMatrix-511 laminin fragment (0.5 ⁇ g/ ⁇ l) (Nippi Corporation) was added.
• day 14 the medium was replaced with PBS twice to wash off the remaining medium.
• 30 ml of protein-free medium hereinafter referred to as 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).
• MSC medium A was supplemented with the imidazole dipeptide L-carnosine (Fujifilm Wako Pure Chemical Corporation) at no addition (0 mM), or at 1 mM, 10 mM, or 30 mM.
• the cells were added to MSC medium A supplemented with carnosine and cultured for three days (production culture). This culture is referred to as the "first production culture.”
• the culture supernatant of MSC medium A was collected.
• the amount of cytokines contained in the culture supernatant was analyzed using an ELISA analysis kit (R&D Systems) or a Milliplex kit (Merck Millipore).
• the cytokines analyzed were Hepatocyte growth factor (HGF), Granulocyte colony stimulating factor (G-CSF), Monocyte chemotactic protein 1 (MCP-1), Vascular endothelial growth factor-C (VEGF-C), Transforming growth factor- ⁇ 1 (TGF- ⁇ 1), Interleukin-6 (IL-6), Interleukin-7 (IL-7), and Interleukin-8 (IL-8).
• the amount of exosomes contained in the culture supernatant was analyzed using a CD9/CD63 ELISA kit (Cosmo Bio) with exosome marker protein (CD9/CD63 fusion protein) as an indicator.
• the amounts of cytokines and exosome marker proteins refer to values measured by ELISA using specific antibodies.
• the medium was returned to MSC medium B and cultured for two days (recovery culture). After the two-day recovery culture, the medium was replaced with MSC medium A supplemented with carnosine as above and cultured for three days (the carnosine concentration was the same as in the first production culture). This culture is referred to as the "second production culture.”
• the culture supernatant of MSC medium A supplemented with carnosine was collected.
• the cytokines contained in the culture supernatant were analyzed using an ELISA analysis kit (R&D Systems).
• the medium was returned to MSC medium B and cultured for two days (recovery culture). After the two-day recovery culture, the medium was replaced with MSC medium A supplemented with carnosine as above and cultured for three days (the carnosine concentration was the same as in the first production culture). This culture is referred to as the "third production culture.”
• the culture supernatant of MSC medium A supplemented with carnosine was collected.
• the cytokines contained in the culture supernatant were analyzed using an ELISA analysis kit (R&D Systems).
• the medium was returned to MSC medium B and cultured for two days (recovery culture). After the two-day recovery culture, the medium was replaced with MSC medium A supplemented with carnosine as above and cultured for three days (the carnosine concentration was the same as in the first production culture). This culture is referred to as the "fourth production culture.”
• the culture supernatant of MSC medium A supplemented with carnosine was collected.
• the cytokines contained in the culture supernatant were analyzed using an ELISA analysis kit (R&D Systems).
• FIGS. 1 to 4 are micrographs of MSCs after culturing in carnosine-containing or -free media. There was no significant cell death, and no obvious difference in cell morphology was observed at each carnosine concentration.
• Cell count Figure 5 shows the results of counting the number of MSC cells after culturing in a medium containing or not containing carnosine. There was no significant change in the number of cells due to the addition of carnosine to the medium.
• Figure 6 shows the results of examining the amount of exosome marker in the culture supernatant. The addition of carnosine to the medium showed a tendency for the exosome marker to increase.
• FIG. 7 to 14 show the results of examining the amounts of HGF (HGF, 3082), G-CSF (CSF3, 1440), MCP-1 (CCL2, 6347), VEGF-C (VEGFC, 7424), TGF- ⁇ 1 (TGFB1, 7040), IL-6 (IL6, 3569), IL-7 (IL7, 3574), and IL-8 (CXCL8, 3576) in the culture supernatant.
• HGF HGF, 3082
• G-CSF CSF3, 1440
• MCP-1 CCL2, 6347
• VEGF-C VEGFC, 7424
• TGF- ⁇ 1 TGFB1, 7040
• IL-6 IL6, 3569
• IL-7 IL7, 3574
• IL-8 CXCL8, 3576
• G-CSF, MCP-1, VEGF-C, TGF- ⁇ 1, IL-6, IL-7, and IL-8 showed an increasing tendency (each number in parentheses indicates the official symbol and gene ID of NCBI). In particular, the increase rate of G-CSF and IL-6 was remarkable.
• the G-CSF concentration increased 14.7-fold (approximately 1621 pg/mL) and the IL-6 concentration increased more than 22-fold (more than 7000 pg/mL) when 10 mM carnosine was added.
• Example 2 Inhibitory effect of culture supernatant on bone differentiation 2.1 Experimental method 2.1.1 Culture of MSC using imidazole dipeptide-containing medium and collection of culture supernatant Using human umbilical cord-derived mesenchymal stem cells (CET03 line) established by the present inventor, the culture supernatant was collected according to the experimental method up to the third production culture in Example 1. However, at this time, 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.
• DMEM human umbilical cord-derived mesenchymal stem cells
• IMDM IMDM
• umbilical cord MSC supernatant The six types of umbilical cord MSC supernatants obtained are referred to as DMEM_supernatant, DMEM_C_supernatant, DMEM/F12_supernatant, DMEM/F12_C_supernatant, IMDM_supernatant, and IMDM_C_supernatant (the names of these supernatants are indicated by the name of the medium used on the left side. Supernatants obtained under carnosine-supplemented conditions are indicated by a C to the right of the medium name).
• Human bone marrow MSCs primary cultured human bone marrow MSCs
• ⁇ MEM medium containing 10% FBS (Hyclone)
• trypsin-EDTA solution to separate into single cells.
• the obtained single cells were seeded on a fibronectin-coated 24-well plate at 8 ⁇ 10e4 cells/well in basal medium (DMEM (Sigma) with 0.6 mM CaCl 2 ) containing 10% FBS.
• DMEM basal medium
• Basal medium DMEM (Sigma) with 0.6 mM CaCl 2
• bone differentiation induction components 10% FBS, 0.1 ⁇ M dexamethasone, 50 ⁇ M ascorbic acid, and 10 mM ⁇ -glycerophosphate
• 0 or 10 mM carnosine 0 or 10 mM carnosine at a final concentration
• DMEM_medium_cells The cells obtained are referred to as DMEM_medium_cells and DMEM_C_medium_cells (the name of the medium used is written on the left side of the cell names. Cells obtained under carnosine-supplemented conditions are marked with a C to the right of the medium name).
• Step 2 was carried out under the condition that the basal medium was changed to DMEM/F12 (Sigma) containing 0.6 mM CaCl 2.
• the obtained cells are designated as DMEM/F12_medium_cells and DMEM/F12_C_medium_cells (the names of these cells are indicated by the name of the medium used on the left side.
• the cells obtained under the condition of adding carnosine are indicated by C to the right of the medium name).
• Step 3 was repeated except that the basal medium was changed to IMDM (Sigma) containing 2 mM L-glutamin.
• the cells obtained are referred to as IMDM_medium_cells and IMDM_C_medium_cells (the names of these cells are indicated by the name of the medium used on the left. Cells obtained under carnosine-added conditions are indicated by a C to the right of the medium name).
• the same procedure as step 3 was also carried out separately, and calcium deposition images were taken of the cells after bone differentiation induction culture using an Alizarin Red S staining kit (Cosmo Bio).
• Step 4 was carried out under the following conditions: the bone differentiation induction medium was replaced with the umbilical cord MSC culture supernatant (DMEM_supernatant or DMEM_C_supernatant) obtained in 2.1.1 above, supplemented with components for bone differentiation induction (10% FBS, 0.1 ⁇ M dexamethasone, 50 ⁇ M ascorbic acid, and 10 mM ⁇ -glycerophosphate).
• the cells obtained are referred to as DMEM_supernatant_cells and DMEM_C_supernatant_cells (the names of these cells are indicated by the name of the medium used on the left side.
• the cells obtained under carnosine-added conditions are indicated by C to the right of the medium name).
• Step 2 was carried out under the following conditions: the bone differentiation induction medium was replaced with the umbilical cord MSC culture supernatant obtained in 2.1.1 above (DMEM/F12_supernatant or DMEM/F12_C_supernatant) supplemented with bone differentiation induction components (10% FBS, 0.1 ⁇ M dexamethasone, 50 ⁇ M ascorbic acid, and 10 mM ⁇ -glycerophosphate).
• the cells obtained are referred to as DMEM/F12_supernatant_cells and DMEM/F12_C_supernatant_cells (the names of these cells are indicated by the name of the medium used on the left side.
• the cells obtained under carnosine-added conditions are indicated by C to the right of the medium name).
• Step 6 the bone differentiation induction medium was replaced with the umbilical cord MSC culture supernatant obtained in 2.1.1 above (IMDM_supernatant or IMDM_C_supernatant) supplemented with bone differentiation induction components (10% FBS, 0.1 ⁇ M dexamethasone, 50 ⁇ M ascorbic acid, and 10 mM ⁇ -glycerophosphate).
• the cells obtained are referred to as IMDM_supernatant_cells and IMDM_C_supernatant_cells (the names of these cells are indicated by the name of the medium used on the left. For cells obtained under carnosine-added conditions, C is indicated to the right of the medium name).
• the same procedure as step 6 was separately performed, and calcium deposition images were taken of the cells after bone differentiation induction culture using an Alizarin Red S staining kit (Cosmo Bio).
• FIG. 15 shows the quantitative results of calcium deposition in bone marrow MSCs.
• the names of the medium in the figure are the names of the basal medium used.
• the amount of calcium was significantly lower in cells obtained using the supernatant of MSCs obtained by culturing in a medium containing carnosine (supernatant_C_10mM in the figure; corresponding samples are DMEM_C_supernatant_cells, DMEM/F12_C_supernatant_cells, and IMDM_C_supernatant_cells) than in cells obtained using a medium without carnosine (medium_C_0mM in the figure; corresponding samples are DMEM_medium_cells, DMEM/F12_C_supernatant_cells, and IMDM_medium_cells) and cells obtained using the supernatant of MSCs obtained by culturing in a medium without carnosine (medium_C_0mM in the figure; corresponding samples are
• the amount of calcium was also significantly lower than that of cells obtained using carnosine-supplemented medium (medium_C_10mM in the figure.
• the corresponding samples are DMEM_C_medium_cells, DMEM/F12_C_medium_cells, and IMDM_C_medium_cells.)
• the supernatant of MSCs obtained by culturing in carnosine-supplemented medium was found to have a significant inhibitory effect on bone differentiation.
• Figure 16 shows images of bone marrow MSCs stained with Alizarin Red S.
• DMEM_medium_cells cells obtained using a medium without carnosine
• DMEM_supernatant_cells cells obtained using the supernatant of MSCs cultured in a medium without carnosine
• DMEM_C_medium_cells cells obtained using a medium containing carnosine
• DMEM_C_supernatant_cells did not show any staining with Alizarin Red.
• the supernatant of MSCs cultured in a medium containing carnosine was found to have a significant inhibitory effect on bone differentiation.
• Example 3 Inhibitory effect of MSC on bone differentiation 3.1
• Experimental method 3.1.1 Cultivation of umbilical cord MSC using medium containing imidazole dipeptide
• Human umbilical cord MSC established by the present inventors were cultured in DMEM/F12 medium (hereinafter also referred to as MSC medium C) containing 10 ng/ml bFGF (Peprotech, AF-100-18C), 10 ⁇ g/ml insulin (Nacalai, 12878-44), 1000 ⁇ g/ml albumin (Sigma, A9511), 10 ug/ml transferrin (Nacalai, 12879-34), and 1 ng/ml LIF (Peprotech, AF-300-05), and then treated with trypsin-EDTA solution to dissociate into 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 to a final concentration of 0.25 ⁇ g/ml, and the cells were seeded in a T25 flask (Corning). After 2 weeks of culture, the cells were washed with PBS to remove carnosine, and 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.
• Bone marrow MSCs primary cultured human bone marrow MSCs
• ⁇ MEM medium containing 10% FBS Hyclone
• trypsin-EDTA solution to separate into single cells.
• These bone marrow MSCs were mixed with umbilical cord MSCs obtained by culturing in the above 3.1.1 medium containing 0 or 5 mM carnosine at a ratio of 1:0, 0:1, 1:1, and 3:1, respectively.
• the total number of cells of each MSC combined was 8.0x10e4/well, and the mixture was seeded on a 24-well plate in ⁇ MEM medium containing 10% FBS.
• the medium in the wells for each co-culture condition was replaced with either (1) 10% FBS ⁇ MEM medium supplemented with osteogenic differentiation components (0.1 ⁇ M dexamethasone, 50 ⁇ M ascorbic acid, and 10 mM ⁇ -glycerophosphate) or (2) 10% FBS ⁇ MEM medium without osteogenic differentiation components, and the medium was replaced with the same medium every 2-3 days for 9 days. Osteogenic differentiation was evaluated by calcium assay and Alizarin Red S staining.
• FIG. 17 shows the quantitative results of calcium deposition during co-culture of bone marrow MSCs and umbilical cord MSCs.
• a large amount of calcium was deposited in the bone differentiation condition of 1:1 co-culture of bone marrow MSCs and umbilical cord MSCs (BM:UC(1:1)), but calcium deposition was significantly suppressed in the bone differentiation condition of 1:1 co-culture of bone marrow MSCs and umbilical cord MSCs pre-cultured with carnosine (BM:UC_preC(1:1)). It was shown that umbilical cord MSCs pre-cultured with carnosine have the effect of suppressing bone differentiation of MSCs.
• BM Non-co-culture conditions using bone marrow MSC 8X10 ⁇ 4 cells/well.
• ⁇ MEM medium conditions with osteogenic differentiation inducer.
• BM_NC Non-co-culture conditions using bone marrow MSC 8X10 ⁇ 4 cells/well.
• ⁇ MEM medium conditions without osteogenic differentiation inducers.
• UC Non-co-culture conditions using umbilical cord MSCs (8X10 ⁇ 4 cells/well) pre-cultured for 2 weeks in MSC medium C without carnosine. ⁇ MEM medium conditions with osteogenic differentiation inducer.
• UC_preC Non-co-culture conditions using umbilical cord MSCs (8X10 ⁇ 4 cells/well) pre-cultured for 2 weeks in MSC medium C containing 5mM carnosine. ⁇ MEM medium conditions with osteogenic differentiation induction.
• BM UC (1:1): Bone marrow MSCs and umbilical cord MSCs pre-cultured for 2 weeks in MSC medium C without carnosine were mixed and seeded at 4X10 ⁇ 4 cells/well in a co-culture condition.
• BM:UC_preC (1:1) Bone marrow MSCs and umbilical cord MSCs pre-cultured for 2 weeks in CET original medium containing 5mM carnosine were mixed and seeded at 4X10 ⁇ 4 cells/well.
• ⁇ MEM medium conditions with osteogenic differentiation inducer are examples of bone marrow MSCs (6X10 ⁇ 4 cells/well) and umbilical cord MSCs (2X10 ⁇ 4 cells/well) pre-cultured for 2 weeks in MSC medium C without carnosine were mixed and seeded.
• Figure 18 shows an Alizarin Red S stained image of bone marrow MSCs and umbilical cord MSCs co-cultured.
• a large amount of calcium was deposited in the bone differentiation conditions (BM:UC(1:1)) of bone marrow MSCs co-cultured with umbilical cord MSCs, but calcium deposition was significantly suppressed in the bone differentiation conditions (BM:UC_preC(1:1)) of bone marrow MSCs co-cultured with umbilical cord MSCs pre-cultured with carnosine. It was shown that umbilical cord MSCs pre-cultured with carnosine have the effect of suppressing the bone differentiation of MSCs.
• Example 4 Analysis of MSC 4.1 Experimental method 4.1.1 Recovery of RNA from MSC cultured using imidazole dipeptide-containing medium Using human umbilical cord-derived mesenchymal stem cells (CET03 line) established by the present inventor, the culture supernatant was recovered according to the experimental method up to the third production culture in Example 1 described above. However, at this time, the proliferation medium (MSC medium B), the supernatant recovery medium (MSC medium A), the carnosine concentration, and the culture vessel used in Example 1 were changed as shown in FIG. 19. The supernatant recovery medium was ⁇ MEM (Sigma), DMEM (Sigma), or DMEM/F12 (Sigma). The proliferation medium was MSC medium B or MSC medium C (see Example 3 for composition).
• RNAseq analysis Cells were collected on day 27 after supernatant collection, and RNA was collected using miRNeasy Mini Kit (Qiagen) and used for RNAseq analysis. NextSeq500 (Illumina) was used as the sequencing analysis device. The sequence read length was 75 bp for a single read, equivalent to 1 million reads.
• the analysis program was StrandNGS ver4.0 (Strand Life Sciences Pvt Ltd), and fastq reads after removal of low-reliability bases were used to map to the human reference genome hg38, and RPM expression level correction was performed. The RPM correction value of each gene was calculated for a total of 19 samples.
• the top 21 genes encoding secreted proteins were selected as follows: The top three are: 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, and Dickkopf WNT signaling pathway inhibitor 1.
• RNAseq analysis identified the top 21 secretory protein-encoding genes whose expression levels were increased by carnosine. Of these, the following 10 have been previously reported to be involved in the inhibition of bone formation.
• Gremlin 1 DAN family BMP antagonist (GREM1, 26585, Gremlin 1), KIT ligand (KITLG, 4254, SCF), R-spondin 2 (RSPO2, 340419), semaphorin 3B (SEMA3B, 7869), fibroblast growth factor 11 (FGF11, 2256), TNF receptor superfamily member 11b (TNFRSF11B, 4982, Osteoprotegerin), ADAM metallopeptidase domain 8 (ADAM8, 101), interleukin 34 (IL34, 146433, IL-34), insulin-like growth factor binding protein 2 (IGFBP2, 3485), dickkopf WNT signaling pathway inhibitor 1 (DKK1, 22943).
• GREM1, 26585, Gremlin 1 DAN family BMP antagonist
• Figures 20(a) to 20(j) show the changes in the % increase values of the above 10 types of 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.
• Figures 20 to 22 show the results when DMEM/F12 (Sigma) was used as the medium for supernatant recovery and MSC medium B was used as the growth medium.
• the concentrations in the figures indicate the concentration of carnosine added.
• Figure 21 is a graph showing the RPM values of interleukin 34 expression levels in umbilical cord MSCs. When 10mM or 30mM carnosine was added, interleukin 34 expression was observed, but no expression was observed when no carnosine was added (0mM).
• Figure 22 is a graph showing the RPM values of ADAM metallopeptidase domain 8 expression levels in umbilical cord MSCs. When 10mM, 20mM, or 30mM carnosine was added, ADAM metallopeptidase domain 8 expression was observed, but no expression was observed when no carnosine was added (0mM).
• Example 5 Analysis of culture supernatant and MSC 5.1 Culture of MSC using imidazole dipeptide-containing medium and collection of culture supernatant 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 in Example 1 described above. However, at this time, the MSC medium A used in Example 1 was replaced with ⁇ MEM (Sigma), DMEM (Sigma), or DMEM/F12 (Sigma). In addition, the concentration of carnosine added was changed to 0 or 30 mM (500 mM carnosine solution in distilled water was used when adding.
• ⁇ MEM Sigma
• DMEM DMEM/F12
• 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 types of 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.
• qPCR Quantitative PCR analysis
• Cells were harvested on day 27 after supernatant collection, and RNA was extracted using the miRNeasy Mini Kit (Qiagen).
• the expression levels of the osteoprotegerin gene TNFRSF11B and the M-CSF gene CSF1 were analyzed using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as an internal standard by quantitative PCR (PowerUp SYBR Green Master Mix, Thermo Fisher).
• GPDH glyceraldehyde-3-phosphate dehydrogenase
• FIG. 23 to 25 show the results of ELISA analysis of Osteoprotegerin
• Figures 26 to 28 show the results of ELISA analysis of M-CSF.
• the concentration (mM) in the figures indicates the carnosine concentration.
• ⁇ MEM was used in Figures 23 and 26, DMEM in Figures 24 and 27, and DMEM/F12 in Figures 25 and 28.
• the amount of Osteoprotegrin and M-CSF secreted tended to increase in a concentration-dependent manner by adding carnosine in the first, second, and third collection times, respectively.
• Figure 29 shows the results of qPCR analysis of Osteoprotegerin
• Figure 30 shows the results of qPCR analysis of M-CSF.
• the concentration (mM) in the figures indicates the carnosine concentration, and the medium name indicates the medium used for collecting the supernatant. Under each condition, the amount of Osteoprotegrin and M-CSF gene expression tended to increase in a concentration-dependent manner by adding carnosine.
• Example 6 Analysis of culture supernatant and MSC 6.1 Cultivation of MSC using imidazole dipeptide-containing medium and recovery of culture supernatant Using human umbilical cord-derived mesenchymal stem cells (CET03 line) or human bone marrow-derived mesenchymal stem cells (ATCC) established by the present inventor, the culture supernatant was recovered according to the experimental method up to the third production culture in Example 1. However, at this time, MSC medium B or MSC medium C (see Example 3 for composition) was used as the proliferation medium (MSC medium B in Example 1). In addition, DMEM/F12 (Sigma) was used as the supernatant recovery medium (MSC medium A in Example 1).
• Figure 31 shows the results of ELISA analysis of osteoprotegerin.
• Figure 31(a) shows the results when carnosine was added
• Figure 31(b) shows the results when anserine was added.
• umbilical cord MSCs were used as the cells
• MSC medium B was used as the growth medium.
• Figures 32-33 show the results of qPCR analysis of osteoprotegerin.
• Figures 32(a)-(c) show the conditions with carnosine addition
• Figures 33(a)-(c) show the conditions with anserine addition.
• UC_B_C means that umbilical cord MSCs were used as cells
• MSC medium B was used as the growth medium
• carnosine was used as an additive.
• BM_B_C means that bone marrow MSCs were used as cells
• MSC medium B was used as the growth medium
• carnosine was used as an additive.
• BM_C_C means that bone marrow MSCs were used as cells, MSC medium C was used as the growth medium, and carnosine was used as an additive.
• UC_B_A means that umbilical cord MSCs were used as cells, MSC medium B was used as the growth medium, and anserine was used as an additive.
• BM_B_A means that bone marrow MSCs were used as cells, MSC medium B was used as the growth medium, and anserine was used as an additive.
• BM_C_A means that bone marrow MSCs were used as the cells, MSC medium C was used as the growth medium, and anserine was used as the additive. Under each condition, the addition of carnosine or anserine tended to increase the expression level of the osteoprotegin gene.
• Figure 34 shows the results of ELISA analysis of M-CSF.
• Figure 34(a) shows the condition with the addition of carnosine
• Figure 34(b) shows the condition with the addition of anserine.
• umbilical cord MSCs were used as the cells
• MSC medium B was used as the growth medium.
• Figures 35-36 show the results of qPCR analysis of M-CSF.
• Figures 35(a)-(b) show the conditions with the addition of carnosine
• Figures 36(a)-(b) show the conditions with the addition of anserine.
• UC_B_C means that umbilical cord MSCs were used as cells
• MSC medium B was used as the growth medium
• carnosine was used as an additive.
• BM_B_C means that bone marrow MSCs were used as cells
• MSC medium B was used as the growth medium
• carnosine was used as an additive.
• UC_B_A means that umbilical cord MSCs were used as cells, MSC medium B was used as the growth medium, and anserine was used as an additive.
• BM_B_A means that bone marrow MSCs were used as cells, MSC medium B was used as the growth medium, and anserine was used as an additive. Under each condition, a tendency was observed for the addition of carnosine or anserine to increase the expression level of the M-CSF gene.
• Example 7 Proliferation-promoting effect of MSCs 7.1 Experimental method 7.1.1 MSC expansion culture Human umbilical cord-derived mesenchymal stem cells established by the present inventors were detached using TrypLE Select and seeded in a 24-well plate at a cell number 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 (Peprotech), 10 ⁇ g/ml insulin (Nacalai), 1000 ⁇ g/ml albumin (Sigma), 10 ug/ml transferrin (Nacalai), and 1 ng/ml LIF (Peprotech) (hereinafter also referred to as MSC medium D), or (3) a medium obtained by removing LIF from MSC medium D (hereinafter also referred to as MSC medium E).
• iMatrix-511 laminin fragment (Nippi Corporation) was added to each medium at a final concentration of 0 or 0.1%, and carnosine or anserine was added to each medium at a final concentration of 0, 0.2, 0.5, 1, 2, or 5 mM.
• Each sample was cultured for 4 to 7 days in the same medium as used, with the medium being replaced every 2 to 3 days. After that, the samples were stained with DAPI, phalloidin, and CD44 antibody, and fluorescent images of each were taken using a confocal quantitative image cytometer CellVoyager CQ-1 (Yokogawa Electric Corporation), and DAPI cell counts and phalloidin (actin filament marker) and CD44 (MSC marker) fluorescent areas were measured.
• Table 1 The experimental conditions are summarized in Table 1.
• the umbilical cord MSCs established by the inventors of the present 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) MSC medium D without LIF (hereinafter also referred to as MSC medium E).
• MSC medium E MSC medium D without LIF
• iMatrix-511 laminin fragment Nappi Corporation
• carnosine was added to each medium at a final concentration of 0, 0.2, 1, or 5 mM, and seeded.
• Each sample was cultured for 7 days in the same medium as used, with the medium being replaced every 2 to 3 days.
• Figures 37-38 show the results of cell counting for groups 1-4.
• the addition of carnosine tended to increase the number of cells, especially in group 3.
• Figure 39 shows the results of measuring the fluorescent area of phalloidin for groups 1-4.
• the addition of carnosine tended to increase the phalloidin area, especially in group 3.
• Figure 40 shows the results of measuring the fluorescent area of CD44 for groups 1-4.
• the addition of carnosine tended to increase the CD44 area, especially in group 3.
• Figure 41 shows the results of taking fluorescent images of groups 1 and 3 (cell nucleus staining). There was a tendency for the number of cells to increase, especially in group 3, where carnosine was 1 mM.
• Figure 42 shows the results of taking fluorescent images of groups 1 and 3 (phalloidin staining). There was a tendency for the number of cells and the phalloidin area to increase, especially in groups 1 and 3, where carnosine was 1 mM. No significant differences were observed in cell morphology.
• Figure 43 shows the results of taking fluorescent images of groups 1 and 3 (CD44 staining). There was a tendency for the number of cells and the CD44 area to increase, especially in group 3, where carnosine was 1 mM. No significant differences were observed in the marker expression pattern.
• Figures 44-45 show the results of cell counting for groups 5-8.
• the addition of anserine tended to increase the number of cells, particularly in group 7.
• Figures 46-47 show the results of measuring the fluorescent area of phalloidin for groups 5-8.
• the addition of anserine tended to increase the phalloidin area, particularly in group 7.
• Figures 48-49 show the results of measuring the fluorescent area of CD44 for groups 5-8.
• the addition of anserine tended to increase the CD44 area, particularly in group 7.
• Figure 50 shows the results of taking fluorescent images (phalloidin staining) of groups 5 and 7. Particularly in group 7, where anserine was 1 mM, there was a tendency for the cell number and phalloidin area to increase. No significant differences were observed in cell morphology.
• Figure 51 shows the results of taking fluorescent images (CD44 staining) of groups 5 and 7. Particularly in group 7, where anserine was 1 mM, there was a tendency for the cell number and CD44 area to increase. No significant differences were observed in the marker expression pattern.
• Figure 52 shows the results of measuring the positivity rate of MSC markers after culturing umbilical cord MSCs in MSC medium D. While the cell number increased with the addition of carnosine, each MSC positive and negative marker did not change with the addition of carnosine.
• Figure 53 shows the results of cell counting for groups 9 and 10. In group 9, the addition of carnosine tended to increase the number of cells, while in group 10, no increase was observed.
• Figure 54 shows the results of measuring the phalloidin fluorescence area for groups 9 and 10. In group 9, the addition of carnosine tended to increase the phalloidin area, while in group 10, no increase was observed.
• Figure 55 shows the results of taking fluorescent images (phalloidin staining) of groups 9 and 10.
• group 9 where carnosine was 1 mM
• group 10 where carnosine was 1 mM
• no tendency for an increase was observed.

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