JP2019532081A - Composition for promoting stem cell activity comprising a histone deacetylation inhibitor and a priming factor as active ingredients - Google Patents

Abstract

The present invention relates to a composition for promoting stem cell activity comprising a histone deacetylation inhibitor and a priming factor as active ingredients, and when stem cells are treated with a low concentration of histone deacetylation inhibitor and priming factor, the stem cell The activity of was significantly improved. In particular, primed MSCs promote cell migration, colony-forming activity and anti-inflammatory ability in cell culture conditions, which improves the therapeutic effect of hypertension, inflammatory disease or immune disease, and is therefore a useful therapeutic strategy It is expected that

Description

  The present invention relates to a composition for promoting stem cell activity comprising a histone deacetylation inhibitor and a priming factor as active ingredients.

  Mesenchymal stem-cells (MSCs) are separated from bone-marrow (BM), fat, dental pulp, umbilical cord (UC) and umbilical cord blood (UC-blood; UCB) Multipotent progenitor cells. Administration of MSC repairs the organ tissue of the damaged organ and provides a therapeutic effect. The efficacy of MSCs is due to cytoprotective effects regulated by cytokines and paracrine factors such as pro-angiogenic and pro-arterogenic effects . However, MSC treatment currently has limitations due to its therapeutic effectiveness, particularly the low engraftment and viability of transplanted cells. Most (≧ 99%) inject MSCs intravenously near the lung, but only 2-3% of these are released into the circulatory system. Therefore, it is necessary to understand the exact mechanism for the migration and engraftment of MSCs during the tissue repair process.

  Some chemokines, such as stromal cell derived factor-1 (SDF-1) and growth factors, such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (Platelet-derived growth-factor; PDGF) or hepatocyte growth-factor (HGF) plays an important role in the migration and engraftment of adult MSCs. Of these, SDF-1 and its receptor, CXCR4 axis (axis), are important factors for homing / engraftment of stem cells. Indeed, in osteoclasts, the engraftment of hematopoietic stem / progenitor cells (HSPCs) by the SDF-1 gradient is preconditioning for transplantation (eg whole body irradiation and osteotomy). After anti-cancer treatment), it is adjusted upward in the urn. The sensitivity / reactivity of HSPCs to SDF-1 gradients has a positive impact (“primed”) on the damaged tissue due to abundant molecular assembly. This includes bioactive lipids [eg, sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P)], cations derived from neutrophils And the soluble peptides cathelicidin (LL-37), β2-defensin, and a soluble membrane attack complex (sMAC) (C5b-9).

  Recently, the present inventors have confirmed that the priming phenomenon observed in the HSPCs process occurs similarly in MSCs primed with S1P and CP1 bioactive lipids and the cationic peptide LL-37. In particular, primed MSCs promote cell migration, colony forming activity and anti-inflammatory ability in cell culture conditions, which improves the therapeutic effect of pulmonary arterial hypertension (PAH). However, primed MSCs still exhibit limited in vivo engraftment with damaged tissue. In addition, prior to MSC administration, despite the intensive lavage process, it failed to completely remove the residual priming factor that triggered a negative inflammatory response. This requires the development of priming strategies at low concentrations.

  An object of the present invention is to provide a composition for promoting stem cell activity comprising a histone deacetylation inhibitor and a priming factor as active ingredients, and a method for promoting stem cell activity therethrough.

  Another object of the present invention is to provide a pharmaceutical composition for preventing or treating hypertension, which comprises, as an active ingredient, a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture solution thereof.

  Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases or immune diseases comprising, as active ingredients, stem cells treated with a histone deacetylation inhibitor and a priming factor or a culture solution thereof. That is.

  The present invention provides a composition for promoting stem cell activity comprising a histone deacetylation inhibitor and a priming factor as active ingredients.

  The present invention also provides a method for promoting stem cell activity, comprising the step of treating the isolated stem cells with a histone deacetylation inhibitor and a priming factor.

  The present invention also provides a pharmaceutical composition for preventing or treating hypertension, comprising as an active ingredient a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture solution thereof.

  The present invention also provides a pharmaceutical composition for preventing or treating inflammatory diseases or immune diseases comprising, as active ingredients, stem cells treated with a histone deacetylation inhibitor and a priming factor or a culture solution thereof.

  The present invention also provides use of a histone deacetylation inhibitor and a priming factor for promoting stem cell activity.

  In addition, the present invention provides use of a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture solution thereof in the manufacture of a drug for preventing or treating hypertension.

  In addition, the present invention provides use of a stem cell or a culture solution thereof treated with a histone deacetylation inhibitor and a priming factor in the manufacture of a drug for the prevention or treatment of inflammatory diseases or immune diseases.

  The present invention relates to a composition for promoting stem cell activity comprising a histone deacetylation inhibitor and a priming factor as active ingredients, and when stem cells are treated with a low concentration of histone deacetylation inhibitor and priming factor, the stem cell The activity of was improved significantly. In particular, primed MSCs promote cell migration, colony-forming activity and anti-inflammatory ability in cell culture conditions, which improves the therapeutic effect of hypertension, inflammatory disease or immune disease, and is therefore a useful therapeutic strategy It is expected that

Results are shown for the negative effects of 5-azacytidine (5-Aza) on UC-MSCs primed with S1P. (A) RQ-PCR analysis results for CXCR4 in human UC-MSCs treated with 1 μM 5-Aza or 0.5 mM VPA for 24 hours. Relative expression levels of the indicated genes are shown as fold change compared to untreated MSCs (NT) values, means ± SEM (n = 4), ^*** p <0.001, one-way Indicated by ANOVA test. (B) The expressed MSC surface (CD29, CD73 and CD90) and hematopoietic lineage (CD14, CD34 and CD45) protein expression in UC-MSCs primed for 24 hours with 1 μM 5-Aza and 50 nM S1P It is the result of analysis. (C) Representative images of chondrocyte, bone cell or adipocyte differentiation analysis using UC-MSCs primed with 5-Aza + S1P. Chondrocyte generation, bone cell generation and adipocyte generation were measured through Alcian Blue, Alizarin Red S and Oil Red O staining, respectively. UC-MSCs primed with 5-Aza + S1P for 24 hours (D) Cell proliferation (n = 3), (E) Chemotaxis to SDF-1 (n = 6) and (F) CFU-F analysis (n ≧ The result of 6) is shown. All results are expressed as means ± SEM. ^** p <0.01, ^*** p <0.001, compared to non-primed cells (non-parametric Mann Whitney test). Representative images for migrated cells (E) or stained colonies (F) are shown in the right panel. Results for the effect of VPA on UC-MSCs primed with S1P are shown. (A and B) Parenchyma analysis results and pluripotency for surface antigen (A) of UC-MSCs primed for 24 hours with 0.5 mM VPA alone (VPA) or in combination with 50 nM S1P (VPA + S1P) (B) The analysis results are shown. Results are shown for improved reactivity to SDF-1 with UC-MSCs primed with VPA + S1P. (A and B) Analysis of chemotaxis of MSCs exposed to 150 ng / ml SDF-1 for 24 hours in 0.5 mM VPA alone (VPA), 50 nM S1P alone (S1P) or in combination with 50 nM S1P (VPA + S1P) Results are shown. (A) Representative images of transwell inserts (insets) in migration analysis. (B) The relative amount with respect to migration was quantified as the fold change with respect to the number of migrated cells, and expressed as mean ± SEM (n = 6). ^** p <0.01, ^*** P <0.001 compared to nonprime cells (NT) (one-way ANOVA with Bonferroni post-test). (C) shows the results of Western blot analysis of phosphorylated- (p-) or whole AKT and MAPK ^{p42 / 44} . MSCs primed with VPA + S1P were treated for 12 hours in serum-deficient state and then for the time indicated on 150 ng / ml SDF-1. Results for the effect of VPA on UC-MSCs primed with S1P are shown. (A and B) Cell proliferation analysis (A, n = 3) and CFU-F analysis (B, n = 6) results for UC-MSCs primed with VPA, S1P alone or VPA + S1P for 24 hours. For CFU-F analysis, 60 cells were seeded in 6-well culture plates, cultured for 14 days, and the number of colonies was quantified. A representative stained colony of adherent cells is shown in the right panel. (C) TNF-α protein (n = 4) secreted from rat alveolar macrophage strain stimulated with LPS for 5 hours depending on the presence or absence of conditioned medium (CM) harvested from the indicated cells The quantitative result of is shown. All ^{results, means ± SEM, * p <} 0.05, ** p <0.01, *** p <0.001 compared to nonprime cells (NT), # p <0.05, ### p <0.001 compared to VPA + S1P primed cells one-way ANOVA test. Figure 3 shows VPA + S1P up-regulated genes associated with therapeutic effects of MSCs. (AD) MMP12 (A), growth factor (B) in UC-MSCs primed for 24 hours with 0.5 mM VPA alone (VPA), 50 nM S1P alone (S1P) or in combination with 50 nM S1P (VPA + S1P) ), Angiogenesis (C) and anti-inflammatory (D) related gene expression levels. The expression level was calculated as a numerical ratio of primed UC-MSCs to unprimed UC-MSCs (NT). The ^{results, means ± SEM (n ≧ 4} ); * p <0.05, ** p <0.01, *** p <0.001 compared to NT group, # p <0.05, ### p <0.001 compared to VPA + S1P (one-way ANOVA with Bonferroni post-test). Chemotaxis to SDF-1a with various VPA and S1P concentrations (Chemo) The results show improved therapeutic potential of VPA + S1P primed UC-MSC in association with bladder function recovery in the HCl-IC rat model. (A) The result of the urinary bladder pressure measurement under anesthesia is shown. (B) Control group (naive) or VPA + S1P primed UC-MSCs (5 independent animals per group) were injected at 2.5 × 10 ⁵ (250K) cell number (K = 1,000). The quantitative result of the bladder urination parameter after a week is shown. IVP; intravesical pressure, IAP; intra-abdominal pressure. Sham: sham-operated group. All ^{results, means ± SEM; * p <} 0.05, ** p <0.01, *** p <0.001 compared to the HCl-IC group; # p <0.05, ## p < 0.001, ^### p <0.001 compared to the native UC-MSC group with Bonferroni post-test. The result of the histological analysis with respect to the effect of UC-MSC administration in HCl-induced bladder injury is shown. (A) One week after administration of PBS or 250K cell count, (i) cytokeratin immunostaining (magnification x40, scale bar = 100 μm) in bladder tissue of UC-MSCs HCl-IC rats, ii) Toluidine blue (magnification × 200, scale bar = 100 μm), (iii) Masson's trichrome (magnification × 200, scale bar = 100 μm) and (iv) TUNEL analysis (magnification) × 400, scale bar = 100 μm). Arrows indicate infiltrated mast cells. Sham: Sham operation group. Nuclei were stained with Mayer's hematoxylin (i, ii and iii) or DAPI (blue, iv). (B) Results of quantification of histological experiments. Data were normalized from sham group (n = 15). The ^{results, mean ± SEM, * p <} 0.05, ** p <0.01, *** p <0.001 compared to the HCl-IC group; # p <0.05, ## p <0 001, ^## p <0.001 compared to the naïve UC-MSC group with Bonferroni post-test. In the MCT-induced PAH rat model, the effects of UCB-MSCs and VPA + S1P primed MSCs (VPA + S1P-MSC) are shown. (A) MCT induced an increase in RVSP levels, MCT infusion and administration of VPA + S1P primed UC-MSC 2 weeks later greatly reduced such elevation (left panel). MCT increased the weight ratio of RV / (LV + S) and VPA + S1P-UC-MSC greatly reduced the effect (right panel). (B) MCT increased lung tissue inflammation and medial wall thickness index (vessel wall thickness per vessel diameter), VPA + S1P-MSC greatly reduced the increase (upper panel) . Unprimed UCB-MSCs showed little improvement in RV / (LV + S) and median thickness index. The result of immunofluorescence staining of α-SMA ⁺ smooth muscle cells in the pulmonary artery is shown (lower panel) (magnification 400 ×). RVSP, right ventricular systolic pressure; CTL, control group; MSC, human cord blood mesenchymal stem cell; S1P-MSC, sphingosine-1-phosphoric acid human umbilical cord Mesenchymal stem cells; RV, right ventricle; LV + S, left ventricle and interventricular septum. ^* P <0.05, ^** p <0.01 compared to MCT alone (one-way ANOVA with Bonferroni post-test).

  The expression of CXCR4, the main target of stem cell priming, is upregulated by the DNA demethylating agent 5-azacytidine (5-Aza) and the histone deacetylation inhibitor valproic acid (VPA). Thus, the present inventors have confirmed the role of the epigenetic regulator in a strategy for improving MSC priming and promoting a therapeutic effect, thereby completing the present invention.

  The present invention provides a composition for promoting stem cell activity comprising a histone deacetylation inhibitor and a priming factor as active ingredients.

  Desirably, the histone deacetylation inhibitor may be, but is not limited to, valproic acid (VPA), sodium butyrate (NaB), nicotinamide (NAD), or searchinol. It is not a thing.

  Preferably, the priming factor may be, but is not limited to, sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), cathelicidin (LL-37) or pioglitazone. is not.

Desirably, the VPA may be included at a concentration of 0.1 to 1.0 mM, and the S1P may be included at a concentration of 10 to 50 nM, but is not limited thereto.

  The term “priming” used herein refers to a phenomenon in which reactivity (activity) is improved in order to enhance the therapeutic efficacy of stem cells. In the present invention, the histone deacetylation inhibitor that induces priming is used. And the activity of the said stem cell is promoted using a priming factor.

  In the present invention, “stem cell activity” means cell mobility, cell proliferation ability, pluripotency (in vitro differentiation into chondrocytes, bone cells or adipocyte lineage), colony forming ability or anti-inflammatory activity, etc. To do.

  In the present invention, the activity promotion or priming induction is not limited to the induction of stem cell priming when the histone deacetylation inhibitor and the priming factor are directly treated to the stem cell, but the priming treatment (pretreatment) causes the stem cell priming. Also included are those in which other differentiation is induced using stem cells with improved activity.

  In addition, the composition for promoting activity not only enhances the in vivo effect of the cell therapeutic agent by mixing it with a therapeutic cell therapeutic agent, but also in vivo treatment of the cell therapeutic agent. After that, it is also used in a method of transplanting a cell therapeutic agent with increased function in vivo.

  For example, priming of the present invention improves stem cell-related functions including stem cell mobility, colony forming ability and anti-inflammatory activity.

  As used herein, the term “stem cell” refers to a cell that has the ability to differentiate into two or more cells while having the ability to self-replicate, and is a universal stem cell or a pluripotent stem cell. cell) and multipotent stem cells.

  The stem cells of the present invention are appropriately selected without limitation depending on the purpose, and are derived from mammals including humans, desirably from any known tissue or cells derived from humans, for example, osteoclasts, umbilical cord blood, From placenta (or placental tissue cells), fat (or adipose tissue cells), etc.

  For example, the stem cells may be osteoclasts, adipose tissue, muscle tissue, ex vivo cultured autologous hepatic lobe stem cells, allogeneic hepatic stem cells, umbilical cord blood, embryonic yolk sac, placenta, umbilical cord, periosteum, fetus and adolescent skin And stem cells obtained without restriction from blood, and can be stem cells from a fetus or a newborn or an adult.

  In a preferred embodiment of the present invention, the stem cells are selected from the group consisting of neural stem cells, hepatic stem cells, hematopoietic stem cells, umbilical cord blood stem cells, epidermal stem cells, gastrointestinal stem cells, endothelial stem cells, muscle stem cells, mesenchymal stem cells and pancreatic intestinal stem cells. More preferably, it can be selected from the group consisting of hepatic stem cells, hematopoietic stem cells, umbilical cord blood stem cells and mesenchymal stem cells, but is not limited thereto.

  The present invention also provides a method for promoting stem cell activity, comprising the step of treating the isolated stem cells with a histone deacetylation inhibitor and a priming factor.

  Desirably, the histone deacetylation inhibitor may be, but is not limited to, valproic acid (VPA), sodium butyrate (NaB), nicotinamide (NAD) or searchinol.

  Desirably, the priming factor may be, but is not limited to, sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), cathelicidin (LL-37), or pioglitazone.

  Desirably, the VPA may be included at a concentration of 0.1 to 1.0 mM, and the S1P may be included at a concentration of 10 to 50 nM, but is not limited thereto.

  The present invention also provides a pharmaceutical composition for preventing or treating hypertension, which comprises, as an active ingredient, a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture solution thereof.

  On the other hand, if stem cells are activated through priming, they have an effect on cardiovascular diseases including hypertension, but there are various reports on the mechanism of action of primed stem cells related to cardiovascular diseases (Stem). Cell Research & Therapy (2015) 6: 218; Stroke. 2011; 42: 2932-2939; J. Cell. Mol. Med. Vol 17, No. 5, 2013 pp. 617-625; Stem Cells International 2015 Art ID 83). .

  The present invention also provides a pharmaceutical composition for preventing or treating inflammatory diseases or immune diseases comprising, as active ingredients, stem cells treated with a histone deacetylation inhibitor and a priming factor or a culture solution thereof.

  On the other hand, if stem cells are activated through priming, they are involved in immune regulation and inflammatory responses and applied to various inflammatory diseases and immune diseases. And various reports (J Orthop. 2016 Apr 6, 1-12; Stem Cell Rev and Rep (2014) 10: 351-375; Stem Cells International 2016 Article ID 9364213).

  Desirably, the histone deacetylation inhibitor may be, but is not limited to, valproic acid (VPA), sodium butyrate (NaB), nicotinamide (NAD) or searchinol.

  Desirably, the priming factor may be, but is not limited to, sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), cathelicidin (LL-37), or pioglitazone.

  In the present invention, the “culture solution” includes a medium that can support stem cell growth and survival in vitro, a secreted product of cultured stem cells contained in the medium, and the like. The medium used for the culture includes any normal medium used in the art suitable for culturing stem cells. The medium and culture conditions can be selected depending on the cell type. The medium used for the culture is desirably a cell culture minimum medium (CCMM), and generally includes a carbon source, a nitrogen source, and a trace element component. Examples of such a cell culture minimal medium include DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimum Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, and αMEM (αMEM). Examples include, but are not limited to, α Minimal Essential Medium), GMEM (Glasgow's Minimal Essential Medium), Iscove's Modified Dulbecco's Medium, and the like.

  The medium may contain an antibiotic such as penicillin, streptomycin, gentamicin.

  On the other hand, the present invention provides the above-described stem cell, its secreted form, a form containing all of the medium components, a form containing only the secreted substance and the medium component, a form in which only the secreted substance is separated and used alone or with the stem cell, or It is also possible to use it in a form in which it is administered alone to produce secretions in the body.

  The stem cells can be obtained using a predetermined method known to those skilled in the art.

  Stem cells treated with the histone deacetylation inhibitors and priming factors of the present invention are used as cell therapeutics for the treatment of specific diseases, which can be direct treatment or pretreatment of the molecule .

  The “cell therapeutic agent” means that living autologous, allogeneic, xenogenic cells are proliferated and sorted outside the body in order to restore cell and tissue functions, or other methods. It means a drug that is used for therapeutic, diagnostic, and preventive purposes through a series of actions such as changing the biological characteristics of cells.

  The cell therapeutic agent is administered to the human body through a predetermined general route as long as it can reach the target tissue.

In a preferred embodiment of the present invention, the hypertension is idiopathic pulmonary arterial hypertension; familial pulmonary arterial hypertension; collagen vascular disease, congenital somatic lung shunt, portal hypertension, HIV infection, pulmonary arterial hypertension associated with drugs or toxins. Pulmonary arterial hypertension associated with thyroid disorders, glycogen storage disease, Gaucher disease, hereditary hemorrhagic telangiectasia, hemochromatosis, osteoproliferative disorder or splenectomy; pulmonary arterial hypertension associated with pulmonary capillary hemangiomatosis Persistent pulmonary hypertension in newborns; chronic obstructive pulmonary disease, interstitial lung disease, hypoxia-induced alveolar hypoventilation disorder, hypoxia-induced sleep disorder breathing or pulmonary hypertension associated with high-level chronic exposure; Selected from the group consisting of pulmonary hypertension associated with developmental abnormalities; and pulmonary hypertension due to thromboembolic closure of the distal pulmonary artery, more preferably idiopathic pulmonary movement Sexual hypertension, a familial pulmonary arterial hypertension, or chronic obstructive pulmonary disease, and most preferably, idiopathic pulmonary arterial hypertension.

  In a preferred embodiment of the present invention, the inflammatory disease or immune disease is osteoarthritis, rheumatoid arthritis, cystitis, interstitial cystitis, asthma, dermatitis, atopy, psoriasis (Psoriasis), Cystic Fibrosis, Late Stage of Solid Organ Transplantation and Chronic Rejection, Posttransplantation Rate and Chronic Solid Organ Rejection, Graft-versusus-rejection, Rejection Multiple sclerosis, systemic lupus erythematosus, scho Renjou syndrome (Sjogren syndrome), Hashimoto thyroid (polymyositis), scleroderma, Addison disease, vitiligo bulb, malignant leukemia (vitiligosphere) Nephritis and pulmonary fibrosis, inflammatory bowel disease, Crohn's disease, autoimmune diabetes, rhinoretinitis, diabetic retinitis ), Ischemia reperfusion Injury (Ischemia-reperfusion injury), Restenosis after angioplasty (Post-angioplasty restenosis), Chronic obstructive lung disease (COPD), Gastrointestinal gastrointestinal gastrointestinal gastrointestinal gastrointestinal gastrointestinal gastrointestinal May be conjunctivitis, atherosclerosis, coronary artery disease, angina, cancer metastasis, small artery disease or mitochondrial disease. What There is no.

  The pharmaceutical composition of the present invention can be produced using a pharmaceutically suitable and physiologically acceptable auxiliary agent in addition to the active ingredient. Examples of the auxiliary agent include excipients, disintegrants, sweeteners, and the like. Solubilizers such as agents, binders, coatings, swelling agents, lubricants, lubricants or flavoring agents can be used. The pharmaceutical composition of the present invention can be desirably formulated as a pharmaceutical composition for administration, containing one or more pharmaceutically acceptable carriers in addition to the active ingredient. In a composition formulated in a liquid solution, acceptable pharmaceutical carriers are those suitable for sterilization and living organisms, including saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution. , Maltodextrin solution, glycerol, ethanol and one or more of these ingredients are mixed and used, and if necessary, other usual additives such as antioxidants, buffers, bacteriostatics, etc. are added Can do. In addition, diluents, dispersants, surfactants, binders and lubricants can be added in addition to injectable dosage forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets. It can be formulated.

  The pharmaceutical formulation of the pharmaceutical composition of the present invention can be composed of granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions and active compounds. It can be a sustained release formulation. The pharmaceutical composition of the present invention can be administered by intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. Can be administered in the usual manner. The effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required for prevention or treatment of diseases. Therefore, the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of dosage form and the age of the patient, body weight, general health, sex and diet, administration time, It is controlled by a variety of factors, including route of administration and composition secretion rate, duration of treatment, and concomitant drugs.

  The present invention also provides use of a histone deacetylation inhibitor and a priming factor for promoting stem cell activity.

  In addition, the present invention provides use of a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture solution thereof in the manufacture of a drug for preventing or treating hypertension.

  In addition, the present invention provides use of a stem cell or a culture solution thereof treated with a histone deacetylation inhibitor and a priming factor in the manufacture of a drug for the prevention or treatment of inflammatory diseases or immune diseases.

  Hereinafter, in order to help understanding of the present invention, examples will be described in detail. However, the following examples illustrate the contents of the present invention, and the scope of the present invention is not limited to the following examples. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art.

<Experimental example>

  The following experimental examples are provided to provide experimental examples that are commonly applied to the respective examples according to the present invention.

1. Culture of human umbilical cord-derived MSCs

Human UC was obtained from a healthy normal full-fledged newborn after receiving parental written consent according to guidelines approved by the Bioethics Committee of Ulsan Hospital in South Korea. Prior to UC collection, we received consent forms from all mothers. UC-derived MSCs (UC-MSCs) are 2 mM L-glutamine, 20 mM 4- (2-hydroxyethyl) -1-piperazine etheric acid (HEPES) (pH 7.3), minimum essential medium (minimum-sentent). medium; MEM) non-essential amino acid solution, penicillin / streptomycin (Corning Cellgro, Pittsburgh, PA), 1 mg / ml ascorbic acid (Sigma-Aldrich), 10% heat-inactivated fetal bovine serum (FBS) (HyClone), 5 ng / mL human epidermal growth factor (Sigma-Aldrich, St. Louis, MO), 10 n Low-glucose Dulbecco's modified Eagle's medium (DMEM) (HyClone, Pittsgight) supplemented with / ml basal fibroblast growth factor and 50 mg / ml long-R3 insulin-like growth factor-1 (ProSpec, Rehovot, Israel) , PA), and cultured at 37 ° C. in 5% CO ₂ atmosphere. In order to retain pluripotency, UC-MSCs that had been subcultured 5 times or less were used. Surface protein expression was analyzed as previously reported (Stem Cells and Dev. 24 (2015) 1658-1671).

2. Cell migration analysis

An 8 μm polycarbonate membrane was coated with 50 μl of 1.0% gelatin (Sigma) for 1 hour. UC-MSCs were primed for 1 day mixed with VPA (0.5 mM; Sigma-Aldrich) or 5-Aza (1 μM; Sigma-Aldrich) alone or 50 nM S1P. UC-MSCs were separated by TrypLE solution (Thermo Scientific, Pittsburgh PA), washed, resuspended in DMEM containing 0.5% bovine serum albumin (BSA) at a density of ³ × 10 ⁴ cells / well. The upper chamber of the Transwell inserts (Corning Costar, Pittsburgh, PA) was inoculated. The lower chamber was filled with 150 ng / ml SDF-1 (R & D Systems) in DMEM containing 0.5% BSA. One day later, the insert was removed from the Transwell plate. The cells remaining in the upper chamber were scraped with absorbent cotton, and the migrated cells were fixed with 4% paraformaldehyde (PFA) solution in phosphate-buffered saline (PBS), and then 0. Stained with 5% crystal violet (Sigma-Aldrich). Digital images were analyzed with Image Pro 5.0 software (Media Cybernetics, Rockville, MD, USA) to quantify stained cells on the lower surface of the membrane.

3. In vitro characterization of MSCs

  Cell proliferation of MSCs, colony-forming unit-fibroblast (CFU-F), pluripotency (in vitro differentiation into chondrocytes, bone cells or adipocyte lineage) and in vitro anti-inflammatory analysis , As previously reported (Stem Cells and Dev. 24 (2015) 1658-1671).

4). Western blot and real-time quantitative PCR (RQ-PCR)

UC-MSCs primed with 50 nM S1P and 0.5 mM VPA were starved for 12 hours at 37 ° C. in DMEM containing 0.5% BSA, and 5, 10, 20 or 150 ng / ml SDF-1 After stimulation for 30 minutes, phosphorylation of mitogen-activated protein kinase (MAPK) ^{p42 / 44} and AKT (Ser473) was analyzed through Western blot using 30 μg-cell extract. For gene expression analysis, total RNA obtained from the displayed cells was reverse transcribed and the displayed transcripts were quantified through RQ-PCR.

5. Statistical analysis

  To confirm statistical significance differences, data were analyzed using non-parametric Mann Whitney test or one-way ANOVA with the Bonferroni post-hoc test. We performed any analysis using GraphPad Prism 6.0 software (GraphPad Software, La Jolla, Calif.), And statistical significance was p <0.05, 0.01, or 0.00. 001.

6). Experimental design

  The object of the present invention is to treat human umbilical cord-derived MSCs (umbilical cord) primed with V1P + S1P to treat interstitial cystitis / bladder pain syndrome (IC / BPS) in a rat model. In addition to measuring the effects of -delivered MSCs (UC-MSCs), we also tried to observe a wide range of in vivo cellular properties of transplanted cells. Control group or V1P + S1P primed UC-MSCs were administered to rats with bladder injury, bladder urination function, urothelium degeneration, mast cell infiltration, tissue fibrosis, cell apoptosis and tumor formation The effect on was measured. In every experiment, two independent experiments were performed on 5 independent animals per group. These were treated by arbitrarily allocating to the injury group, cell transplantation or vehicle administration group and cystometry group. Investigators involved in the surgical process were not informed about the morphology and dose of cells administered. All intravesical pressure measurements, histology and gene expression measurements were performed by researchers who had no information about the treatment group. Any animals that died unexpectedly due to bladder injury or catheterization were excluded from any analysis. All animal experiments were conducted according to the guidelines and regulations of the Animal Experiment Ethics Committee of Ulsan University School of Medicine, Korea (IACUC-2014-14-167).

7). Animal models and transplantation of UC-MSCs

The HCl infusion IC / BPS rat model has been previously reported (Stem Cells and Development 24, 2015, 1648-1657). One week after HCl injury, the lower abdomen was dissected and 2.5 × 10 ⁵ (250K) UC-MSCs or PBS were administered directly to the entire bladder outer wall and ceiling using a 500 μm syringe and 26 cage needle. One day prior to stem cell administration, indomethacin (indomethacin; PMG Pharm Co., Ltd. Ansan, Korea; every 12 hat 2.5 mg / kg) or gefitinib (Gefitinib) to block Wnt or IGF-mediated signaling (Santa Cruz Biotechnology, Santa Cruz, CA, USA; every day at 5 mg / kg) was injected subcutaneously.

8). Unanesthetized and unrestored intravesical pressure measurement collection (intravesical pressure measurement without anesthesia)

  Intravesical pressure measurements (Cytomometrics) were performed in unanesthetized and unsuppressed rats in metabolic cages. For recording measurements of intravesical pressure (IVP) and intraperitoneal pressure (IAP), a catheter was simultaneously inserted 3 days before the measurement of intravesical pressure. Briefly, after induction of anesthesia, a polyethylene catheter (PE-50; Becton-Dickinson, Parsippany, NJ, USA) with a cuff was inserted through the abdominal incision into the bladder ceiling. To record the IAP, a catheter tip near cuff abdominal cavity (Latex; Daewoo Medical, Incheon, Korea) was positioned proximal to the bladder and tied to another catheter with a silk thread. A polyethylene catheter (PE-50) was heated with warm water, and the tip of the insertion section was stretched about 1.5 times the original length and filled with heparinized saline (100 IU / mL). Once the bladder catheter was implanted, the extended catheter was inserted into the femoral vein. The catheter then pierced through the subcutaneous space, exited through the animal's back and attached to the skin on the back. After surgery, each rat was individually housed and held in the same manner.

  Pressure transducer (Research Grade blood pressure transducer, HD, P, H, and H), for non-anesthetized intravesical pressure measurement analysis (Research Grade blood pressure transducer, HD, P, and H). An indwelling catheter was connected to the bladder with a double valve through a T-tube connected to the Apparatus. In order to record the IAP, another indwelling catheter to which a peritoneal balloon filled with body fluid was connected was connected to another pressure transducer. Sterilized saline was infused into the bladder at a rate of 0.4 mL / min and urine output continued to be recorded through a fluid collector connected with a force displacement transducer (Research Grade Isotopic Transducer; Harvard Apparatus). MPP data acquisition system (Biopac Systems, Goleta, CA, USA) equipped with Acq Knowledge 3.8.1 software at a sampling rate of 50 Hz was used to continue recording IVP, IAP and urine output. All micturition values measured for 8 minutes from each animal were used for evaluation.

If the IVP increased from the reference value to exceed 15 cm H ₂ O without urine excretion, it was measured by non-voiding contraction (NVC). BP means minimum bladder pressure while the bladder is filled, MP means maximum bladder pressure during the micturition cycle, MV means urine output at urine output, and RV is after urine output Means residual urine volume. BC is defined as MV + RV and MI means the interval between micturition contractions.

9. Pulmonary arterial hypertension (PAH) animal model

Monocrotaline (MCT) induced PAH rat model was established by previous reports (Stem Cells and Development 24, 2015, 1658-1671). Male free pathogenic free Lewis rats (8 weeks old, 250-280 g) were raised without food and water restrictions under the control of breeding temperature and light (12-hour light-dark cycle). PAH was induced through subcutaneous administration of MCT (60 mg / kg, Sigma). Control rats received the same volume of PBS. Two weeks after MCT or PBS administration, unprimed UC-MSCs or UC-MSCs pretreated with VPA + S1P were administered through the tail vein at a density of 2.5 × 10 ⁵ cells per 200 μl PBS. A control group treated with solvent alone (vehicle control) received 200 μl PBS without cells. Prior to injection, cells were washed twice with warm PBS and the viability of the administered cells was observed using FACS analysis through 7-AAD (BD Biosciences) exclusion staining.

10. Measurement of systolic right ventricular pressure (RVSP) and right ventricular hypertrophy (RVH)

  4 weeks after administration of MCT or PBS, patient monitoring (respiratory maintenance through ventilator (Harvar Apparatus, Holliston, Mass.)) And anesthesia with zoletil (40 mg / kg) and rompun (10 mg / kg). Systolic right ventricular pressure was measured through direct puncture of the diaphragm using a 26G needle coupled with MDE Escort II patient monitor (Arletta). Right ventricular pressure (RVP) allowed breathing through the ventilator. To measure RVH, the right ventricle of the heart was separated from the ventricular septum and the right ventricle (RV) and the left ventricle (LV + S) including the ventricular septum were weighed.

11. Histology and gene expression analysis

  For histological analysis of bladder tissue, epithelial cell detachment through immunostaining for cytokeratin, toluidine blue staining (8544-4125; Daejung Chemicals & Metals, Seoul, Korea) mast cell infiltration, Masson trichrome Cell death was measured through tissue fibrosis and TUNEL staining (1 684 795; Roche, Mannheim, Germany) through staining (Masson's trichrome staining; Junsei Chemical, Tokyo, Japan). For lung tissue, H & E- or α-smooth muscle actin (α-SMA)-arbitrarily selected with a thickness of 4 μm and a diameter of 25-100 μm at the stained site Captured at 400 × magnification at least 5 times for arbitrarily selected areas of blood vessels. Anti-α-SMA (1: 100, Abcam) was applied and reacted according to the manufacturer's recommended protocol. Nuclei were counterstained with 4 ', 6'-diamino-2-phenylindole (4', 6'-diamino-2-phenylindole; D9542; DAPI, Sigma-Aldrich). The thickness of the media was measured using the NIH ImageJ program (http://rsbweb.nih.gov/ij/). The media thickness index is defined as the ratio of the outer diameter to the inner diameter with respect to the outer diameter.

  For gene expression analysis, total RNA was prepared using RNeasy Mini Kit (Qiagen Inc., Valencia, Calif.), Reverse transcription using TaqMan Reverse Transcription Reagents (Applied Biosystems), and Rek. -Real-time quantitative PCR (RQ-PCR) was performed using Time PCR System (Thermo Scientific) and iQ SYBR Green PCR Master Mix (Bio-Rad, Hercules, CA). In 5 independent animals per treatment group, a three-part area arbitrarily selected from each slide (n = 15) was used for quantification of digital images. Similarly, RQ-PCR was repeated twice from 5 animals arbitrarily selected per group (n = 10) to obtain gene expression data.

<Example 1> Effect of 5-Aza on human UC-MSCs priming by S1P

  Low concentrations of 5-Aza and VPA (1 μM and 0.5 mM) greatly increase CXCR4 expression in UC-MSCs (FIG. 1A). The inventors then confirmed the effect of the epigenetic regulator on the basic characteristics of UC-MSCs. Independent of priming 50 nM S1P, 5-Aza and VPA did not significantly affect the expression of surface marker proteins (CD29, CD73 and CD90 positive, CD34 and CD45 negative) (FIG. 1B and FIG. 2A). In addition, S1P priming treated with 5-Aza (5-Aza + S1P) or VPA (VPA + S1P) had little effect on multilineage differentiation ability, but multilineage differentiation ability was not limited to glycosaminoglycans (glycosaminoglycans; Alcian blue), mineral deposition (alizarin red S) and lipid accumulation (oil red O staining) levels are measured to measure the in vitro differentiation of chondrocytes, bone cells and adipocyte lineages, respectively. Based on the analysis (FIGS. 1C and 2B).

  Contrary to the up-regulation of CXCR4 (FIG. 1A), in Transwell migration analysis, UC-MSCs primed with 5-Aza + S1P are about 30% more than cells that are not primed for migration activity for the SDF-1 response. It decreased to a certain extent (FIG. 1E). In addition, 5-Aza + S1P priming severely damaged the activity of clonogenic CFU-F, which shows the frequency of self-renewal in clonogenic progenitor cells (FIG. 1F). Cell growth of MSCs was almost unchanged by priming 5-Aza + S1P (FIG. 1D).

Example 2 VPA that enhances UC-MSCs priming during low concentration S1P treatment

Next, the inventors investigated the effect of VPA on the priming of UC-MSCs during treatment with low concentrations of S1P. To this end, we applied 50 nM S1P, 4 times less than the optimal capacity (200 nM) used for priming of fat- and UCB-derived MSCs. Unlike VPA alone and S1P alone, UC-MSCs priming increased chemotaxis to SDF-1 2-3 times higher than unprimed cells when treated with VPA + S1P. (FIGS. 3A and 3B). The response to SDF-1 enhanced by VPA + S1P priming appeared completely different from that of 5-Aza + S1P priming. The inventors then confirmed the state of the signal transmission path associated with the movement of HSPCs. Consistent with chemotaxis analysis, UC-MSCs exposed to VPA + S1P increased phosphorylation of MAPK ^{p42 / 44} and AKT proteins, indicating activation of the signaling pathway (FIG. 3C). The results show that unlike 5-Aza, VPA can activate MAPK ^{p42 / 44} and AKT signaling pathways and promote UC-MSCs priming even at low concentrations of S1P .

<Example 3> Improvement of therapeutic ability of UC-MSCs primed with VPA + S1P

  We tested the effect of VPA + S1P priming on other cellular activities associated with the therapeutic effects of MSCs. Unlike 5-Aza + S1P (FIG. 1), UC-MSCs primed with VPA + S1P improved both cell proliferation (FIG. 4A) and clonogenic CFU-F activity (FIG. 4B). However, in UC-MSCs treated with VPA alone and S1P alone, cell proliferation ability and CFU-F activity increase are not observed (FIGS. 4A and 4B). Since MSCs affect anti-inflammatory and immunomodulatory capabilities, we tested whether VPA + S1P priming affects the anti-inflammatory effect of UC-MSCs. This allows us to prepare conditioned media collected from unprimed UC-MSCs or VPA alone or UC-MSCs primed with VPA + S1P, where the conditioned media is lipopolysaccharide (lipopolysaccharide); Whether or not secretion of tumor necrosis factor-α (TNF-α) from MH-S cells, which are alveolar macrophages cells stimulated with LPS, can be suppressed. Was tested. Conditioned media collected from UC-MSCs was effective in reducing TNF-α secretion from LPS-stimulated MH-S cells (FIG. 4C). In particular, conditioned media collected from VPA + S1P-primed UC-MSCs further suppressed TNF-α secretion than unprimed UC-MSCs or UC-MSCs primed with VPA alone. We used CM collected with IMR90, a human lung fibroblast, as a control group, which was shown to further increase TNF-α secretion in an in vitro anti-inflammatory assay.

  To confirm the molecular mechanism for the effects of VPA + S1P priming, we tested the expression of growth factors, pro-inflammatory cytokines and anti-inflammatory factors secreted by MSCs. Consistent with the experimental results of cytological properties described above, UC-MSCs primed with VPA + S1P are growth factors and their receptors [e. g. , PDGFB, PDGFRB, cMET] (FIG. 5B), pre-angiogenesis— [e. g. , VEGFB, VEGFC, ANGPT1, ANGPT2] (FIG. 5C), anti-inflammatory-[e. g. , LIF, TSG6, IDO1, IDO2] (FIG. 5D) and stem cell migration— [e. g. , MMP12] (FIG. 5A) highly upregulated the expression of related factors. However, in UC-MSCs treated with VPA alone and S1P alone, no significant change was observed in the gene expression described above (FIGS. 4A and 4B). In addition, 5-Aza + S1P priming down-regulated the aforementioned genes that were affected by VPA + S1P priming. Taken together, the results show that priming of MSCs through treatment with minimal concentrations of VPA and S1P effectively promotes MSCs migration, proliferation, self-renewal and anti-inflammatory capacity, Plays an important role in therapeutic potential.

<Example 3> Analysis of chemotaxis to SDF-1a with various VPA and S1P concentrations

  The inventors attempted to determine the lowest concentrations of VPA and S1P by performing chemotaxis analysis on SDF-1a with various VPA and S1P concentrations.

  As shown in FIG. 6, VPA was fixed at 0.5 mM and the S1P concentration was tested at 5 to 50 nM. As a result, the lowest concentration of S1P was determined to be 10 nM (FIG. 6 a). In addition, S1P was fixed at 50 nM and the VPA concentration was experimented at 0.05 to 0.5 mM. As a result, the lowest concentration of VPA was determined to be 0.1 nM (FIG. 6b).

<Example 4> Confirmation of in vivo treatment possibility of VPA + S1P primed UC-MSC in IC / BPS treatment

The inventors then confirmed the in vivo effects of UC-MSCs using an IC / BPS animal model established with HCl infusion. As a result of bladder function analysis using urinary bladder pressure measurement without anesthesia, HCl infusion-induced IC / BPS rats (HCl-IC group) were more irregular than control-group (sham-operated; sham) rats. Not only decreased micturition interval (MI), but also decreased micturition volume (MV), maximum pressure, micturition pressure (MP) and bladder capacity (BC) (FIGS. 7a and 7b). Single transplantation of 2.5 × 10 ⁵ cell number control UC-MSCs (UC-MSC 250K) or VPA + S1P primed cells (UC-MSC 250K + VPA + S1P) improved impaired micturition parameters . Importantly, VPA + S1P primed UC-MSCs showed a better effect than control cells (FIGS. 7a and 7b). In particular, animals in the HCl-IC group showed increased contraction frequency during non-voiding periods (NVC), but such symptoms were greatly improved with UC-MSCs primed with VPA + S1P. However, the control group UC-MSCs did not improve much (FIGS. 7a and 7b). The results of urinary bladder pressure measurement under anesthesia were also consistent with the function improvement effect, but serious urothelial detachment in the rat group by administration of the control group or 2.5 × 10 ⁵ UC-MSCs primed with VPA + S1P, It was shown that abnormal histological phenomena such as mast cell infiltration, fibrosis, and cell death were recovered (FIGS. 8a and 8b). Such an abnormal histological phenomenon is a characteristic that appears also in human IC / BPS bladder. In particular, restoration of histological deformation showed an even better improvement effect in bladder tissue transplanted with UC-MSC primed with VPA + S1P compared to control group cells. Taken together, the results indicated that VPA + S1P priming can greatly improve the therapeutic potential of UC-MSCs in IC bladder treatment.

Example 5 Improved VPA + S1P-mediated therapeutic potential of UC-MSC in PAH animal model

The inventors then confirmed the effect of VPA + S1P priming under in vivo conditions using an MCT-induced PAH animal model. Similar to previous reports, RVSP was greatly increased 4 weeks after MCT administration. Unlike the control UC-MSC (PAH + MSC group), administration of UC-MSC primed with VPA + S1P (PAH + VPA + S1P-MSC group) greatly reduced the increase in MCT-induced RVSP (FIG. 9a). ). Also, RV / (LV + S) increased after MCT administration, but VPA + S1P-MSC greatly reduced RV hypertrophy, and unprimed UC-MSC was less effective (FIG. 9a). ). In addition, VPA + S1P-MSC administration decreased lung tissue inflammation, medial thickness index and α-SMA ⁺ smooth muscle cell increase induced by MCT (FIG. 9b). Taken together, the results indicated that priming of UC-MSC with VPA + S1P can induce a microenvironment that promotes neovascularization and suppresses inflammatory responses.

  As described above, specific portions of the present invention have been described in detail. For those skilled in the art, such specific descriptions are merely desirable embodiments, and the scope of the present invention is not limited thereby. Is obvious. Accordingly, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (20)

  A composition for promoting stem cell activity comprising a histone deacetylation inhibitor and a priming factor as active ingredients.   The histone deacetylation inhibitor is any one or more selected from the group consisting of valproic acid (VPA), sodium butyrate (NaB), nicotinamide (NAD), and searchinol. Item 10. The composition for promoting stem cell activity according to Item 1.   The priming factor is any one or more selected from the group consisting of sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), cathelicidin (LL-37), and pioglitazone. The composition for promoting stem cell activity according to claim 1, wherein the composition is for promoting stem cell activity.   The composition for promoting stem cell activity according to claim 2, wherein the VPA is contained at a concentration of 0.1 to 1.0 mM.   The composition for promoting stem cell activity according to claim 3, wherein the S1P is contained at a concentration of 10 to 50 nM.   The stem cells are selected from the group consisting of neural stem cells, hepatic stem cells, hematopoietic stem cells, umbilical cord blood stem cells, epidermal stem cells, gastrointestinal stem cells, endothelial stem cells, muscle stem cells, mesenchymal stem cells, and pancreatic intestinal stem cells. The composition for promoting stem cell activity according to any one of claims 1 to 5.   The composition for promoting stem cell activity according to any one of claims 1 to 5, wherein the stem cell activity is cell mobility, colony forming ability, and anti-inflammatory activity.   A method for promoting stem cell activity, comprising a step of treating a separated stem cell with a histone deacetylation inhibitor and a priming factor.   The histone deacetylation inhibitor is any one or more selected from the group consisting of valproic acid (VPA), sodium butyrate (NaB), nicotinamide (NAD), and searchinol. Item 9. The method for promoting stem cell activity according to Item 8.   The priming factor is any one or more selected from the group consisting of sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), cathelicidin (LL-37), and pioglitazone. The method for promoting stem cell activity according to claim 8, which is characterized by:   The method for promoting stem cell activity according to claim 9, wherein the VPA is treated at a concentration of 0.1 to 1.0 mM.   The method for promoting stem cell activity according to claim 10, wherein the S1P is treated at a concentration of 10 to 50 nM.   A pharmaceutical composition for preventing or treating hypertension, comprising a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture solution thereof as an active ingredient.   The histone deacetylation inhibitor is any one or more selected from the group consisting of valproic acid (VPA), sodium butyrate (NaB), nicotinamide (NAD), and searchinol. Item 14. A pharmaceutical composition for preventing or treating hypertension according to Item 13.   The priming factor is any one or more selected from the group consisting of sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), cathelicidin (LL-37), and pioglitazone. The pharmaceutical composition for prevention or treatment of hypertension according to claim 13.   Said hypertension is idiopathic pulmonary arterial hypertension; familial pulmonary arterial hypertension; collagen vascular disease, congenital somatic lung shunt, portal hypertension, HIV infection, pulmonary arterial hypertension associated with drugs or toxins; thyroid disorder, glycogen storage disease, Gaucher disease, hereditary hemorrhagic telangiectasia, hemochromosis, pulmonary arterial hypertension associated with osteoproliferative disorders or splenectomy; pulmonary arterial hypertension associated with pulmonary capillary hemangiomatosis; persistent neonatal pulmonary hypertension; chronic obstructive Lung disease, interstitial lung disease, hypoxia-induced alveolar hypoventilation disorder, hypoxia-induced sleep disorder breathing or pulmonary hypertension associated with chronic exposure at high altitude; pulmonary hypertension associated with developmental abnormalities; and distal 14. The pharmaceutical composition for preventing or treating hypertension according to claim 13, wherein the pharmaceutical composition is selected from the group consisting of pulmonary hypertension due to thromboembolic closure of the pulmonary artery.   A pharmaceutical composition for preventing or treating inflammatory diseases or immune diseases, comprising as an active ingredient a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture solution thereof.   The histone deacetylation inhibitor is any one or more selected from the group consisting of valproic acid (VPA), sodium butyrate (NaB), nicotinamide (NAD), and searchinol. Item 18. A pharmaceutical composition for preventing or treating an inflammatory disease or immune disease according to Item 17.   The priming factor is any one or more selected from the group consisting of sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), cathelicidin (LL-37), and pioglitazone. The pharmaceutical composition for prevention or treatment of inflammatory disease or immune disease according to claim 17 characterized by the above.   The inflammatory diseases or immune diseases include osteoarthritis, rheumatoid arthritis, cystitis, interstitial cystitis, asthma, dermatitis, atopy, psoriasis, cystic fibrosis, solid organ transplant late stage and chronic rejection, graft pair Host disease, transplant rejection, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, Hashimoto's thyroid, polymyositis, scleroderma, Addison's disease, leukoplakia, pernicious anemia, glomerulonephritis and pulmonary fibrosis, inflammation Enteropathy, Crohn's disease, autoimmune diabetes, diabetic retinopathy, rhinitis, ischemia-reperfusion injury, restenosis after angioplasty, chronic obstructive pulmonary disease (COPD), Graves' disease, gastrointestinal allergy, conjunctivitis, 18. The prevention of inflammatory disease or immune disease according to claim 17, wherein the disease is selected from the group consisting of atherosclerosis, coronary artery disease, angina pectoris, cancer metastasis, small artery disease and mitochondrial disease. Therapeutic pharmaceutical compositions.