Agent for suppressing tissue stem cell aging

Abstract

The present disclosure provides an agent that contains a substance which inhibits signaling from CXCL5 and that is for suppressing tissue stem cell aging.

Description

Anti-aging agent for tissue stem cells

[0001] The present invention relates to an agent for inhibiting cellular senescence of tissue stem cells, which contains a substance that inhibits signal transduction from CXCL5.

[0002] Various stem cells are used in regenerative medicine, but because ES cells and iPS cells require time and cost to culture and have a high risk of cancer, the use of tissue stem cells, especially mesenchymal stem cells (MSCs), is increasing. Regenerative medicine using MSCs requires extracting a small amount of tissue from a patient and culturing the MSCs contained therein. However, the number of stem cells in tissue is low, and repeated subculture causes stem cells to gradually age and stop dividing, making it a major challenge to secure the number of cells needed for treatment. Furthermore, hematopoietic stem cell transplants are used to treat blood disorders such as leukemia, but, like MSCs, securing the number of cells needed for treatment is a major challenge.

[0003] The G protein-coupled receptor CXCR2 is a chemokine receptor. Chemokines such as CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8 (also known as IL-8) are known to act as CXCR2 ligands. CXCL5 is known to interact with CXCL2 and induce neutrophil chemotaxis and activation.

[0004] WO2018 / 056461

[0005] Acosta, JC et al. Chemokine signaling via the CXCR2 receptor reinforces senescence. Cell 133, 1006-1018 (2008). Li JJ et al. Knockdown of IL-8 provoked premature senescence of Placenta-Derived mesenchymal stem cells. Stem Cells Dev. 2017; 26:912-931.

[0006] An object of the present invention is to provide a means for preventing the aging of tissue stem cells.

[0007] It has been previously disclosed that inhibiting signal transduction from CXCL5 can improve the decline in preimplantation embryo quality due to aging and other factors (Patent Document 1). It has also been reported that knockdown of the chemokine receptor CXCR2 (IL8RB) in fibroblasts alleviates both replicative senescence and oncogene-induced senescence (OIS) and reduces the DNA damage response (Non-Patent Document 1). However, the effects of inhibiting signal transduction from CXCL5 on stem cells are unknown, as are the effects of knockdown of CXCR2 on stem cells. On the other hand, it has been reported that knockdown of IL-8, a ligand of CXCR2, induces cellular senescence in mesenchymal stem cells (Non-Patent Document 2).

[0008] Surprisingly, the present inventors have found that adding a substance that inhibits signal transduction from CXCL5 to a culture medium can suppress cellular senescence of tissue stem cells and increase the total passage number of the tissue stem cells. Even more surprisingly, they have found that tissue stem cells that have been repeatedly passaged in a medium containing a substance that inhibits signal transduction from CXCL5 still retain their differentiation potential. Based on this finding, the present inventors have completed the present invention.

[0009] Specific embodiments of the present invention are exemplified below.

[0010] [Item 1] An agent for suppressing cellular senescence of tissue stem cells, comprising a substance that inhibits signal transduction from CXCL5. [Item 2] The agent of Item 1, wherein the substance that inhibits signal transduction from CXCL5 is an anti-CXCL5 antibody or a fragment thereof, an anti-CXCR2 antibody or a fragment thereof, or a CXCR2 antagonist. [Item 3] The agent of Item 1 or 2, wherein the substance that inhibits signal transduction from CXCL5 is an anti-CXCL5 antibody or a fragment thereof, or a CXCR2 antagonist. [Item 4] The agent of Item 2 or 3, wherein the CXCR2 antagonist is SB225002 or SB265610. [Item 5] The agent of any one of Items 1 to 4, wherein the substance that inhibits signal transduction from CXCL5 is an anti-CXCL5 antibody or a fragment thereof. [Item 6] The agent according to any one of Items 1 to 5, wherein the agent is for suppressing cellular senescence of tissue stem cells in vitro or ex vivo. [Item 7] The agent according to any one of Items 1 to 6, wherein the agent is to be added to a culture medium for tissue stem cells. [Item 8] A culture medium for tissue stem cells, comprising the agent according to any one of Items 1 to 7. [Item 9] A culture medium for tissue stem cells, comprising a substance that inhibits signal transduction from CXCL5. [Item 10] The culture medium according to Item 9, wherein the substance that inhibits signal transduction from CXCL5 is an anti-CXCL5 antibody or a fragment thereof, an anti-CXCR2 antibody or a fragment thereof, or a CXCR2 antagonist. [Item 11] The culture medium according to Item 9 or 10, wherein the substance that inhibits signal transduction from CXCL5 is an anti-CXCL5 antibody or a fragment thereof, or a CXCR2 antagonist. [Item 12] The medium according to Item 10 or 11, wherein the CXCR2 antagonist is SB225002 or SB265610. [Item 13] The medium according to any one of Items 9 to 12, wherein the substance that inhibits signaling from CXCL5 is an anti-CXCL5 antibody or a fragment thereof. [Item 14] A method for culturing tissue stem cells, comprising the step of culturing tissue stem cells in the medium according to any one of Items 8 to 13. [Item 15] A method for suppressing cellular senescence of tissue stem cells, comprising the step of contacting tissue stem cells with a substance that inhibits signaling from CXCL5.[Item 16] A method for producing a pharmaceutical containing tissue stem cells, comprising the step of contacting tissue stem cells with a substance that inhibits signal transduction from CXCL5. [Item 17] A substance that inhibits signal transduction from CXCL5, for use in suppressing cellular senescence of tissue stem cells. [Item 18] Use of a substance that inhibits signal transduction from CXCL5 for suppressing cellular senescence of tissue stem cells.

[0011] According to the present invention, cellular senescence of tissue stem cells can be suppressed.

[0012] 1 is a graph showing the relationship between antibody concentration and maximum fluorescence intensity in Test Example 1.

[0034] FIG. 1 is a graph showing the ND50 value of each antibody in Test Example 1.

[0035] FIG. 1 is a graph showing the number of days required for hBMSCs to become semi-confluent at each passage when a CXCL5 antibody (FCX0803) is added.

[0036] FIG. 1 is a graph showing the number of days required for hBMSCs to become semi-confluent at each passage when a CXCL5 antibody (FCX0511) is added.

[0037] FIG. 1 is a graph showing the number of days required for hBMSCs to become semi-confluent at each passage when a CXCL5 antibody (Mab254) is added.

[0038] FIG. 1 is a graph showing the number of days required for hBMSCs to become semi-confluent at each passage when a CXCR2 antagonist (SB225002) is added.

[0039] FIG. 2 is a graph showing the number of days required for hBMSCs to become semi-confluent at each passage when a CXCR2 antagonist (SB265610) is added. 1 is a graph showing the number of days required for hAMSCs to become semi-confluent at each passage when a CXCL5 antibody (FCX0511) is added. 2 is a graph showing the number of days required for hAMSCs to become semi-confluent at each passage when a CXCR2 antagonist (SB225002) is added. 3 is a graph showing the number of days required for hAMSCs to become semi-confluent at each passage when a CXCR2 antagonist (SB265610) is added. 4 is a graph showing the cell proliferation rate of hHSCs at each passage when a CXCL5 antibody (FCX0511) is added. 5 is a graph showing the cell proliferation rate of hHSCs at each passage when a CXCR2 antagonist (SB225002) is added. 6 is a graph showing the cell proliferation rate of hHSCs at each passage when a CXCR2 antagonist (SB265610) is added. Graphs showing the expression levels of senescence-associated marker genes (P16 and P21) in hBMSCs at each passage when CXCL5 antibodies (FCX0511 or FCX0803) were added.Graph showing the expression level of the senescence-associated marker gene (P53) in hBMSCs at each passage when a CXCL5 antibody (FCX0511 or FCX0803) was added. Graph showing the expression level of the senescence-associated marker gene (IL-6) in hBMSCs at each passage when a CXCL5 antibody (FCX0511 or FCX0803) was added. Graph showing the expression level of the senescence-associated marker gene (IL-8) in hBMSCs at each passage when a CXCL5 antibody (FCX0511 or FCX0803) was added. Graph showing the expression level of the senescence-associated marker gene (TNF-α) in hBMSCs at each passage when a CXCL5 antibody (FCX0511 or FCX0803) was added. Graph showing the expression level of the senescence-associated marker gene (PAI-1) in hBMSCs at each passage when a CXCL5 antibody (FCX0511 or FCX0803) was added. 1 is a graph showing SA-β-gal activity in hBMSCs at passage 10 when a CXCL5 antibody (FCX0511 or FCX0803) is added. 2 is a graph showing the differentiation ability of hBMSCs at passage 10 into adipocytes when a CXCL5 antibody (FCX0511 or FCX0803) is added. 3 shows that hBMSCs at passage 10 differentiated into chondrocytes when a CXCL5 antibody (FCX0511 or FCX0803) was added.

[0013] In one aspect, the present disclosure provides an agent (sometimes referred to as the "agent of the present invention") containing a substance that inhibits signal transduction from CXCL5. The agent of the present invention can be used to suppress cellular senescence in tissue stem cells. The agent of the present invention can be used, for example, as a raw material for producing a culture medium, or as a supplement that is appropriately added to a basal culture medium for tissue stem cells. The agent of the present invention can be used as an agent for suppressing cellular senescence in tissue stem cells, as an additive to a pharmaceutical containing tissue stem cells. The agent of the present invention may be the substance that inhibits signal transduction from CXCL5 itself, or may contain the substance that inhibits signal transduction from CXCL5 in an amount of, for example, 0.1 to 99% by weight, or 0.1 to 85% by weight, based on the total weight of the agent. The form of the agent of the present invention is not particularly limited, and can be, for example, a liquid mixed with an appropriate solvent (e.g., water, buffer solution), or a lyophilized solid. The agent of the present invention may contain known additives, such as culture medium components, as long as they do not adversely affect tissue stem cells.

[0014] In the present disclosure, cellular senescence refers to the persistent arrest of cell proliferation induced by DNA damage, activation of oncogenes, stress, and the like.

[0015] In the present disclosure, examples of substances that inhibit signal transduction from CXCL5 (hereinafter sometimes referred to as "substances of the present invention") include substances that inhibit the interaction between CXCL5 and CXCR2. Examples of substances that inhibit the interaction between CXCL5 and CXCR2 include anti-CXCL5 antibodies or fragments thereof, anti-CXCR2 antibodies or fragments thereof, and CXCR2 antagonists. These can be used alone or in combination of two or more.

[0016] In the present disclosure, an anti-CXCL5 antibody refers to an antibody that inhibits the interaction between CXCL5 and CXCR2 by binding to CXCL5. In the present disclosure, an anti-CXCL5 antibody may also be referred to as a CXCL5 neutralizing antibody or a CXCL antibody. In the present disclosure, a fragment of an anti-CXCL5 antibody refers to a fragment derived from an anti-CXCL5 antibody that inhibits the interaction between CXCL5 and CXCR2 by binding to CXCL5. Alternatively, if an anti-CXCL5 antibody or a fragment thereof has the effect of suppressing cellular senescence of tissue stem cells, it can be used as a substance that inhibits signal transduction from CXCL5 in the present disclosure.

[0017] In the present disclosure, an anti-CXCR2 antibody refers to an antibody that inhibits the interaction between CXCL5 and CXCR2 by binding to CXCR2. In the present disclosure, an anti-CXCR2 antibody may also be referred to as a CXCR2 neutralizing antibody. In the present disclosure, a fragment of an anti-CXCR2 antibody refers to a fragment derived from an anti-CXCR2 antibody that inhibits the interaction between CXCL5 and CXCR2 by binding to CXCR2. Alternatively, if an anti-CXCR2 antibody or a fragment thereof has the effect of suppressing cellular senescence of tissue stem cells, it can be used as a substance that inhibits signal transduction from CXCL5 in the present disclosure.

[0018] Known anti-CXCL5 antibodies and anti-CXCR2 antibodies can be used. Commercially available anti-CXCL5 antibodies and anti-CXCR2 antibodies typically inhibit the interaction between CXCL5 and CXCR2, and therefore, commercially available anti-CXCL5 antibodies and anti-CXCR2 antibodies can be used in the present invention. Examples of anti-CXCL5 antibodies include, but are not limited to, anti-CXCL5 antibodies (Abcam, R and D systems, LifeSpan Biosciences, ThermoFisher SCIENTIFIC), such as Mab254 (sold by R&D systems). Examples of anti-CXCR2 antibodies include, but are not limited to, Anti-CXCR2 antibody (Abcam), Human CXCR2 / IL-8RB Mab (R and D systems), Mouse Monoclonal CXCR2 / IL-8RB Antibody (Novuss Biologicals), and CXCR2 / IL8RB antibody (ThermoFisher Scientific).

[0019] Anti-CXCL5 antibodies may be prepared by standard methods using CXCL5 or a fragment thereof as an antigen. Anti-CXCR2 antibodies may be prepared by standard methods using CXCR2 or a fragment thereof as an antigen.

[0020] The antibodies that can be used in the present invention are preferably isolated antibodies. Furthermore, the antibodies that can be used in the present invention may be either polyclonal or monoclonal antibodies, and can be prepared by conventional methods.

[0021] Antibody fragments that can be used in the present invention include, for example, antibody fragments capable of specifically binding to an antigen, such as Fab (fragment of antigen binding), F(ab')2, Fab', Fv, scFv (single chain Fv), dsFv (disulfide stabilized Fv), and CDR (complementarity determining region). Human chimeric antibodies, humanized antibodies, and the like can also be used. The antibody fragments can be produced by methods known in the art.

[0022] In the present disclosure, the CXCR2 antagonist may be, for example, a substance that inhibits the interaction between CXCL5 and CXCR2 by binding to CXCR2 in competition with CXCL5, but does not itself have the ability to activate CXCR2. Alternatively, if the CXCR2 antagonist has the effect of suppressing cellular senescence of tissue stem cells, it can be used as a substance that inhibits signal transduction from CXCL5 in the present disclosure.

[0023] Known CXCR2 antagonists can be used. Examples of CXCR2 antagonists include SB225002 (1-(2-bromophenyl)-3-(2-hydroxy-4-nitrophenyl)urea; CAS Number: 182498-32-4), SB265610 (1-(2-bromophenyl)-3-(7-cyano-2H-benzotriazol-4-yl)urea; CAS Number: 211096-49-0), reparixin ((2R)-2-[4-(2-methylpropyl)phenyl]-N-methylsulfonylpropanamide; CAS Number: 266359-83-5), and ladarixin (methylsulfonyl-[(2R)-2-[4-(trifluoromethylsulfonyloxy)phenyl]propanoyl]azanide; CAS Number: 865625-56-5), Navarixin (2-hydroxy-N,N-dimethyl-3-[[2-[[(1R)-1-(5-methylfuran-2-yl)butyl]amino]-3,4-dioxocyclobuten-1-yl]methyl]benzamide;hydrate; CAS Number: 862464-58-2), Danirixin (1-[4-chloro-2-hydroxy-3-[(3S)-piperidin-3-yl]sulfonylphenyl]-3-(3-fluoro-2-methylphenyl)urea; CAS Number: 954126-98-8), Vimnerixin (N-[6-[(2R,3S)-3,4-dihydroxybutan-2-yl]oxy-2-[(4-fluorophenyl)methylsulfanyl]pyrimidin-4-yl]-3-methylazetidine-1-sulfonamide;CAS Number: 1418112-77-2)、AZD-5069 (N-[2-[(2,3-difluorophenyl)methylsulfanyl]-6-[(2R,3S)-3,4-dihydroxybutan-2-yl]oxypyrimidin-4-yl]azetidine-1-sulfonamide; CAS Number: 878385-84-3)、AZD-8309 (5-[(2,3-difluorophenyl)methylsulfanyl]-7-[[(2R)-1-hydroxypropan-2-yl]amino]-3H-[1,3]thiazolo[4,5-d]pyrimidin-2-one; CAS Number: 333742-48-6)、AZ-10397767 (5-[(3-chloro-2-fluorophenyl)methylsulfanyl]-7-[[(2R)-1-hydroxypropan-2-yl]amino]-3H-[1,3]thiazolo[4,5-d]pyrimidin-2-one; CAS Number: 333742-63-5)、SB-656933 (1-(2-chloro-3-fluorophenyl)-3-(4-chloro-2-hydroxy-3-piperazin-1-ylsulfonylphenyl)urea; CAS Number: 688763-64-6)、SB-332235 (1-(4-chloro-2-hydroxy-3-sulfamoylphenyl)-3-(2,3-dichlorophenyl)urea; CAS Number: 276702-15-9)、SCH-527123 (2-hydroxy-N,N-dimethyl-3-[[2-[[(1R)-1-(5-methylfuran-2-yl)propyl]amino]-3,4-dioxocyclobuten-1-yl]amino]benzamide; CAS Number: 473727-83-2)、SX-682 ([2-[[5-[(4-fluorophenyl)carbamoyl]pyrimidin-2-yl]sulfanylmethyl]-4-(trifluoromethoxy)phenyl]boronic acid;CAS Number: 1648843-04-2);

[0024] In some embodiments, the CXCR2 antagonist is SB225002 or SB265610.

[0025] Whether an anti-CXCL5 antibody or a fragment thereof, an anti-CXCR2 antibody or a fragment thereof, or a CXCR2 antagonist has the effect of inhibiting the interaction between CXCL5 and CXCR2 can be evaluated by a conventional method, for example, by the Ready-to-Assay method (Eurofins) or the Hunter eXpress method (Eurofins DiscoverX).

[0026] Whether an anti-CXCL5 antibody or a fragment thereof, an anti-CXCR2 antibody or a fragment thereof, or a CXCR2 antagonist has the effect of suppressing cellular senescence in tissue stem cells can be evaluated by a conventional method, for example, by improving the cell proliferation ability of tissue stem cells, reducing the expression level of senescence-related marker genes, suppressing the activation of senescence-associated β-galactosidase (SA-β-gal), etc., according to the methods described in the Examples of the present application.

[0027] In some embodiments, inhibiting cellular senescence of tissue stem cells comprises increasing the total number of passages of the tissue stem cells.

[0028] In some embodiments, the inhibition of cellular senescence of tissue stem cells comprises reducing the expression level of a senescence-associated marker gene, for example, at least one senescence-associated marker gene selected from the group consisting of P16, P21, P53, IL-6, IL-8, TNF-α, and PAI-1.

[0029] In some embodiments, the inhibition of cellular senescence of tissue stem cells comprises the inhibition of senescence-associated β-galactosidase (SA-β-gal) activation.

[0030] In the present disclosure, tissue stem cells refer to stem cells that have the self-renewal ability and pluripotency necessary for the maintenance and regeneration of the tissue of origin. Examples of tissue stem cells include hematopoietic stem cells and mesenchymal stem cells.

[0031] In some embodiments, the tissue stem cells are hematopoietic stem cells or mesenchymal stem cells.

[0032] Examples of mesenchymal stem cells include bone marrow-derived mesenchymal stem cells, umbilical cord tissue-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, and adipose tissue-derived mesenchymal stem cells.

[0033] In some embodiments, the tissue stem cells are hematopoietic stem cells, bone marrow-derived mesenchymal stem cells, or adipose tissue-derived mesenchymal stem cells.

[0034] In some embodiments, the substance of the present invention is used to inhibit cellular senescence of in vitro or ex vivo tissue stem cells. In the present disclosure, in vitro tissue stem cells may be, for example, cells in cell culture. In the present disclosure, ex vivo tissue stem cells may be, for example, a part of a tissue sample removed from an organism (e.g., a mammal).

[0035] In some embodiments, the substances of the present invention are used to inhibit cellular senescence of tissue stem cells in vitro, for example, in tissue stem cell culture media (e.g., culture solutions).

[0036] In some embodiments, the substance of the present invention is used to inhibit cellular senescence of tissue stem cells ex vivo. For example, in the production of pharmaceuticals using tissue stem cells, tissue (e.g., bone marrow fluid, blood, adipose tissue) is collected from a subject (e.g., a patient), tissue stem cells are isolated, and then mass-cultured to produce the pharmaceuticals. Adding the substance of the present invention to a culture medium used for washing, suspending, preserving, etc., of tissue collected from a subject (e.g., a patient) can inhibit cellular senescence of tissue stem cells contained in the tissue.

[0037] In one aspect, the present disclosure provides a culture medium for tissue stem cells (hereinafter, sometimes referred to as the "culture medium of the present invention") containing a substance of the present invention. By culturing tissue stem cells in the culture medium of the present invention, cellular senescence of tissue stem cells can be suppressed, making it possible to repeat subculture more frequently. The substance of the present invention can suppress the decrease in proliferation rate of tissue stem cells due to cellular senescence, making it possible to proliferate tissue stem cells more efficiently. Furthermore, because tissue stem cells with suppressed cellular senescence can be produced, the quality of pharmaceuticals containing tissue stem cells can be improved or stabilized. In addition to being used for culturing tissue stem cells, the culture medium of the present invention can also be used as a washing solution, suspension, or preservation solution.

[0038] The form of the medium of the present invention is not particularly limited, and may be selected appropriately depending on the intended use of the medium, such as a solid medium (e.g., agar medium), a liquid medium, or a powdered medium (e.g., dissolved in water and used as a liquid medium), and can be produced using conventional methods. As with the agent of the present invention described above, the medium of the present invention may contain known additives as medium components in known concentration ranges, as long as they do not adversely affect tissue stem cells. For example, the medium of the present invention can be produced by adding the substance of the present invention to a known medium known for use in tissue stem cells (e.g., Human Mesenchymal Stem Cell Growth Medium 2, Mesenchymal Stem Cell Growth Medium XF, Hematopoietic Progenitor Cell Expansion Medium XF, all manufactured by PromoCell).

[0039] The final concentration of the substance of the present invention contained in the medium of the present invention (i.e., the concentration of the substance of the present invention contained in the medium used for contact with tissue stem cells) can be appropriately selected depending on the form of the medium, the type of substance of the present invention, the type of tissue stem cells, etc. When the substance of the present invention is an antibody or a fragment thereof, for example, the final concentration in the medium can be 0.0001 to 10 μg / mL (e.g., 0.001 to 7 μg / mL, 0.001 to 6 μg / mL, 0.003 to 5 μg / mL). When the substance of the present invention is a CXCR2 antagonist, for example, the final concentration in the medium can be 0.0001 to 10 nM (e.g., 0.001 to 5 nM, 0.002 to 3 nM, 0.003 to 1 nM).

[0040] In one embodiment, when the tissue stem cells are human bone marrow-derived mesenchymal stem cells, the final concentration of the anti-CXCL5 antibody (the concentration of the anti-CXCL5 antibody contained in the culture medium used for contact with the tissue stem cells) is, for example, 0.003 to 7 μg / mL, more preferably 0.01 to 6 μg / mL, and even more preferably 0.3 to 5 μg / mL.

[0041] In one embodiment, when the tissue stem cells are human bone marrow-derived mesenchymal stem cells, the final concentration of the CXCR2 antagonist (SB225002) (the concentration of the CXCR2 antagonist (SB225002) contained in the culture medium used for contact with the tissue stem cells) is, for example, 0.001 to 0.05 nM, more preferably 0.001 to 0.03 nM.

[0042] In one embodiment, when the tissue stem cells are human bone marrow-derived mesenchymal stem cells, the final concentration of the CXCR2 antagonist (SB265610) (the concentration of the CXCR2 antagonist (SB265610) contained in the culture medium used for contact with the tissue stem cells) is, for example, 0.001 to 0.02 nM, more preferably 0.001 to 0.01 nM, and even more preferably 0.002 to 0.01 nM.

[0043] In one embodiment, when the tissue stem cells are human adipose-derived mesenchymal stem cells, the final concentration of the anti-CXCL5 antibody (the concentration of the anti-CXCL5 antibody contained in the culture medium used for contact with the tissue stem cells) is, for example, 0.003 to 7 μg / mL, more preferably 0.01 to 6 μg / mL, and even more preferably 0.01 to 5 μg / mL.

[0044] In one embodiment, when the tissue stem cells are human adipose-derived mesenchymal stem cells, the final concentration of the CXCR2 antagonist (SB225002) (the concentration of the CXCR2 antagonist (SB225002) contained in the culture medium used for contact with the tissue stem cells) is, for example, 0.001 to 0.05 nM, more preferably 0.001 to 0.03 nM.

[0045] In one embodiment, when the tissue stem cells are human adipose-derived mesenchymal stem cells, the final concentration of the CXCR2 antagonist (SB265610) (the concentration of the CXCR2 antagonist (SB265610) contained in the culture medium used for contact with the tissue stem cells) is, for example, 0.001 to 0.005 nM, more preferably 0.001 to 0.004 nM, and even more preferably 0.001 to 0.003 nM.

[0046] In one embodiment, when the tissue stem cells are human hematopoietic stem cells, the final concentration of the anti-CXCL5 antibody (the concentration of the anti-CXCL5 antibody contained in the culture medium used for contact with the tissue stem cells) is, for example, 0.003 to 7 μg / mL, 0.005 to 5 μg / mL, 0.007 to 3 μg / mL, or 0.01 to 1.5 μg / mL.

[0047] In one embodiment, when the tissue stem cells are human hematopoietic stem cells, the final concentration of the CXCR2 antagonist (SB225002) (the concentration of the CXCR2 antagonist (SB225002) contained in the culture medium used for contact with the tissue stem cells) is, for example, 0.003 to 7 nM, 0.005 to 5 nM, 0.007 to 3 nM, or 0.01 to 1.5 nM.

[0048] In one embodiment, when the tissue stem cells are human hematopoietic stem cells, the final concentration of the CXCR2 antagonist (SB265610) (the concentration of the CXCR2 antagonist (SB265610) contained in the culture medium used for contact with the tissue stem cells) is, for example, 0.003 to 2 nM, 0.005 to 1.5 nM, 0.007 to 1 nM, or 0.01 to 1 nM.

[0049] In the present disclosure, tissue stem cells can be cultured by any known culture method suitable for the type of tissue stem cells, except for using the medium of the present invention.

[0050] Generally, repeated subculture of tissue stem cells leads to cellular senescence and a decrease in cell proliferation ability, and therefore, for culture using the medium of the present invention, it is desirable to use tissue stem cells that have been subcultured a small number of times in a substance-free medium of the present invention, preferably 9 times or less, more preferably 5 times or less, and even more preferably 3 times or less.

[0051] In the present disclosure, tissue stem cells are, for example, tissue stem cells derived from mammals (humans and non-human mammals (e.g., cows, horses, pigs, dogs, cats, mice, rats, rabbits, and monkeys)), and more preferably tissue stem cells derived from humans.

[0052] In one aspect, the present disclosure provides a method for inhibiting cellular senescence of tissue stem cells, comprising the step of contacting the tissue stem cells with a substance of the present invention.

[0053] In one aspect, the present disclosure provides a method for producing a medicament containing tissue stem cells, comprising the step of contacting a substance of the present invention with tissue stem cells.

[0054] Methods for contacting the substance of the present invention with tissue stem cells include, for example, adding the substance of the present invention to a culture medium (culture solution) and culturing, preserving, washing, and / or suspending the tissue stem cells in the same medium.

[0055] In the present disclosure, pharmaceuticals containing tissue stem cells can be used in regenerative medicine, for example, for the treatment of intractable diseases (e.g., cancer, traumatic tissue injury, neurological diseases, liver dysfunction, kidney diseases, ischemic diseases, heart diseases, aplastic anemia, malignant blood diseases, etc.).

[0056] Test Example 1. Creation of CXCL5 Monoclonal Antibodies (1) Creation of Recombinant CXCL5 Protein The CXCL5 gene was artificially synthesized and inserted into the GST (Glutathione S-transferase) tag expression vector pGEX-4T-1. This plasmid was transformed into Escherichia coli BL21(DE3) pLysS (BioDynamics Laboratories) to express GST-CXCL5 protein. Soluble GST-CXCL5 was prepared by lysing the E. coli, recovering inclusion bodies, denaturing with guanidine, reducing with 2-mercaptoethanol, and refolding by dialysis. The GST tag was then cleaved with thrombin, and the recombinant CXCL5 protein (hereinafter referred to as rCXCL5) was purified sequentially using a cation exchange column, Hitrap SP (Cytiva), a gel filtration column, Sephacryl S100 (GE Healthcare), and a cation exchange column, Hitrap SP (Cytiva), all connected to an AKTA explorer 10S.

[0057] (2) Establishment of CXCL5 monoclonal antibody-producing hybridomas. a. Immunization of mice. Six-week-old BALB / c female mice (CLEA Japan) were subcutaneously inoculated with the emulsion (approximately 100 μg / 100 μL / mouse) of rCXCL5 (Fuso Pharmaceutical Industries) obtained above and TiterMax Gold (TiterMax USA) as adjuvant. Booster inoculations were performed every two weeks, and three days before splenocyte collection, rCXCL5 (Fuso Pharmaceutical Industries) diluted in PBS (30 μg / 100 μL / mouse) was intraperitoneally inoculated. b. Generation of mouse hybridomas. Splenocytes from rCXCL5-immunized mice were fused with mouse myeloma cells, and HAT selection and cloning of the fused cells were performed using the ClonaCell-HY cloning kit (Veritas) according to the kit's instructions. A summary of the procedure is provided below. 1) One week before cell fusion: Pre-culture of mouse myeloma cells (SP2 cells) was started (37°C, 5% CO2). 2) On the day of cell fusion: The immunized mice were subjected to abdominal sectioning, and splenocytes were collected from the spleen. 2 x 10 7 1 x 10 SP2 cells and 1 x 10 8Splenocytes were fused using polyethylene glycol and cultured statically at 37°C, 5% CO2. 3) Cloning and HAT Selection: The fused cells were collected, resuspended in selective medium, and placed in a 10-cm dish for static culture at 37°C, 5% CO2. 4) Harvesting: Colony formation began to appear around 10 days after culture in selective medium, and individual colonies were harvested into a 96-well culture plate containing growth medium at 37°C, 5% CO2. 5) Screening: After 3-4 days of culture, the culture supernatant was collected and used for screening. 100 μL of hybridoma culture supernatant, diluted 2-fold with diluent (PBS / 0.5% BSA / 0.05% Tween 20), was added to a 96-well plate coated with rCXCL5 and incubated. Anti-mouse IgG-HRP (betyl) was used as the secondary antibody. TMB substrate (SeraCare) was added, and the absorbance (450 nm) was measured. Clones with an absorbance of 2.0 or higher were considered positive. 6) Recloning of Positive Clones: Positive clones were resuspended in selective medium, and the cloning, HAT selection, and screening procedures were repeated twice as described above. The third screening was performed using a 96-well plate coated with rCXCL5 (R&D Systems), and the reaction to rCXCL5 (R&D Systems) was confirmed. Positive clones were identified as CXCL5 antibody-producing hybridomas. 7) Purification of Monoclonal Antibodies: The two hybridoma cell lines obtained above, FCX0511 and FCX0803, were adapted to Hybridoma SFM serum-free medium (Gibco) and cultured for 5 days. The IgG fraction was purified from the culture supernatant using an affinity column, Hitrap Protein G (Cytiva), to obtain monoclonal antibodies CXCL5 (FCX0511) and CXCL5 (FCX0803).

[0058] (3) Evaluation of the neutralizing activity of monoclonal antibodies. Using Ready-to-Assay® CXCR2 Chemokine Receptor Frozen Cells (Eurofins), the 50% neutralizing dose (the antibody concentration required to neutralize 50% of CXCL5 activity, hereafter referred to as ND50) of the CXCL5 antibodies (FCX0511, FCX0803) obtained above and the commercially available CXCL5 antibody Mab254 (R & D Systems) was measured. A normal mouse antibody, mouse IgG (ZYMED), was used as a control. The following is an overview. 1) Cell thawing and culturing. After thawing the CXCR2-expressing Chem-1 cells provided with the kit, the cell pellet was suspended in the medium provided with the kit. The cell suspension was seeded (100 μL / well) into an assay plate (96-well clear-bottom black plate, Corning), incubated at room temperature for 30 minutes, and then cultured in a CO2 incubator (37°C, 5% CO2) for 24 hours. 2) Preparation of Loading Buffer and Recording Buffer. The following day, loading buffer and recording buffer (x1) were prepared using the reagents provided with the Calcium kit-Fluo 4 (Dojindo Laboratories) according to the kit's instructions. The prepared solutions were warmed to 37°C until use. 3) Loading of Cells with Fluo-4-AM. After 24 hours of culture, the medium was removed from the assay plate, and the cells were washed with warmed PBS. 100 μL / well of loading buffer was added to the assay plate, and the plate was incubated at 37°C for 1 hour. The loading buffer was removed from the assay plate, washed with warmed PBS, and 100 μL / well of recording buffer (x1) was added to the assay plate. 4) Preparation of antigen-antibody solution One hour before adding recording buffer (x1) to the assay plate, the antibody was serially diluted with recording buffer (x1) using a sample plate (ProteoSave SS Plate 96U, Sumitomo Bakelite) to prepare antibody solutions (5.0-160.0 μg / mL). As a control, wells containing recording buffer only were also prepared.Next, an equal volume of CXCL5 (BioLegend) diluted to 8.0 μg / mL in recording buffer was added to the antibody solution. The sample plate was incubated at room temperature for 1 hour to prepare the antigen-antibody solution. 5) Measurement: The assay plate and sample plate were placed in a microplate reader with an autoinjector (FlexStation 3, Molecular Devices), and the fluorescence intensity of the assay plate (excitation wavelength: 495 nm, emission wavelength: 518 nm) was measured at 5-second intervals for 120 seconds. 25 seconds after the start of measurement, 10 μL of the antigen-antibody solution taken from the sample plate was added to the assay plate at a rate of 2 μL / second. This procedure was repeated for every 8 wells. 6) Data Analysis: Fluorescence intensity was baseline-corrected using the values ​​measured before the addition of the antigen-antibody solution. Figure 1 shows the relationship between antibody concentration and maximum fluorescence intensity for each antibody. In addition, the ND50 value for each antibody was calculated, defining the antibody concentration required for 50% inhibition as ND50, where the fluorescence intensity in a well containing only recording buffer (no antibody added) was set at 100 (Figure 2). As shown in Figures 1 and 2, the CXCL5 antibodies FCX0511 (Fuso Pharmaceutical Industries), FCX0803 (Fuso Pharmaceutical Industries), and Mab254 (R&D Systems) were confirmed to have neutralizing activity.

[0059] Test Example 2: Evaluation of the Effect of Inhibition of CXCL5 Signaling on Cell Proliferation Potential We evaluated whether cellular senescence of tissue stem cells can be suppressed by neutralizing CXCL5 or inhibiting its receptor, CXCR2, through the control of CXCL5 signaling. Culture experiments using various tissue stem cells were performed to evaluate the efficacy of CXCL5 signaling. Human bone marrow-derived mesenchymal stem cells (hBMSCs, human bone-marrow mesenchymal stem cells, Promocell), human adipose-derived mesenchymal stem cells (hAMSCs, human adipose-derived mesenchymal stem cells, Promocell), and human hematopoietic stem cells (hHSCs, human hematopoietic stem cells, Promocell) were used. CXCL5 neutralizing antibodies FCX0803, FCX0511, or Mab254 were used, and CXCR2 antagonists SB225002 and SB265610 (Cayman Chemical) were used. The ability of CXCL5 signaling inhibition to inhibit cell proliferation and cell senescence was evaluated by assessing cell proliferation and measuring gene expression levels of senescence-related markers. Unless otherwise specified, the "control group" refers to the group to which neither CXCL5 antibody nor CXCR2 antagonist was added.

[0060] Test Example 2-(a) Evaluation of cell proliferation ability of hBMSCs hBMSCs at passage 3 were plated on a 96-well plate at 2 × 10 4 Cells were seeded at a concentration of 1000 cells / mL and culture was initiated (37°C, 5% CO2). The culture medium was changed every 48 hours, and culture was continued until the cells reached semi-confluence. The cells were divided into a control group, a CXCL5 antibody (FCX0803, FCX0511, Mab254)-added group, and a CXCR2 antagonist-added group, and the number of days required to reach semi-confluence from the start of culture at each passage and the total number of passages were compared. Cells were cultured in three wells in each group. Cells were detached from semi-confluent wells using Accutase, and the cell number was counted using a Countess II. After that, 2 x 10 4The cells were passaged at a concentration of 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, and 5.0 μg / mL. The CXCL5 antibody (FCX0803) was added at concentrations of 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, and 5.0 μg / mL. The CXCL5 antibody (FCX0511) was added at concentrations of 0.03, 0.1, 0.3, 1.0, 3.0, and 5.0 μg / mL. The CXCL5 antibody (Mab254) was added at concentrations of 1.0, 3.0, and 10.0 μg / mL. The CXCR2 antagonists were added at concentrations of 0.003, 0.01, 0.03, 0.1, 0.3, and 1.0 nM. The results are shown in Tables 1 to 5 and Figures 3 to 7. The number of days in the tables is the average value. A "-" in the tables indicates that cell proliferation to semi-confluence was not observed. As shown in Tables 1 to 5 and Figures 3 to 7, it was suggested that the addition of CXCL5 antibody or CXCR2 antagonist increased the total passage number of hBMSCs.

[0061]

[0062] Test Example 2-(b) Evaluation of cell proliferation ability of hAMSCs hAMSCs at passage 3 were plated on a 96-well plate at 2 × 10 4 Cells were seeded at a concentration of 1000 cells / mL and culture was initiated (37°C, 5% CO2). The culture medium was changed every 48 hours, and culture was continued until the cells reached semi-confluence. The cells were divided into a control group, a CXCL5 antibody (FCX0511)-added group, and a CXCR2 antagonist-added group, and the number of days required to reach semi-confluence from the start of culture at each passage, as well as the total number of passages, were compared. Cells were cultured in three wells in each group. Cells were detached from semi-confluent wells using Accutase, and the cell number was counted using a Countess II. After that, 2 x 10 4The cells were passaged at a concentration of 0.003, 0.01, 0.03, 0.1, 0.3, and 1.0 μg / mL. Cultures were continued until no cell proliferation was observed. All groups were cultured three times. In the antibody-added group, a CXCL5 antibody (FCX0511) was added at concentrations of 0.003, 0.01, 0.03, 0.1, 0.3, and 1.0 μg / mL. In the CXCR2 antagonist-added group, both antagonists were added at concentrations of 0.003, 0.01, 0.03, 0.1, 0.3, and 1.0 nM. The results are shown in Tables 6–8 and Figures 8–10. A "-" in the table indicates that cell proliferation to semiconfluence was not observed. As shown in Tables 6–8 and Figures 8–10, the addition of a CXCL5 antibody or a CXCR2 antagonist increased the total passage number of hAMSCs.

[0063]

[0064] Test Example 2-(c) Evaluation of cell proliferation ability of hHSCs Cryopreserved hHSCs were thawed and immediately plated in a 96-well plate at 1 × 10 4 Cells were seeded at a concentration of 1000 cells / mL and cultured at 37°C, 5% CO2. Every 48 hours, the cells were harvested along with the culture medium, centrifuged, the supernatant removed, and the cells were resuspended in fresh medium and seeded into 96-well plates for further culture. Cells were cultured in standard culture medium (HPC expansion medium XF: Promocell) for 28 days. Cell numbers were measured in each well and proliferation rates were compared. All cultures were performed in triplicate. In the antibody-treated group, the CXCL5 antibody (FCX0511) was added at concentrations of 0.01, 0.1, or 1.0 μg / mL. In the CXCR2 antagonist-treated group, both antagonists were added at concentrations of 0.01, 0.1, or 1.0 nM. The results are shown in Figures 11–13. As shown in Figures 11 to 13, it was suggested that the addition of a CXCL5 antibody or a CXCR2 antagonist increased the cell proliferation rate of hHSCs.

[0065] Experimental Example 3: Measurement of Senescence-Associated Marker Expression. For hBMSCs, cells at passages 4, 6, 8, and 10 from the CXCL5 antibody (FCX0511 or FCX0803)-treated groups cultured as described in Experimental Example 2-(a) were used as samples. Cells at the end of culture were used as samples to measure the expression of senescence-associated markers in each experimental group. 70 μL of Buffer RLT (QIAGEN) was added to each well to collect cell lysates. RNA was extracted from the collected lysates using an RNeasy Micro Kit (QIAGEN). cDNA was synthesized by reverse transcription of the extracted RNA using PrimeScript RT Master Mix (TaKaRa Bio). The expression levels of senescence marker genes (P16, P21, P53) and inflammatory marker genes (IL-6, IL-8, TNF-α, PAI-1) in the synthesized cDNA were measured using FastStart Essential DNA Green Master (Roche). All procedures were performed according to the kit instructions. The results are shown in Figures 14 to 20. As shown in Figures 14 to 20, it was suggested that the addition of CXCL5 antibody reduced the expression levels of senescence-related marker genes.

[0066] Test Example 4: Evaluation of Cellular Senescence by Senescence-Associated β-Galactosidase Staining. The degree of cellular senescence in hBMSC cells was compared by staining with senescence-associated β-galactosidase (SA-β-gal). The 10th-passage wells of each group containing CXCL5 antibodies (FCX0511 or FCX0803), cultured as described in Test Example 2-(a), were stained using the Cellular Senescence Plate Assay Kit-SPiDER-βGal (Dojindo Laboratories), and fluorescence intensity was measured using a Varioskan Flash microplate reader (ThermoFisher Scientific). For the control group, wells at passages 4 and 10 were measured. All procedures were performed according to the kit manual. The results are shown in Figure 21. As shown in Figure 21, a significant increase in SA-β-gal activity was observed at passage 10 in the non-additive group, but the increase was suppressed at passage 10 in the CXCL5 antibody-added group, suggesting that the addition of CXCL5 antibody suppresses cellular senescence of hBMSC cells.

[0067] Test Example 5: Evaluation of cell differentiation potential Whether or not cells that had been subcultured for a long period of time maintained their differentiation potential was evaluated by differentiating them into adipocytes and chondrocytes. 1) Differentiation into adipocytes hBMSCs were cultured in the same manner as in Test Example 2-(a) in the CXCL5 antibody (FCX0511 or FCX0803)-added and non-added groups, respectively. At the 10th passage, 2.0 x 10 cells were plated onto a 96-well plate coated with fibronectin at a concentration of 5.0 μg / mL. 5Cells were seeded at a concentration of 100 μL / mL. When the cell area reached 50-60% of the well, 100 μL of MSCgo Adipogenic XF medium (Sartorius Stedim Japan), an adipocyte differentiation medium, was added to wells and cultured for 14 days. The culture medium was changed every 48 hours, and on day 14, the lipid droplet-staining fluorescent dye Lipi-Green (Dojindo Laboratories) was added to each well. After 2 hours of culture, the cells were observed under a BZ-X800 (KEYENCE) fluorescence microscope. After observation, the fluorescence intensity was measured using a Varioskan Flash and compared between groups. The results are shown in Figure 22. As shown in Figure 22, the CXCL5 antibody-treated group retained the ability to differentiate into adipocytes even at passage 10. Furthermore, the CXCL5 antibody-treated group had a higher number of cells differentiating into adipocytes and significantly higher luminescence intensity compared to the control group. 2) Differentiation into chondrocytes hBMSCs were cultured in the CXCL5 antibody (FCX0511 or FCX0803)-treated group and the non-treated group (control) in the same manner as in Test Example 2-(a). At the 10th passage, 2.0 × 10 cells were plated onto a spheroid microplate (Corning). 5 Cells were seeded at a concentration of 100 μL / mL. After 48 hours of culture, 100 μL of chondrogenic differentiation medium, MSCgo Chondrogenic XF medium (Sartorius Stedim Japan), was added to the wells where hBMSC spheroids had formed, and the cells were cultured for 21 days. The culture medium was changed every 48 hours, and on day 21, the cells were fixed and the chondrocytes were stained with Alcian blue (Nacalai Tesque). The results are shown in Figure 23. As shown in Figure 23, the CXCL5 antibody-treated group retained the ability to differentiate into chondrocytes even at passage 10. In contrast, the non-treated group (control) showed lower spheroid formation ability than the CXCL5 antibody-treated group, even though differentiation induction was initiated using the same number of cells as the CXCL5 antibody-treated group. Since only a portion of the seeded cells formed spheroids, this suggests that the differentiation ability was reduced due to cell senescence.

[0068] The present invention can be used in cell culture for regenerative medicine using tissue stem cells.

Claims

1. An agent for suppressing cellular senescence of tissue stem cells, comprising a substance that inhibits signal transduction from CXCL5.

2. The agent according to claim 1, wherein the substance that inhibits signal transduction from CXCL5 is an anti-CXCL5 antibody or a fragment thereof, an anti-CXCR2 antibody or a fragment thereof, or a CXCR2 antagonist.

3. The agent according to claim 1 or 2, wherein the substance that inhibits signal transduction from CXCL5 is an anti-CXCL5 antibody or a fragment thereof, or a CXCR2 antagonist.

4. The agent according to claim 2 or 3, wherein the CXCR2 antagonist is SB225002 or SB265610.

5. The agent according to any one of claims 1 to 4, wherein the substance that inhibits signal transduction from CXCL5 is an anti-CXCL5 antibody or a fragment thereof.

6. The agent according to any one of claims 1 to 5, which is an agent for suppressing cellular senescence of tissue stem cells in vitro or ex vivo.

7. The agent according to any one of claims 1 to 6, which is used by adding to a culture medium for tissue stem cells.

8. A culture medium for tissue stem cells containing the agent according to any one of claims 1 to 7.

9. A culture medium for tissue stem cells containing a substance that inhibits signal transduction from CXCL5.

10. The medium according to claim 9, wherein the substance that inhibits signal transduction from CXCL5 is an anti-CXCL5 antibody or a fragment thereof, an anti-CXCR2 antibody or a fragment thereof, or a CXCR2 antagonist.

11. The medium according to claim 9 or 10, wherein the substance that inhibits signal transduction from CXCL5 is an anti-CXCL5 antibody or a fragment thereof, or a CXCR2 antagonist.

12. The medium of claim 10 or 11, wherein the CXCR2 antagonist is SB225002 or SB265610.

13. The medium according to any one of claims 9 to 12, wherein the substance that inhibits signal transduction from CXCL5 is an anti-CXCL5 antibody or a fragment thereof.

14. A method for culturing tissue stem cells, comprising the step of culturing tissue stem cells in the medium according to any one of claims 8 to 13.

15. A method for suppressing cellular senescence of tissue stem cells, comprising the step of contacting the tissue stem cells with a substance that inhibits signal transduction from CXCL5.

16. A method for producing a pharmaceutical containing tissue stem cells, comprising the step of contacting tissue stem cells with a substance that inhibits signal transduction from CXCL5.

17. A substance that inhibits signal transduction from CXCL5 for use in suppressing cellular senescence of tissue stem cells.

18. Use of a substance that inhibits signal transduction from CXCL5 to suppress cellular senescence of tissue stem cells.

Images