Mesenchymal stem cell with improved Anti-inflammatory and immune regulatory capabilities or conditioned medium thereof

Abstract

To provide a mesenchymal stem cell with improved anti-inflammatory and immune regulatory capabilities or a conditioned medium thereof.SOLUTION: A method for producing a mesenchymal stem cell with improved anti-inflammatory and immune regulatory capabilities or a conditioned medium thereof includes culturing a mesenchymal stem cell in the presence of a YAP / TAZ mechano-signal cascade inhibitor.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present application relates to mesenchymal stem cells or their culture supernatants having improved anti-inflammatory and immunoregulatory capabilities. [Background technology]

[0002] Mesenchymal stem cells (MSCs) have high self-proliferation ability and the ability to differentiate into bone, cartilage, fat, etc. Therefore, research into regenerative medicine, in which MSCs from the body are isolated and cultured and transplanted into patients with tissue loss diseases, has flourished. Meanwhile, it has been revealed that MSCs not only have the ability to differentiate into multiple tissues, but also have anti-inflammatory and immunoregulatory capabilities, and great expectations are being placed on them as a cell therapy for inflammatory immune diseases. In Japan, based on their anti-inflammatory and immunoregulatory capabilities, MSCs have been approved as a cell therapy for the treatment of acute graft-versus-host disease (GVHD) and traumatic spinal cord injury.

[0003] Furthermore, basic research has suggested that MSCs may be effective not only for these two diseases, but also for many other inflammatory immune diseases such as sepsis, enteritis, hepatitis, nephritis, and rheumatoid arthritis, raising hopes for clinical application. However, there is a large variation in the results of basic research and clinical trials using MSCs as a cell therapy, and there remain issues regarding the reproducibility and certainty of the therapeutic effect.

[0004] One possible solution to this problem would be to administer MSCs in a state that allows them to maximize their cellular properties. Methods that have been investigated include pre-treating MSCs with inflammatory cytokines such as IFNγ to enhance their immune regulatory ability before administering them. However, this type of priming only has a temporary effect and cannot be said to fundamentally control the properties of MSCs. In fact, even with this method of pre-priming with inflammatory cytokines, the therapeutic effect varies.

[0005] The differentiation fate of MSCs is strictly controlled by mechanosignals that convert and sense the extracellular microenvironment, such as the hardness of the field, into biochemical signals. For example, when MSCs are placed on a hard scaffold, F-actin expands, which is accompanied by the nuclear translocation of the transcriptional coactivators YAP / TAZ, promoting osteogenic differentiation. On the other hand, when placed on a soft scaffold, F-actin contracts and YAP / TAZ is exported from the nucleus, reducing its signaling activity, promoting a tendency toward adipose and chondrogenic differentiation (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Dupont et al., Nature. 2011 Jun 8; 474(7350): 179-83. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present application is to provide mesenchymal stem cells or their culture supernatants having improved anti-inflammatory and immunoregulatory capabilities. [Means for solving the problem]

[0008] The present application provides a method for producing mesenchymal stem cells or their culture supernatant having improved anti-inflammatory and immunoregulatory capabilities, comprising culturing mesenchymal stem cells in the presence of a YAP / TAZ mechano-signaling cascade inhibitor.

[0009] The present application also provides a composition comprising mesenchymal stem cells with improved anti-inflammatory and immunoregulatory capabilities, produced by the method of the present application, or a culture supernatant thereof.

[0010] The present application also provides a method for improving the anti-inflammatory and immunoregulatory capabilities of mesenchymal stem cells, comprising culturing the mesenchymal stem cells in the presence of a YAP / TAZ mechano-signaling cascade inhibitor.

[0011] The present application also provides a composition for producing mesenchymal stem cells with improved anti-inflammatory and immunoregulatory capabilities, comprising a YAP / TAZ mechano-signaling cascade inhibitor.

[0012] The present application also provides a kit for producing mesenchymal stem cells with improved anti-inflammatory and immunoregulatory capabilities, comprising a YAP / TAZ mechano-signaling cascade inhibitor and mesenchymal stem cells. Effect of the Invention

[0013] The present application provides a method for producing mesenchymal stem cells or a culture supernatant thereof with improved anti-inflammatory and immunoregulatory capabilities. [Brief description of the drawings]

[0014] [Figure 1] Schematic diagram showing the relationship between YAP / TAZ mechanosignaling and anti-inflammatory and immunoregulatory abilities in mesenchymal stem cells (MSCs). Mesenchymal stem cells sense and respond to the state of the extracellular microenvironment, such as field stiffness, through mechanosignals mediated by F-actin extension / contraction and nuclear translocation of the transcriptional co-regulatory factor YAP / TAZ. MSCs in a state of reduced YAP / TAZ mechanosignaling exhibit high anti-inflammatory and immunoregulatory abilities. [Diagram 2] IDO mRNA and TNFAIP6 mRNA expression in MSCs transfected with YAP siRNA and / or TAZ siRNA. The expression levels of IDO mRNA and TNFAIP6 mRNA were further enhanced by TNF-α stimulation. [Diagram 3] IDO mRNA and TNFAIP6 mRNA expression in MSCs transfected with YAP siRNA and / or TAZ siRNA. The expression levels of IDO mRNA and TNFAIP6 mRNA were further enhanced by IFN-γ stimulation. [Figure 4]IDO mRNA and TNFAIP6 mRNA expression in MSCs derived from iPS cells transfected with YAP siRNA and TAZ siRNA (YAP / TAZ siRNAs). The expression levels of IDO mRNA and TNFAIP6 mRNA were further increased by TNF-α stimulation. [Diagram 5] IDO and TNFAIP6 mRNA expression in TEAD siRNA-introduced MSCs. The expression levels of IDO and TNFAIP6 mRNA were further increased by TNF-α stimulation. The IDO and TNFAIP6 mRNA expressions in TEAD siRNA-introduced MSCs were comparable to those in MSCs transfected with YAP siRNA and TAZ siRNA. [Figure 6] IDO mRNA and TNFAIP6 mRNA expression in MSCs treated with the F-actin extension inhibitor Y27632 or Blebbist. The expression levels of IDO mRNA and TNFAIP6 mRNA were further increased by TNF-α stimulation. [Figure 7] Suppressive effect of culture supernatant (CM) of MSCs transfected with YAP / TAZ siRNAs on M1 macrophages. Culture supernatant (CM) of MSCs transfected with YAP / TAZ siRNAs reduced the abundance of CD86+ and CD80+ macrophages. [Figure 8] Suppressive effect of culture supernatant (CM) from MSCs transfected with YAP / TAZ siRNAs on M1 macrophages. Culture supernatant (CM) from MSCs transfected with YAP / TAZ siRNAs reduced the activity of M1 macrophages. [Figure 9] Schematic diagram of non-contact co-culture of YAP / TAZ siRNA-introduced MSCs and T cells. [Figure 10] Suppressive effect of YAP / TAZ siRNA-introduced MSCs on T cells. Co-culture with YAP / TAZ siRNA-introduced MSCs suppressed T cell activation induced by CD3 / CD28 stimulation. [Figure 11] Schematic diagram of non-contact co-culture of T cells and MSCs transfected with TEAD siRNA or YAP / TAZ siRNAs. [Figure 12] Suppressive effect of TEAD siRNA-introduced MSCs on T cells. Co-culture with TEAD siRNA-introduced MSCs suppressed T cell activation induced by CD3 / CD28 stimulation. [Figure 13] Protective effect of YAP / TAZ siRNA-introduced MSCs against septic shock. Administration of YAP / TAZ siRNA-introduced MSCs alleviated sepsis. [Figure 14] Protective effect of TEAD siRNA-introduced MSCs against colitis. Administration of TEAD siRNA-introduced MSCs suppressed weight loss in a mouse colitis model. [Figure 15] Protective effect of TEAD siRNA-introduced MSCs against colitis. Administration of TEAD siRNA-introduced MSCs suppressed colon shortening in a mouse colitis model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] In this disclosure, when a numerical value is accompanied by the term "about," it is intended to include a range of ±10% of that value. For example, "about 20" is intended to include "18-22." A range of numerical values ​​includes all values ​​between and at the endpoints. "About" in reference to a range applies to both endpoints of the range. Thus, for example, "about 20-30" is intended to include "18-33."

[0016] MESSEMBLING STEM CELLS HAVING IMPROVED ANTI-INFLAMMATORY AND IMMUNOREGULATING ABILITY OR CULTURE SURFACE THEREOF - Patent application The present application provides a method for producing mesenchymal stem cells or their culture supernatant having improved anti-inflammatory and immunoregulatory capabilities, comprising culturing mesenchymal stem cells in the presence of a YAP / TAZ mechano-signaling cascade inhibitor.

[0017] In the present application, there is no limitation on the animal from which the mesenchymal stem cells are derived, and examples thereof include mammals such as mice, rats, hamsters, guinea pigs, cows, horses, pigs, sheep, monkeys, orangutans, chimpanzees, dogs, cats, and humans, and are preferably primates, more preferably humans. Mesenchymal stem cells can be derived from, for example, bone marrow, fat, umbilical cord, nerves, gums, dental pulp, peripheral blood, skin, hair roots, muscle tissue, endometrium, placenta, or pluripotent stem cells. Mesenchymal stem cells can be isolated from the above tissues by known methods. For example, mesenchymal stem cells can be isolated from tissues such as bone marrow or fat by flow cytometry or FACS using a mesenchymal stem cell marker as an indicator. Commercially available mesenchymal stem cells may be used. Bone marrow-derived mesenchymal stem cells can be purchased, for example, from Lonza.

[0018] Methods for inducing mesenchymal stem cells from pluripotent stem cells are known, and any known method may be used. For example, the method of Kamiya et al. (Kamiya et al., 2020 Sneak Peek, http: / / dx.doi.org / 10.2139 / ssrn.3741231) can be used. Examples of pluripotent stem cells include, but are not limited to, induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos obtained by nuclear transfer (ntES cells), spermatogonial stem cells (GS cells), embryonic germ cells (EG cells), cultured fibroblasts, and pluripotent cells derived from bone marrow stem cells (Muse cells). The pluripotent stem cells are preferably ES cells or iPS cells. The pluripotent stem cells may be produced by a conventionally known method, or a commonly available cell line may be used.

[0019] In some embodiments, the mesenchymal stem cells are derived from bone marrow, adipose, embryonic stem cells or induced pluripotent stem cells. In some embodiments, the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells or adipose tissue-derived mesenchymal stem cells.

[0020] In the present application, mesenchymal stem cells with improved anti-inflammatory and immunoregulatory capabilities refer to mesenchymal stem cells with improved anti-inflammatory and immunoregulatory capabilities compared to mesenchymal stem cells before being cultured in the presence of a YAP / TAZ mechano-signaling cascade inhibitor. Mesenchymal stem cells with improved anti-inflammatory and immunoregulatory capabilities can be confirmed by measuring the expression levels of genes related to anti-inflammation and immunoregulation or genes related to the YAP / TAZ mechano-signaling cascade. Genes related to anti-inflammation and immunoregulation include, but are not limited to, the TNFAIP6 gene, the IDO gene, the IL-10 gene, the PGES gene, the COX-2 gene, the TGF-β gene, the IGFBP3 gene, the SDF-1 gene, the PDL-1 gene, the PDL-2 gene, and the IL-15 gene. Genes related to anti-inflammation and immunoregulation are, for example, the TNFAIP6 gene or the IDO gene. YAP / TAZ mechano-signal cascade-related genes include, but are not limited to, CTGF gene, CYR61 gene, and ANKDR gene, which are YAP / TAZ target genes. YAP / TAZ mechano-signal cascade-related genes are, for example, CTGF gene.

[0021] In one embodiment, the mesenchymal stem cells with improved anti-inflammatory and immunoregulatory capabilities have an increased expression level of the TNFAIP6 gene compared to mesenchymal stem cells before being cultured in the presence of a YAP / TAZ mechano-signaling cascade inhibitor. The mesenchymal stem cells with improved anti-inflammatory and immunoregulatory capabilities have, for example, 2- to 10-fold higher TNFAIP6 mRNA expression level compared to mesenchymal stem cells before being cultured in the presence of a YAP / TAZ mechano-signaling cascade inhibitor.

[0022] In one embodiment, the mesenchymal stem cells with improved anti-inflammatory and immunoregulatory capabilities have an increased expression level of IDO gene compared to mesenchymal stem cells before being cultured in the presence of a YAP / TAZ mechano-signaling cascade inhibitor. The mesenchymal stem cells with improved anti-inflammatory and immunoregulatory capabilities have, for example, 2- to 5-fold higher IDO mRNA expression level compared to mesenchymal stem cells before being cultured in the presence of a YAP / TAZ mechano-signaling cascade inhibitor.

[0023] In one embodiment, the mesenchymal stem cells with improved anti-inflammatory and immunoregulatory capabilities have a reduced expression level of the CTGF gene compared to the mesenchymal stem cells before being cultured in the presence of a YAP / TAZ mechano-signaling cascade inhibitor. The mesenchymal stem cells with improved anti-inflammatory and immunoregulatory capabilities have, for example, 10- to 100-fold higher CTGF mRNA expression level compared to the mesenchymal stem cells before being cultured in the presence of a YAP / TAZ mechano-signaling cascade inhibitor.

[0024] The expression of each gene can be confirmed by methods well known in the art. For example, the expression of mRNA can be confirmed by quantitative PCR (qPCR), RT-PCR, real-time RT-PCR, microarray, or Northern blotting, and the expression of protein can be confirmed by flow cytometry, ELISA, antibody immunoprecipitation, Western blotting, or mass spectrometry.

[0025] In the present application, the culture supernatant of mesenchymal stem cells with improved anti-inflammatory and immunoregulatory capabilities means a culture solution of mesenchymal stem cells with improved anti-inflammatory and immunoregulatory capabilities from which the mesenchymal stem cells have been removed. The culture supernatant can be collected by a method known to those skilled in the art. The culture supernatant can be collected, for example, by aspirating the culture solution with a dropper or pipette. Alternatively, the culture supernatant may be collected by centrifuging the culture solution containing mesenchymal stem cells and / or filtering it through a filter with an appropriate pore size.

[0026] The collected culture supernatant may be appropriately treated, for example, by centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, and storage. As used herein, the culture supernatant includes collected culture supernatant and culture supernatant that has been subjected to one or more treatments.

[0027] In the present application, the medium can be prepared by appropriately adding necessary factors to a basal medium used for culturing animal cells. Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, MEM Zinc Option medium, IMEM Zinc Option medium, Dulbecco's modified Eagle's Medium (DMEM) medium, DMEM / F12 medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. The basal medium may contain serum (e.g., fetal bovine serum (FBS)) or may be serum-free. If necessary, the medium may contain one or more serum substitutes, such as albumin, transferrin, KnockOut Serum Replacement (KSR) (serum replacement for ES cell culture) (Thermo Fisher Scientific), N2 supplement (Thermo Fisher Scientific), B27 supplement (Thermo Fisher Scientific), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, or may contain one or more substances, such as lipids, amino acids, L-glutamine, GlutaMAX (Thermo Fisher Scientific), non-essential amino acids (NEAA), vitamins, growth factors, antibiotics (e.g., streptomycin, penicillin, puromycin, mitomycin), antioxidants, pyruvic acid, buffers, inorganic salts, and the like, or one or more substances that are typically added to other animal culture media. The basal medium used in the present application may be, for example, DMEM medium.

[0028] The YAP / TAZ mechano-signal cascade inhibitor is not particularly limited as long as it is a substance that inhibits the YAP / TAZ mechano-signal cascade. The "YAP / TAZ mechano-signal cascade" refers to a series of signal transduction in which mechanical stimuli such as scaffold stiffness promote actin extension or polymerization, which causes YAP or TAZ to migrate into the nucleus and interact with transcription factors such as TEAD. Examples of YAP / TAZ mechano-signal cascade inhibitors include YAP inhibitors, TAZ inhibitors, TEAD inhibitors, substances that inhibit the interaction between YAP or TAZ and TEAD, and actin extension or polymerization inhibitors. Also included are integrin receptor inhibitors or anti-integrin antibodies for sensing extracellular matrix.

[0029] The YAP / TAZ mechano-signaling cascade inhibitor may be added to the culture medium or may be introduced into the mesenchymal stem cell. When the YAP / TAZ mechano-signaling cascade inhibitor is added to the culture medium, the concentration of the YAP / TAZ mechano-signaling cascade inhibitor can be appropriately selected by those skilled in the art depending on the YAP / TAZ mechano-signaling cascade inhibitor used.

[0030] The YAP inhibitor, TAZ inhibitor, or TEAD inhibitor is not particularly limited as long as it is a substance that inhibits YAP, TAZ, or TEAD, respectively. TEAD1, TEAD2, TEAD3, and TEAD4 are known as TEAD, and the TEAD inhibitor may be a substance that inhibits any of TEAD1, TEAD2, TEAD3, and TEAD4, and is preferably a substance that inhibits TEAD1. The YAP inhibitor, TAZ inhibitor, or TEAD inhibitor may be a nucleic acid molecule that suppresses the expression of YAP, TAZ, or TEAD, respectively.

[0031] The YAP inhibitor can be a nucleic acid molecule that suppresses the expression of the YAP gene. The YAP inhibitor includes, for example, YAP siRNA, YAP shRNA, and YAP gene antisense nucleic acid.

[0032] siRNA is a double-stranded RNA consisting of an RNA strand (antisense strand) having a sequence complementary to a part or the whole of a target mRNA, and an RNA strand (sense strand) having a sequence complementary thereto. The antisense strand does not have to be completely complementary to the target gene, as long as it can inhibit the expression of the target mRNA. siRNA may include a single-stranded portion (overhang).

[0033] shRNA is a single-stranded RNA in which an RNA strand (antisense strand) having a sequence complementary to a part or all of a target mRNA is linked to an RNA strand (sense strand) having a sequence complementary thereto by a short spacer sequence. The antisense strand does not have to be completely complementary to the target gene as long as it can inhibit the expression of the target mRNA.

[0034] Antisense nucleic acid is a single-stranded nucleic acid that has a base sequence that is complementary to a part or the whole of a target gene.Antisense nucleic acid does not have to be completely complementary to a target gene as long as it can obtain the effect of inhibiting the expression of the target gene.Antisense nucleic acid can be DNA, RNA or DNA-RNA chimera.

[0035] siRNA, shRNA and antisense nucleic acid can be designed and synthesized by known methods in the art.For example, based on the base sequence information of target gene, the base sequence of siRNA, shRNA and antisense nucleic acid can be designed by conventional methods, and then can be synthesized by known nucleic acid synthesis reaction based on the design.For example, siSNIPER or miRDeep2 can be used to design the base sequence of siRNA, shRNA and antisense nucleic acid.

[0036] Nucleotide molecules constituting siRNA, shRNA and antisense nucleic acids may contain various chemical modifications to improve stability and activity. For example, to prevent degradation by hydrolases such as nucleases, phosphate residues may be replaced with chemically modified phosphate residues such as phosphorothioate (PS), methylphosphonate, phosphorodithioate, etc. Also, at least a portion of them may be composed of a nucleic acid analog such as peptide nucleic acid (PNA).

[0037] The mRNA sequence of human YAP gene can be, for example, the base sequence shown in NCBI Reference Sequence: NM_001130145.3 (SEQ ID NO: 1). YAP siRNA, YAP shRNA, and YAP gene antisense nucleic acid can be designed and synthesized by standard methods based on the base sequence shown in NCBI Reference Sequence: NM_001130145.3. In one embodiment, the YAP inhibitor is YAP siRNA or YAP shRNA.

[0038] The YAP inhibitor may be, for example, a YAP siRNA or YAP shRNA comprising an RNA strand of 15 to 30 nucleotides having a sequence complementary to a portion of SEQ ID NO: 1. The YAP inhibitor may be, for example, a YAP siRNA or YAP shRNA comprising an RNA strand of 15 to 30 nucleotides having a sequence complementary to a portion of SEQ ID NO: 1, which includes 1 to 5, 1 to 3, for example, 1 or 2 modifications. In this specification, "modification" means substitution, deletion, addition, insertion, or a combination thereof of a base. In one embodiment, the sequence complementary to a portion of SEQ ID NO: 1 is the sequence of positions 1 to 19 of SEQ ID NO: 3.

[0039] The sense strand of YAP siRNA may, for example, comprise the sequence of positions 1 to 19 of SEQ ID NO: 2. As long as YAP siRNA can inhibit expression of the YAP gene, the sense strand of YAP siRNA may comprise a sequence containing 1 to 5, 1 to 3, for example 1 or 2 modifications in the sequence of positions 1 to 19 of SEQ ID NO: 2. The sense strand of YAP siRNA may further comprise a DNA overhang (e.g., dTdT) at the 3' end.

[0040] The antisense strand of YAP siRNA may, for example, comprise the sequence of positions 1 to 19 of SEQ ID NO: 3. As long as YAP siRNA can inhibit expression of the YAP gene, the antisense strand of YAP siRNA may comprise a sequence containing 1 to 5, 1 to 3, for example 1 or 2 modifications in the sequence of positions 1 to 19 of SEQ ID NO: 3. The antisense strand of YAP siRNA may further comprise a DNA overhang (e.g., dTdT) at the 3' end.

[0041] In one embodiment, the YAP siRNA comprises a sense strand comprising the sequence of positions 1 to 19 of SEQ ID NO: 2 or a sequence comprising 1 to 5, 1 to 3, for example 1 or 2 modifications in the sequence of positions 1 to 19 of SEQ ID NO: 2, and an antisense strand comprising the sequence of positions 1 to 19 of SEQ ID NO: 3 or a sequence comprising 1 to 5, 1 to 3, for example 1 or 2 modifications in the sequence of positions 1 to 19 of SEQ ID NO: 3. In one embodiment, the YAP siRNA comprises a sense strand comprising the sequence of positions 1 to 19 of SEQ ID NO: 2, and an antisense strand comprising the sequence of positions 1 to 19 of SEQ ID NO: 3. In one embodiment, the YAP siRNA comprises a sense strand comprising the sequence shown in SEQ ID NO: 2, and an antisense strand comprising the sequence shown in SEQ ID NO: 3.

[0042] The TAZ inhibitor can be a nucleic acid molecule that suppresses the expression of the TAZ gene. The TAZ inhibitor includes, for example, TAZ siRNA, TAZ shRNA, and antisense nucleic acid of the TAZ gene. The mRNA sequence of the human TAZ gene includes, for example, the base sequence shown in NCBI Reference Sequence: NM_001168278.3 (SEQ ID NO: 4). The TAZ siRNA, TAZ shRNA, and antisense nucleic acid of the TAZ gene can be designed and synthesized by conventional methods based on, for example, the base sequence shown in NCBI Reference Sequence: NM_001168278.3. In an embodiment, the TAZ inhibitor is TAZ siRNA or TAZ shRNA.

[0043] The TAZ inhibitor may be, for example, a TAZ siRNA or TAZ shRNA comprising an RNA strand of 15 to 30 nucleotides having a sequence complementary to a portion of SEQ ID NO: 4. The TAZ inhibitor may be a TAZ siRNA or TAZ shRNA comprising an RNA strand of 15 to 30 nucleotides having a sequence complementary to a portion of SEQ ID NO: 4, which includes 1 to 5, 1 to 3, for example, 1 or 2 modifications. In this specification, "modification" means substitution, deletion, addition, insertion, or a combination thereof of bases. In one embodiment, the sequence complementary to a portion of SEQ ID NO: 4 is the sequence of positions 1 to 19 of SEQ ID NO: 6.

[0044] The sense strand of TAZ siRNA may, for example, comprise the sequence of positions 1 to 19 of SEQ ID NO: 5. As long as TAZ siRNA can inhibit expression of the TAZ gene, the sense strand of TAZ siRNA may comprise a sequence containing 1 to 5, 1 to 3, for example 1 or 2 modifications in the sequence of positions 1 to 19 of SEQ ID NO: 5. The sense strand of TAZ siRNA may further comprise a DNA overhang (e.g., dTdT) at the 3' end.

[0045] The antisense strand of TAZ siRNA may, for example, comprise the sequence of positions 1 to 19 of SEQ ID NO: 6. As long as TAZ siRNA can inhibit expression of the TAZ gene, the antisense strand of TAZ siRNA may comprise a sequence containing 1 to 5, 1 to 3, for example 1 or 2 modifications in the sequence of positions 1 to 19 of SEQ ID NO: 6. The antisense strand of TAZ siRNA may further comprise a DNA overhang (e.g., dTdT) at the 3' end.

[0046] In one embodiment, the TAZ siRNA comprises a sense strand comprising the sequence of positions 1 to 19 of SEQ ID NO:5 or the sequence of positions 1 to 19 of SEQ ID NO:5 containing 1 to 5, 1 to 3, for example 1 or 2 modifications, and an antisense strand comprising the sequence of positions 1 to 19 of SEQ ID NO:6 or the sequence of positions 1 to 19 of SEQ ID NO:6 containing 1 to 5, 1 to 3, for example 1 or 2 modifications. In one embodiment, the TAZ siRNA comprises a sense strand comprising the sequence of positions 1 to 19 of SEQ ID NO:5 and an antisense strand comprising the sequence of positions 1 to 19 of SEQ ID NO:6. In one embodiment, the TAZ siRNA comprises a sense strand comprising the sequence shown in SEQ ID NO:5 and an antisense strand comprising the sequence shown in SEQ ID NO:6.

[0047] The TEAD inhibitor can be a nucleic acid molecule that suppresses the expression of the TEAD gene. The TEAD inhibitor includes, for example, TEAD siRNA, TEAD shRNA, and antisense nucleic acid of the TEAD gene. For example, the mRNA sequence of the human TEAD1 gene includes the base sequence shown in NCBI Reference Sequence: NM_021961.6 (SEQ ID NO: 7). The TEAD siRNA, TEAD shRNA, and antisense nucleic acid of the TEAD gene can be designed and synthesized by a conventional method based on the base sequence shown in NCBI Reference Sequence: NM_021961.6. In an embodiment, the TEAD inhibitor is a TEAD siRNA or a TEAD shRNA.

[0048] The TEAD inhibitor may be, for example, a TEAD siRNA or TEAD shRNA comprising an RNA strand of 15 to 30 nucleotides having a sequence complementary to a portion of SEQ ID NO: 7. The TEAD inhibitor may be a TEAD siRNA or TEAD shRNA comprising an RNA strand of 15 to 30 nucleotides having a sequence complementary to a portion of SEQ ID NO: 7, and including 1 to 5, 1 to 3, for example, 1 or 2 modifications. In this specification, "modification" means substitution, deletion, addition, insertion, or a combination thereof of bases. In one embodiment, the sequence complementary to a portion of SEQ ID NO: 7 is the sequence of positions 1 to 21 of SEQ ID NO: 9, as long as it can inhibit the expression of the TEAD gene.

[0049] The sense strand of the TEAD siRNA may, for example, comprise the sequence of positions 1 to 21 of SEQ ID NO: 8. As long as the TEAD siRNA can inhibit expression of the TEAD gene, the sense strand of the TEAD siRNA may comprise a sequence containing 1 to 5, 1 to 3, for example 1 or 2 modifications in the sequence of positions 1 to 21 of SEQ ID NO: 8. The sense strand of the TEAD siRNA may further comprise a DNA overhang (e.g., dTdT) at the 3' end.

[0050] The antisense strand of TEAD siRNA may, for example, comprise the sequence of positions 1 to 21 of SEQ ID NO: 9. As long as the TEAD siRNA can inhibit expression of the TEAD gene, the antisense strand of TEAD siRNA may comprise a sequence containing 1 to 5, 1 to 3, for example 1 or 2 modifications in the sequence of positions 1 to 21 of SEQ ID NO: 9. The antisense strand of TEAD siRNA may further comprise a DNA overhang (e.g., dTdT) at the 3' end.

[0051] In one embodiment, the TEAD siRNA is composed of a sense strand comprising the sequence of positions 1 to 21 of SEQ ID NO: 8 or a sequence comprising 1 to 5, 1 to 3, for example 1 or 2 modifications in the sequence of positions 1 to 21 of SEQ ID NO: 8, and an antisense strand comprising the sequence of positions 1 to 21 of SEQ ID NO: 9 or a sequence comprising 1 to 5, 1 to 3, for example 1 or 2 modifications in the sequence of positions 1 to 21 of SEQ ID NO: 9. In one embodiment, the TEAD siRNA is composed of a sense strand comprising the sequence of positions 1 to 21 of SEQ ID NO: 8, and an antisense strand comprising the sequence of positions 1 to 21 of SEQ ID NO: 9. In one embodiment, the TEAD siRNA is composed of a sense strand comprising the sequence shown in SEQ ID NO: 8, and an antisense strand comprising the sequence shown in SEQ ID NO: 9.

[0052] Substances that inhibit the interaction of YAP or TAZ with TEAD include, for example, verteporfin, CA3, YAP-TEAD Inhibitor 1, (R)-PFI 2 hydrochloride, and Super-TDU.

[0053] The actin progression or polymerization inhibitor is not particularly limited as long as it is a substance that inhibits the progression or polymerization of actin, and examples thereof include ROCK inhibitors Y27632, blebbistatin, cytochalasin D, latrunculin, and mycalolide B. The actin progression or polymerization inhibitor is, for example, Y27632 or blebbistatin. When Y27632 is added to the medium, its concentration may be 1 nM to 100 μM, 10 nM to 50 μM, 100 nM to 20 μM, or 1 μM to 10 μM, for example, about 5 μM. When blebbistatin is added to the medium, its concentration may be 1 nM to 100 μM, 10 nM to 100 μM, 100 nM to 50 μM, or 1 μM to 20 μM, for example, about 10 μM.

[0054] In one embodiment, the YAP / TAZ mechano-signaling cascade inhibitor is introduced into a mesenchymal stem cell. A known method can be used as a means for introducing the YAP / TAZ mechano-signaling cascade inhibitor. When the YAP / TAZ mechano-signaling cascade inhibitor is a nucleic acid molecule, an expression vector encoding the YAP / TAZ mechano-signaling cascade inhibitor may be introduced into a cell by a known method. The type of expression vector is not particularly limited, and known expression vectors can be used. Examples of the expression vector include episomal vectors, artificial chromosome vectors, plasmid vectors, and viral vectors.

[0055] Known methods for introducing an expression vector into a somatic cell include, for example, the infection method for a viral vector, and the calcium phosphate method, lipofection method, retronectin method, or electroporation method for a plasmid vector.

[0056] Alternatively, when the YAP / TAZ mechano-signaling cascade inhibitor is a nucleic acid molecule, the YAP / TAZ mechano-signaling cascade inhibitor may be introduced into cells by the calcium phosphate method, the lipofection method, or the electroporation method.

[0057] When the YAP / TAZ mechano-signaling cascade inhibitor is a protein, the YAP / TAZ mechano-signaling cascade inhibitor can be introduced into cells, for example, by direct protein injection (e.g., a needle method, a lipofection method, or an electroporation method).

[0058] In an embodiment, the medium for culturing mesenchymal stem cells may further contain a priming agent. The priming agent is not particularly limited as long as it is a substance that improves the anti-inflammatory ability and immune control ability of mesenchymal stem cells. Examples of the priming agent include inflammatory cytokines such as IFN-γ, TNF-α, IL-1, IL-6, IL-8, IL-12, and IL-18. The priming agent is, for example, IFN-γ or TNF-α. When the priming agent is IFN-γ, the concentration thereof in the medium may be 10 pg / ml to 10 μg / ml, 100 pg / ml to 1 μg / ml, 1 ng / ml to 100 ng / ml, for example, about 10 ng / ml. When the priming agent is TNF-α, the concentration thereof in the medium may be 10 pg / ml to 10 μg / ml, 100 pg / ml to 1 μg / ml, 1 ng / ml to 100 ng / ml, for example, about 10 ng / ml.

[0059] In some embodiments, mesenchymal stem cells may be cultured in an adherent manner. In the present application, "adherent culture" means that cells are cultured in a state where they are attached to a culture substrate. For example, the adherent culture may be performed in a coated culture vessel. Examples of coating agents include Matrigel (BD), Synthemax (Corning), collagen, gelatin, laminin (laminin-511, laminin-111, laminin-411, etc.), heparan sulfate proteoglycan, entactin, fragments thereof, and combinations thereof.

[0060] The culture period is not particularly limited, and is, for example, 1 hour to 14 days. In one embodiment, the culture period for producing mesenchymal stem cells with improved anti-inflammatory and immunoregulatory capabilities may be 1 hour to 14 days, 2 hours to 10 days, 3 hours to 7 days, 5 hours to 5 days, or 12 hours to 3 days, for example, about 1 day. In one embodiment, the culture period for producing a culture supernatant of mesenchymal stem cells with improved anti-inflammatory and immunoregulatory capabilities may be 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, or 1 to 2 days, for example, about 1 day.

[0061] The culture temperature is, but not limited to, about 30 to 40° C., for example, about 37° C., and the culture is performed in an atmosphere of CO2-containing air, with a CO2 concentration of, for example, about 2 to 5%.

[0062] Methods for improving the anti-inflammatory and immunoregulatory capabilities of mesenchymal stem cells The present application also provides a method for improving the anti-inflammatory and immunoregulatory abilities of mesenchymal stem cells, comprising culturing mesenchymal stem cells in the presence of a YAP / TAZ mechano-signal cascade inhibitor. Examples of the YAP / TAZ mechano-signal cascade inhibitor used in this embodiment are as described above. Other culture conditions are also as described above.

[0063] composition The present application also provides a composition comprising mesenchymal stem cells with improved anti-inflammatory and immunoregulatory capabilities produced by the method of the present application, or a culture supernatant thereof. In one embodiment, the composition of the present application comprises mesenchymal stem cells with improved anti-inflammatory and immunoregulatory capabilities.

[0064] The present application also provides a composition comprising a mesenchymal stem cell into which a YAP / TAZ mechano-signaling cascade inhibitor or an expression vector encoding a YAP / TAZ mechano-signaling cascade inhibitor has been introduced. Examples of the YAP / TAZ mechano-signaling cascade inhibitor and the expression vector are as described above.

[0065] The composition of the present application can be used to treat inflammatory immune diseases. Mesenchymal stem cells have been approved as a therapeutic agent for graft-versus-host disease (GVHD) (Temcel®), and are also known to be effective for inflammatory immune diseases such as sepsis, enteritis, hepatitis, nephritis, and rheumatoid arthritis (Song N, et al., Trends in Pharmacological Sciences. 2020; 41: 653-664.). Examples of inflammatory immune diseases include sepsis, enteritis, hepatitis, nephritis, graft-versus-host disease (GVHD), rheumatoid arthritis, systemic inflammatory disease, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, immune-mediated thrombocytopenia, immune-mediated hemolytic anemia, diabetes, systemic lupus erythematosus, atopic dermatitis, collagen disease, steroid-responsive meningoarteritis, multiple sclerosis, psoriasis, autoimmune bullous disease, polyarthritis, pneumonia, infectious osteomyelitis, periodontitis, etc. In one embodiment, the inflammatory immune disease is selected from the group consisting of sepsis, enteritis, hepatitis, nephritis, graft-versus-host disease (GVHD), and rheumatoid arthritis.

[0066] The composition of the present application can also be used to treat spinal cord injury or ischemic disease. Mesenchymal stem cells have been approved as a therapeutic agent for spinal cord injury (Stemilac (registered trademark)), and it is known that they can be used to treat spinal cord injury. In addition, it has been suggested that mesenchymal stem cells can be used to treat ischemic disease because they have a cytoprotective effect. Ischemic disease is, for example, angina pectoris, myocardial infarction or cerebral infarction.

[0067] The composition of the present application may be frozen. The freezing and thawing method of cells and culture supernatant can be performed by known methods. When the composition of the present application contains mesenchymal stem cells, the composition of the present application may contain a cryoprotectant such as DMSO, glycerol, polyvinylpyrrolidone, polyethylene glycol, albumin, dextran, sucrose, etc.

[0068] The composition of the present application may contain a pharma- ceutically acceptable carrier or additive. Examples of such carriers or additives include isotonicity agents, thickeners, sugars, preservatives, pH regulators, stabilizers, gel bases, wetting agents, suspending agents, excipients, buffers, and antioxidants. Specific carriers or additives include, but are not limited to, water for injection, physiological saline, 5% glucose solution, Ringer's solution, lactate Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution, and amino acid solutions. One type of pharma-ceutically acceptable carrier or additive may be used, or two or more types may be mixed together.

[0069] The composition of the present application may be, for example, an injection, a liquid, an oral preparation, a patch, a preparation for implantation, a gel preparation, or the like.

[0070] The administration route of the composition of the present application may be, for example, intravenous administration, subcutaneous administration, intramuscular administration, intra-arterial administration, lymphatic system administration, intrathecal administration, intraperitoneal administration, rectal administration, vaginal administration, transdermal administration, implantation, direct administration to an organ, or local transplantation, and is preferably intravenous administration.

[0071] The dosage and frequency of administration of the composition of the present application can be appropriately determined by a person skilled in the art according to the animal species of the subject, the health condition, age, weight, administration route and administration form of the subject, etc., so that an effective amount of the active ingredient is administered to the subject. For example, the composition of the present application can be administered once to several times a day, or once to several times a day or several days or once a week or several weeks, for example, once every 1 to 4 weeks, but is not limited thereto. The effective amount in a certain situation can be easily determined by routine experimentation and is within the skill and judgment of an ordinary clinician. For example, when the composition of the present application contains mesenchymal stem cells, the effective amount is, but is not limited to, 10 4 pcs / kg body weight or more, 10 5 pcs / kg body weight or more, 10 6 pcs / kg body weight or more, or 2 x 10 6 may be greater than or equal to about 10 4 pieces / kg weight ~ approx. 2×10 6 Pieces / kg body weight.

[0072] As used herein, "treat" or "treatment" means reducing or eliminating the cause of a disease, slowing or halting its progression, and / or reducing, alleviating, ameliorating or eliminating its symptoms in a subject having a disease.

[0073] Subjects for disease treatment include mammals such as mice, rats, hamsters, guinea pigs, cows, horses, pigs, sheep, monkeys, orangutans, chimpanzees, dogs, cats, and humans, preferably primates, and more preferably humans.

[0074] In another embodiment, the present application also provides a composition for producing mesenchymal stem cells with improved anti-inflammatory and immunoregulatory capabilities, comprising a YAP / TAZ mechano-signaling cascade inhibitor. Examples of the YAP / TAZ mechano-signaling cascade inhibitor used in this embodiment are as described above. The composition of this embodiment may further comprise a priming agent, or may be used in combination with a priming agent. Examples of the priming agent are as described above.

[0075] In this embodiment, the composition may contain a pharma- ceutically acceptable carrier or additive. Examples of such carriers or additives include isotonicity agents, thickeners, sugars, sugar alcohols, preservatives, bactericides, antibacterial agents, pH regulators, stabilizers, chelating agents, oily bases, gel bases, wetting agents, surfactants, suspending agents, binders, excipients, lubricants, disintegrants, foaming agents, fluidizing agents, dispersants, emulsifiers, buffers, solubilizers, and antioxidants. Pharmaceutically acceptable carriers or additives may be used alone or in combination of two or more.

[0076] kit The present application also provides a kit for producing mesenchymal stem cells with improved anti-inflammatory and immunoregulatory capabilities, comprising a YAP / TAZ mechano-signaling cascade inhibitor and mesenchymal stem cells.

[0077] Examples of the YAP / TAZ mechano-signal cascade inhibitor and mesenchymal stem cell used in this embodiment are as described above. The kit of the present application may further include a priming agent, or may be used in combination with a priming agent. Examples of the priming agent are as described above. The kit of the present application may further include a buffer solution, a reaction vessel, an instruction manual, etc.

[0078] Treatment method The present application also provides a method for treating inflammatory immune diseases, spinal cord injury, or ischemic disease, comprising administering to a subject in need thereof mesenchymal stem cells or their culture supernatants with improved anti-inflammatory and immune control abilities produced by the method of the present application. The present application also provides a method for treating inflammatory immune diseases, spinal cord injury, or ischemic disease, comprising administering to a subject in need thereof mesenchymal stem cells into which a YAP / TAZ mechano-signaling cascade inhibitor or an expression vector encoding a YAP / TAZ mechano-signaling cascade inhibitor has been introduced. Examples of inflammatory immune diseases and ischemic diseases are as described above.

[0079] The present application also provides use of mesenchymal stem cells or their culture supernatants with improved anti-inflammatory and immunoregulatory capabilities produced by the method of the present application for the manufacture of a pharmaceutical composition for treating an inflammatory immune disease, spinal cord injury, or ischemic disease. The present application also provides use of mesenchymal stem cells into which a YAP / TAZ mechano-signaling cascade inhibitor or an expression vector encoding a YAP / TAZ mechano-signaling cascade inhibitor has been introduced for the manufacture of a pharmaceutical composition for treating an inflammatory immune disease, spinal cord injury, or ischemic disease. Examples of inflammatory immune diseases and ischemic diseases are as described above.

[0080] The present application provides, for example, the following embodiments. [1] A method for producing mesenchymal stem cells or their culture supernatant having improved anti-inflammatory and immunoregulatory capabilities, comprising culturing mesenchymal stem cells in the presence of a YAP / TAZ mechano-signaling cascade inhibitor. [2] The method according to [1] above, wherein the YAP / TAZ mechano-signaling cascade inhibitor is selected from the group consisting of a YAP inhibitor, a TAZ inhibitor, a TEAD inhibitor, and an actin progression or polymerization inhibitor. [3] The method according to [2] above, wherein the YAP inhibitor is YAP siRNA or YAP shRNA. [4] The method according to [2] above, wherein the TAZ inhibitor is TAZ siRNA or TAZ shRNA. [5] The method according to [2] above, wherein the TEAD inhibitor is a TEAD siRNA or a TEAD shRNA. [6] The method according to [2] above, wherein the actin progression or polymerization inhibitor is Y27632 or blebbistatin. [7] The method according to any one of the above [1] to [6], wherein the mesenchymal stem cells are derived from bone marrow, fat, umbilical cord, nerve, gum, dental pulp, peripheral blood, skin, hair root, muscle tissue, endometrium, placenta, or pluripotent stem cells. [8] A composition comprising mesenchymal stem cells with improved anti-inflammatory and immunoregulatory capabilities, produced by the method according to any one of the above [1] to [7], or a culture supernatant thereof. [9] The composition described in [8] above, comprising mesenchymal stem cells.

[10] A composition comprising a mesenchymal stem cell into which a YAP / TAZ mechano-signaling cascade inhibitor or an expression vector encoding a YAP / TAZ mechano-signaling cascade inhibitor has been introduced.

[11] The composition according to any one of the above [8] to

[10] for treating an inflammatory immune disease.

[12] The composition according to

[11] above, wherein the inflammatory immune disease is selected from the group consisting of sepsis, enteritis, hepatitis, nephritis, graft-versus-host disease, and rheumatoid arthritis.

[13] The composition according to any one of the above [8] to

[10] for treating spinal cord injury or ischemic disease.

[14] A method for improving the anti-inflammatory and immunoregulatory capabilities of mesenchymal stem cells, comprising culturing the mesenchymal stem cells in the presence of a YAP / TAZ mechano-signaling cascade inhibitor.

[15] A composition for producing mesenchymal stem cells with improved anti-inflammatory and immunoregulatory capabilities, comprising a YAP / TAZ mechano-signaling cascade inhibitor.

[16] A kit for producing mesenchymal stem cells with improved anti-inflammatory and immune regulatory capabilities, comprising a YAP / TAZ mechano-signaling cascade inhibitor and mesenchymal stem cells.

[0081]

[17] A method for treating inflammatory immune diseases, spinal cord injury, or ischemic diseases, comprising administering to a subject in need thereof mesenchymal stem cells or their culture supernatants having improved anti-inflammatory and immunoregulatory capabilities, the mesenchymal stem cells being produced by the method according to any one of the above [1] to [7].

[18] A method for treating inflammatory immune diseases, spinal cord injury, or ischemic diseases, comprising administering to a subject in need thereof a YAP / TAZ mechano-signaling cascade inhibitor or a mesenchymal stem cell into which an expression vector encoding a YAP / TAZ mechano-signaling cascade inhibitor has been introduced.

[19] The method according to

[17] or

[18] above, wherein the inflammatory immune disease is selected from the group consisting of sepsis, enteritis, hepatitis, nephritis, graft-versus-host disease, and rheumatoid arthritis.

[20] Use of mesenchymal stem cells or their culture supernatants with improved anti-inflammatory and immunoregulatory capabilities, produced by the method described in any one of [1] to [7] above, for the manufacture of a pharmaceutical composition for treating an inflammatory immune disease, spinal cord injury, or ischemic disease. [twenty one] Use of a mesenchymal stem cell into which a YAP / TAZ mechano-signaling cascade inhibitor or an expression vector encoding a YAP / TAZ mechano-signaling cascade inhibitor has been introduced, for the manufacture of a pharmaceutical composition for treating an inflammatory immune disease, spinal cord injury or ischemic disease. [twenty two] The use according to

[20] or

[21] above, wherein the inflammatory immune disease is selected from the group consisting of sepsis, enteritis, hepatitis, nephritis, graft-versus-host disease, and rheumatoid arthritis. EXAMPLES

[0082] The present invention will be described in more detail below by showing examples, but the present invention is not limited to these examples in any way.

[0083] Materials and Methods 1. Cell culture Human bone marrow-derived mesenchymal stem cells (MSCs) purchased from Lonza or MSCs (iMSCs) induced from human iPS cells (1231A line: Center for iPS Cell Research and Application, Kyoto University) according to the method of Kamiya et al. (Kamiya et al., 2020 Sneak Peek, http: / / dx.doi.org / 10.2139 / ssrn.3741231) were cultured in a 24-well culture plate (Corning) at a concentration of 0.5 × 10 5The cells were seeded at a cell density of 100 cells / well and cultured for 5 days in DMEM High glucose (Sigma) containing 10% fetal bovine serum (FBS), 100 U / ml penicillin, and 100 μg / ml streptomycin. Various siRNAs (10 nM) were introduced using the transfection reagent RNAiMAX (Invitrogen) one day after the start of culture. TNF-α (10 ng / ml, peprotech), IFN-γ (10 ng / ml, peptrotech), ROCK inhibitor Y27632 (5 μM, Merck), and actin polymerization inhibitor blebbistatin (10 μM, Sigma) were added 4 days after the start of culture. After the addition of each reagent, the cells were cultured for 24 hours. At the end of the culture, RNA was collected and the mRNA expression levels of the cell protective factor TNFAIP6 and the immune regulatory enzyme IDO were quantified by qPCR. Alternatively, the culture supernatant was collected at the end of the culture and used for culture with macrophages. Alternatively, the cells at the end of the culture were collected using trypsin / EDTA and used for co-culture with T cells or administration to a mouse sepsis model.

[0084] 2. siRNAs The sequences of the siRNAs used in this example are as follows: YAP siRNA (sense strand): GACAUCUUCUGGUCAGAGAdTdT (SEQ ID NO: 2) YAP siRNA (antisense strand): UCUCUGACCAGAAGAUGUCdTdT (SEQ ID NO: 3) TAZ siRNA (sense strand): ACGUUGACUUAGGAACUUUdTdT (SEQ ID NO: 5) TAZ siRNA (antisense strand): AAAGUUCCUAAGUCAACGUdTdT (SEQ ID NO: 6) TEAD siRNA (sense strand): GAUCAACUUCAUCCACAAGCUdTdT (SEQ ID NO: 8) TEAD siRNA (antisense strand): AGCUUGUGGAUGAAGUUGAUCdTdT (SEQ ID NO: 9) Control siRNA (sense strand): UUCUCCGAACGUGUCACGUdTdT (sequence number 10). Control siRNA (antisense strand): ACGUGACACGUUCGGAGAAdTdT (sequence number 11). Here, "dT" refers to the thymine base (thymidine) of the DNA strand.

[0085] 3. qPCR At the end of the culture, total RNA was extracted from the cells using RNA-iso. After cDNA synthesis using ReverTraAce (Toyobo), SYBR green Master Mix (Roche) and TNFAIP6, IDO, or 18S specific primers were prepared, and qPCR was performed using the StepOne System (Applied Biosystems) to quantify mRNA expression.

[0086] 4. M1 macrophage culture using MSCs culture supernatant Human mononuclear cell line (THP-1: ATCC) was cultured in a 24-well culture plate (Corning) at 0.5 × 10 5 The cells were seeded at a cell density of 10 ...

[0087] 5. Flow Cytometry The cells collected in 4 above were treated with mouse anti-human CD86 IgG (BD Biosciences) and rabbit anti-human CD80 IgG (BD Biosciences), and then bound with FITC-labeled goat anti-mouse IgG antibody (BD Biosciences) and PE-labeled goat anti-rabbit IgG antibody (BD Biosciences) as secondary antibodies. After thorough washing with PBS, the percentage of CD86- and CD80-positive cells was quantified using a FACScan flow cytometer (BD Biosciences).

[0088] 6. Co-culture of MSCs and T cells and quantification of T cell activation Human peripheral blood mononuclear cells (PBMCs) were obtained from venous blood of healthy donors by density gradient centrifugation (Lymphoprep (AXS)). The obtained PBMCs were placed in the lower dish of a non-contact co-culture 24-well plate (THINCERT 24well pore 0.41 μm (Greiner)) at 1.0 × 10 6 The cells were seeded at a cell density of 1.0 × 10 cells / well and cultured in RPMI-1640 High glucose medium (FUJIFILM) containing 10% FBS. In addition, CD3 / CD28 antibody-bound agarose beads (ImmunoCult Human CD3 / CD28 T cell activator (STEMCELL Technologies)) were added as a T cell inducer. The human bone marrow-derived MSCs obtained in item 1 above were placed in the upper dish at a density of 2.0 × 10 4 After 3 days of co-culture, BrdU (10 μM) was added, and 2 hours later, the T cells were collected from the bottom dish. The amount of BrdU incorporation was quantified by ELISA using a Cell Proliferation ELISA kit (Roche) to evaluate the degree of T cell activation.

[0089] 7. Mouse Sepsis Model Under the culture conditions described in item 1 above, control siRNA-introduced MSCs and YAP / TAZ siRNA-introduced MSCs were detached and collected with Trypsin / EDTA, and 2.5 × 10 5The cells were suspended in PBS to a cell concentration of 16 mg / kg cells / 200 μl. Eight-week-old male c57BL / 6J mice were intraperitoneally administered with Escherichia coli Lipopolysaccharide (LPS (Sigma)) at a concentration of 16 mg / kg. One hour after LPS administration, the above cell suspension or the same amount of PBS (200 μl) was administered via the tail vein. The survival status of the mice was observed every 8 hours.

[0090] 8. Mouse colitis model Under the culture conditions described in item 1 above, control siRNA-introduced MSCs and TEAD siRNA-introduced MSCs were detached and collected with Trypsin / EDTA, and 5.0 × 10 5 The cells were suspended in PBS to a cell concentration of 100μl cells / 100μl. Eight-week-old male c57BL / 6J mice were allowed to drink sterile distilled water containing 4% dextran sodium sulfate (DSS: MP Biomedicals) ad libitum. A negative control group was given normal sterile distilled water. Drinking water was changed every two days. Two and four days after the start of DSS drinking, 100μl of the cell suspension or the same amount of PBS was administered via the tail vein. In this experimental system, the mice were weighed for 7 days, sacrificed on the 7th day, and the condition of the large intestine was evaluated macroscopically.

[0091] [result] YAP siRNA and / or TAZ siRNA were introduced into human bone marrow-derived MSCs, and the expression of genes involved in anti-inflammation and immune regulation (TNFAIP6 and IDO) was quantified by qPCR. In addition, the responsiveness to stimulation with inflammatory cytokines IFN-γ or TNF-α was also confirmed. Stimulation with inflammatory cytokines reproduces the cytokine storm in an inflammatory state in vivo. Therefore, by quantifying the expression of TNFAIP6 and IDO under inflammatory cytokine stimulation, the anti-inflammatory and immune regulation capabilities of MSCs under inflammatory conditions can be confirmed. As a result, the expression of TNFAIP6 and IDO in MSCs was increased by introduction of YAP siRNA and / or TAZ siRNA. In addition, the expression of anti-inflammation and immune regulation-related genes was further increased by inflammatory cytokine stimulation (Figures 2 and 3). This indicates that MSCs introduced with YAP siRNA and / or TAZ siRNA are highly responsive under inflammatory conditions. Here, the expression level of IDO under IFN-γ stimulation did not change significantly between the control group and the groups introduced with various siRNAs (Figure 3 left). This is thought to be because the expression level of IDO was significantly increased by IFN-γ stimulation, making it impossible to observe the effect of introducing various siRNAs.Similarly, increased expression of TNFAIP6 and IDO was confirmed in iMSCs induced from human iPS cells (Figure 4).

[0092] Furthermore, MSCs transfected with siRNA against TEAD, known to be a target transcription factor of YAP / TAZ, showed properties equivalent to those of the YAP / TAZ siRNA transfected group (Figure 5). A similar tendency was also observed when ROCK inhibitor Y27632 or F-actin polymerization inhibitor blebbistatin were administered (Figure 6). Here, ROCK inhibitor Y27632 degrades F-actin by inhibiting ROCK.

[0093] Next, we investigated the inhibitory effect of the culture supernatant of MSCs transfected with YAP siRNA and TAZ siRNA (YAP / TAZ siRNAs) on inflammatory M1 macrophages. As a result of culturing M1 macrophages using the culture supernatant of MSCs transfected with YAP / TAZ siRNAs, CD86+ Macrophages and CD80 + The presence of macrophages was decreased (Figure 7), and the expression level of TNF-α was also decreased (Figure 8). Therefore, it was confirmed that the culture supernatant of MSCs transfected with YAP / TAZ siRNAs suppressed the activity of inflammatory M1 macrophages.

[0094] We also investigated the T cell suppressive effect of MSCs transfected with YAP / TAZ siRNAs or TEAD siRNAs. When co-cultured with human T cells, YAP / TAZ siRNAs transfected MSCs inhibited T cell activity compared with normal MSCs (Figures 9 and 10). Furthermore, TEAD siRNA transfected MSCs also suppressed T cell activation (Figures 11 and 12). These results demonstrate that MSCs transfected with YAP / TAZ siRNAs or TEAD siRNA have high immune regulatory ability.

[0095] Based on these findings, the present invention, which produces MSCs with high anti-inflammatory and immunoregulatory capabilities by using a culture method that inhibits YAP / TAZ mechanosignaling, suggests the feasibility of a highly reliable cell therapy.

[0096] MSCs exhibiting high anti-inflammatory and immune-regulating abilities obtained by the method of the present application are expected to be used as cell therapy drugs for severe inflammatory immune diseases. As an experimental example, human MSCs transfected with YAP / TAZ siRNAs were intravenously administered to a septic shock model in which E. coli LPS was administered intraperitoneally to c57BL6 mice. As a result, while MSCs transfected with control siRNA showed a protective effect against LPS-induced inflammatory shock, MSCs transfected with YAP / TAZ siRNAs showed an even higher protective effect (Figure 13).

[0097] Furthermore, we intravenously administered TEAD siRNA-introduced human MSCs to c57BL6 mice that were allowed to freely drink DSS as a colitis model. As a result, administration of control siRNA-introduced MSCs did not result in any protective effect against DSS-induced colitis, such as suppression of weight loss, whereas administration of TEAD siRNA-introduced MSCs showed a significant protective effect (Figures 14 and 15).

[0098] These results suggest that YAP / TAZ siRNAs-introduced MSCs can be used as an effective cell therapy for severe inflammatory immune diseases such as sepsis, enteritis, fulminant hepatitis, GVHD, and rheumatoid arthritis, as an example of clinical application. Specifically, when the present MSCs were co-cultured with T cells, the activity of T cells was inhibited (Figures 9 to 12). This result indicates that the present MSCs have high immune control ability, and therefore suggests that the present MSCs are effective for immune diseases such as GVHD and rheumatoid arthritis. In addition, when M1 macrophages were cultured using the culture supernatant of the present MSCs, the activity of inflammatory M1 macrophages was suppressed (Figures 7 and 8). Furthermore, when the present MSCs were intravenously administered to septic shock model mice, the present MSCs exhibited a high protective effect against LPS-induced inflammatory shock (Figure 13). These results indicate that the MSCs of the present application have a high protective effect against inflammation by suppressing the activity of inflammatory macrophages, and therefore suggest that the MSCs of the present application are effective against inflammatory diseases such as sepsis. The MSCs of the present application also exerted a high protective effect against DSS-induced colitis. This result and the above-mentioned experimental results indicate that the MSCs of the present application have both high immune control ability and a protective effect against inflammation, and suggest that the MSCs of the present application are also effective against diseases such as enteritis, hepatitis, nephritis, pneumonia, infectious osteomyelitis, and periodontitis.

[0099] SEQ ID NO:1 (mRNA sequence of human YAP gene)

[0100] SEQ ID NO: 4 (mRNA sequence of human TAZ gene)

[0101] SEQ ID NO: 7 (mRNA sequence of human TEAD1 gene)

Claims

1. A method for producing mesenchymal stem cells or a culture supernatant thereof with improved anti-inflammatory ability and immunomodulatory ability, comprising culturing mesenchymal stem cells in the presence of a YAP / TAZ mechanosignaling cascade inhibitor.

2. The method according to claim 1, wherein the YAP / TAZ mechanosignaling cascade inhibitor is selected from the group consisting of a YAP inhibitor, a TAZ inhibitor, a TEAD inhibitor, and an actin progression or polymerization inhibitor.

3. The method according to claim 2, wherein the YAP inhibitor is YAP siRNA or YAP shRNA.

4. The method according to claim 2, wherein the TAZ inhibitor is TAZ siRNA or TAZ shRNA.

5. The method according to claim 2, wherein the TEAD inhibitor is TEAD siRNA or TEAD shRNA.

6. The method according to claim 2, wherein the actin progression or polymerization inhibitor is Y27632 or blebbistatin.

7. The method according to claim 1, wherein the mesenchymal stem cells are derived from bone marrow, adipose tissue, umbilical cord, nerve, gingiva, dental pulp, peripheral blood, skin, hair follicle, muscle tissue, endometrium, placenta, or pluripotent stem cells.

8. A composition comprising mesenchymal stem cells or a culture supernatant thereof with improved anti-inflammatory ability and immunomodulatory ability produced by the method according to claim 1.

9. The composition according to claim 8, comprising mesenchymal stem cells.

10. A composition comprising mesenchymal stem cells into which a YAP / TAZ mechanosignaling cascade inhibitor or an expression vector encoding a YAP / TAZ mechanosignaling cascade inhibitor has been introduced.

11. The composition according to any one of claims 8 to 10, for treating an inflammatory immune disease.

12. The composition according to claim 11, wherein the inflammatory immune disease is selected from the group consisting of sepsis, enteritis, hepatitis, nephritis, graft-versus-host disease, and rheumatoid arthritis.

13. The composition according to any one of claims 8 to 10, for treating spinal cord injury or ischemic disease.

14. A method for improving the anti-inflammatory ability and immunomodulatory ability of mesenchymal stem cells, comprising culturing mesenchymal stem cells in the presence of a YAP / TAZ mechanosignaling cascade inhibitor.

15. A composition for producing mesenchymal stem cells with improved anti-inflammatory ability and immunomodulatory ability, comprising a YAP / TAZ mechanosignaling cascade inhibitor.

16. A kit for producing mesenchymal stem cells with improved anti-inflammatory ability and immune control ability, comprising a YAP / TAZ mechanosignal cascade inhibitor and mesenchymal stem cells.

Images