KR102097191B1 - Composition for enforcing the priming effect of stem cell comprising histone deacetylase inhibitor and priming factors - 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 an active ingredient, and when a low concentration of a histone deacetylation inhibitor and a priming factor is treated on the stem cell, the activity of the stem cell Excellent improvement. In particular, primed MSCs promote cell migration, colony-forming activity, and anti-inflammatory ability in cell culture conditions, which can be a useful therapeutic strategy, as they improve the effect of treating hypertension, inflammatory disease or immune disease. do.

Description

Composition for enforcing the priming effect of stem cell comprising histone deacetylase inhibitor and priming factors}

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

Mesenchymal stem-cells (MSCs) are isolated from bone-marrow (BM), fat, dental pulp, umbilical-cord (UC) and cord blood (UC-blood; UCB). , A multipotent progenitor cell. The administration of MSC restores the organ tissue of the damaged organ and provides a therapeutic effect. The efficacy of MSC is due to cytokine-controlled cytoprotective effects and paracrine factors such as pro-angiogenic and pro-arteriogenic effects. However, current MSC treatment has limitations due to its low therapeutic efficacy, particularly in vivo engraftment and low survival rate of transplanted cells. Most (≥99%) inject MSCs intravenously near the lungs, of which only 2-3% are released into the circulatory system. Therefore, it is necessary to understand the exact mechanism for the movement and engraftment of MSCs in 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), platelets- The 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. Among them, SDF-1 and its receptor, CXCR4 axis, are important factors for homing / engraftment of stem cells. In fact, engraftment of hematopoietic stem / progenitor cells (HSPCs) according to the SDF-1 gradient in the bone marrow is preconditioning for transplantation (e.g., whole body irradiation and bone marrow excision) After chemotherapy) is up-regulated in the bone marrow. The sensitivity / responsiveness of HSPCs to the SDF-1 gradient is positively influenced by a rich set of molecules in the damaged tissue ("primed"). These include bioactive lipids (eg, sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P)), neutrophil-derived cationic peptide casserici Cathelicidin (LL-37), β2-defensin and soluble membrane attack complex (sMAC) (C5b-9).

The present inventors recently confirmed that the priming phenomenon observed in the course of HSPCs occurs similarly in MSCs primed with S1P and CP1 bioactive lipid and cationic peptide LL-37. In particular, primed MSCs promote cell migration, colony-forming activity and anti-inflammatory ability in cell culture conditions, which enhance the effect of treating pulmonary arterial hypertension (PAH). However, primed MSCs still show limited in vivo engraftment in damaged tissue. Moreover, despite the intensive washing process prior to administration of MSC, it failed to completely remove residual priming factors that triggered a negative inflammatory response. Therefore, it is necessary to develop a priming strategy at a low concentration.

Korean Registered Patent No. 10-1471785 (Registered on Dec. 4, 2014)

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

In addition, another object of the present invention is to provide a pharmaceutical composition for preventing or treating hypertension comprising stem cells treated with a histone deacetylation inhibitor and a priming factor or a culture medium thereof as an active ingredient.

In addition, another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases or immune diseases comprising stem cells treated with a histone deacetylation inhibitor and a priming factor or a culture medium thereof as an active ingredient. Is in

The present invention provides a composition for promoting stem cell activity, including a histone deacetylation inhibitor and a priming factor as an active ingredient.

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

In addition, the present invention provides a pharmaceutical composition for preventing or treating hypertension comprising stem cells treated with a histone deacetylation inhibitor and a priming factor or a culture medium thereof as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease or an immune disease, comprising as an active ingredient a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture medium thereof.

The present invention relates to a composition for promoting stem cell activity comprising a histone deacetylation inhibitor and a priming factor as an active ingredient, and when a low concentration of a histone deacetylation inhibitor and a priming factor is treated on the stem cell, the activity of the stem cell Excellent improvement. In particular, primed MSCs promote cell migration, colony-forming activity, and anti-inflammatory ability in cell culture conditions, which can be a useful therapeutic strategy, as they improve the effect of treating hypertension, inflammatory disease or immune disease. do.

1 shows the results for the negative effect of 5-azacytidine (5-Aza) in UC-MSCs primed with S1P. (A) Results of RQ-PCR analysis for CXCR4 in human UC-MSCs treated with 1-μM 5-Aza or 0.5-mM VPA for 24 hours. The relative expression level of the indicated genes was expressed as a change in magnification compared to the value of untreated MSCs (NT), and represented by means ± SEM (n = 4), *** p <0.001, one-way ANOVA test. (B) Expression of marked MSC surfaces (CD29, CD73 and CD90) and hematopoietic lineage (CD14, CD34, and CD45) proteins in UC-MSCs primed with 1-μM 5-Aza and 50-nM S1P for 24 hours. It is the result of flow cytometry analysis. (C) Representative images of chondrocyte, osteoblast, or adipocyte differentiation analysis using UC-MSCs primed with 5-Aza + S1P. Chondrocyte production, osteoblast production and adipocyte production were measured by Alcian Blue, Alizarin Red S and Oil Red O staining, respectively. (D) Cell proliferation (n = 3), (E) chemotaxis to SDF-1 (n = 6) and (F) CFU-F analysis of UC-MSCs primed with 5-Aza + S1P for 24 hours ( n≥6). All results were 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.
2 shows the results for the effect of VPA in UC-MSCs primed with S1P. (A and B) Flow cytometry results and pluripotency of surface antigens (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.
3 shows the results for improved responsiveness to SDF-1 in UC-MSCs primed with VPA + S1P. (A and B) 150-ng / ml SDF-1 of MSCs exposed for 24 hours to 0.5-mM VPA alone (VPA), 50-nM S1P alone (S1P) or in combination with 50-nM S1P (VPA + S1P) The results of the analysis of chemotaxis for are shown. (A) Representative images of transwell insets in the migration assay. (B) The relative amount for migration was quantified as a change in magnification for the number of migrated cells and expressed as means ± SEM (n = 6). ** p <0.01, *** P <0.001 compared to nonprime cells (NT) (one-way ANOVA with Bonferroni post-test). (C) Western blot analysis of phosphorylated- (p-) or total AKT and MAPK ^{p42 / 44} . MSCs primed with VPA + S1P were treated for 12 hours in a serum-deficient state followed by the time indicated as 150-ng / ml SDF-1.
4 shows the results for the effect of VPA in UC-MSCs primed with S1P. (A and B) Cell proliferation assay of UC-MSCs primed for 24 hours with VPA, S1P alone or VPA + S1P (A, n = 3) and CFU-F analysis (B, n = 6) It shows the result. For CFU-F analysis, 60 cells were seeded in 6-well culture plates, cultured for 14 days, and colony count was quantified. Representative stained colonies of adherent cells are shown in the right panel. (C) Quantitative results of TNF-α protein (n = 4) secreted from rat alveolar macrophage lines stimulated with LPS for 5 hours depending on the presence or absence of conditioned medium (CM) harvested from the indicated cells. . All results are 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.
5 shows VPA + S1P up-regulated genes related to the therapeutic effect of MSCs. (AD) MMP12 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) (A), growth factors (B), angiogenesis (C) and anti-inflammatory (D) related expression levels. Expression levels were calculated as the numerical ratio of primed UC-MSCs to unprimed UC-MSCs (NT). Results were 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) Showed.
6 shows the results of chemotaxis analysis for SDF-1a according to various VPA and S1P concentrations.
7 is a result showing the improvement in the treatment potential of VPA + S1P primed UC-MSC in relation to bladder function recovery in the HCl-IC rat model. (a) Results of awake cystometry under non-anesthesia. (b) Bladder urination parameters 1 week after injecting control group (naive) or VPA + S1P primed UC-MSCs (5 independent animals per group) into 2.5 × 10 ⁵ (250 K) cell numbers (K = 1,000) It shows the quantitative results. IVP; Intravesical pressure, IAP; Intra-abdominal pressure. Sham: Stom-operated. All results were 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 naive UC-MSC group with Bonferroni post-test.
Figure 8 shows the results of histological analysis of the effect of UC-MSC administration in HCl-induced bladder injury. (a) One week after administration of PBS or 250 K cell number, (i) cytokeratin immunostaining (magnification x40, scale bar = 100 μm) in bladder tissue of UC-MSCs HCl-IC mice, (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: Stom-operated. Nuclei were stained with Mayer's hematoxylin (i, ii and iii) or DAPI (blue, iv). (b) It is the result of quantifying histological experiment. Data were normalized from the sham group (n = 15). Results were 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 naive UC-MSC group with Bonferroni post-test.
9 shows the effect of UCB-MSCs and VPA + S1P primed MSCs (VPA + S1P-MSC) in the MCT induced PAH rat model. (a) MCT induced an increase in RVSP levels, and this increase was significantly reduced as a result of administration of VPA + S1P primed UC-MSC 2 weeks after MCT injection (left panel). MCT increased the weight ratio of RV / (LV + S), and VPA + S1P-UC-MSC significantly reduced the effect (right panel). (b) MCT increased lung tissue inflammation and medial wall thickness index (vascular wall thickness per vessel diameter), and VPA + S1P-MSC significantly reduced this increase (top panel). The unprimed UCB-MSC showed little improvement effect of RV / (LV + S) and media thickness index. Immunofluorescence staining results of α-SMA ⁺ smooth muscle cells in the pulmonary artery (lower panel) (magnification 400 ×). RVSP, right ventricular systolic pressure; CTL, control; MSC, human cord blood mesenchymal stem cells; S1P-MSC, sphingosine-1-phosphate primed human umbilical 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).

Expression of CXCR4, a major target for stem cell priming, is up-regulated by the DNA-demethylating agent 5-azacytidine (5-Aza) and histone deacetylation inhibitor valproic acid (VPA). do. Accordingly, the present inventors confirmed the role of the epigenetic modulators in a strategy for improving MSC priming and promoting a therapeutic effect, and completed the present invention.

The present invention provides a composition for promoting stem cell activity, including a histone deacetylation inhibitor and a priming factor as an active ingredient.

Preferably, the histone deacetylation inhibitor may be valproic acid (VPA), sodium butyrate (NaB), nicotinamide (NAD), or sirtinol, but is not limited thereto. It does not work.

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

Preferably, 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" as used herein refers to a phenomenon in which reactivity (activity) is improved to enhance the therapeutic efficacy of stem cells, and in the present invention, a histone deacetylation inhibitor and priming factor that induces the priming. (priming factor) to promote the activity of the corresponding stem cells.

In the present invention, "stem cell activity" refers to stem cell cell mobility, cell proliferation ability, pluripotency (ex vivo differentiation into chondrocytes, bone cells or adipocyte lineage), colony forming ability or anti-inflammatory activity, etc. .

In the present invention, the activity promotion or priming induction is not only induced by priming of the stem cells when the histone deacetylation inhibitor and the priming factor are directly treated on the stem cells, but also by the priming treatment (pre-treatment). This includes inducing differentiation by using stem cells with improved stem cell activity.

In addition, the composition for promoting activity can not only enhance the in vivo effect of the cell therapy agent by injecting it in vivo with a cell therapy agent for treatment, but also a cell therapy agent with increased function after treating the composition on the stem cells themselves. It can also be used as a method of implanting in vivo.

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

The term "stem cell" as used herein refers to a cell having the ability to differentiate into two or more cells while having self-replication ability, totipotent stem cells, pluripotent stem cells , Can be classified as multipotent stem cells.

The stem cells of the present invention may be appropriately selected without limitation depending on the purpose, and may be derived from adult cells such as mammals including humans, preferably all known tissues and cells derived from humans, for example, Bone marrow, umbilical cord blood, placenta (or placental tissue cells), fat (or adipose tissue cells), and the like.

For example, the stem cells are restricted from bone marrow, adipose tissue, muscle tissue, ex vivo cultured autologous mesenchymal stem cells, allogeneic mesenchymal stem cells, umbilical cord blood, embryonic yolk sac, placenta, umbilical cord, periosteum, fetal and adolescent skin, and blood It may be a stem cell obtained without, or may be a stem cell derived from a fetus or immediately after birth or from an adult.

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

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

Preferably, the histone deacetylation inhibitor may be valproic acid (VPA), sodium butyrate (NaB), nicotinamide (NAD), or sirtinol, but is not limited thereto. It does not work.

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

Preferably, 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.

In addition, the present invention provides a pharmaceutical composition for preventing or treating hypertension comprising stem cells treated with a histone deacetylation inhibitor and a priming factor or a culture medium thereof as an active ingredient.

On the other hand, when stem cells are activated through priming, they have an effect on cardiovascular diseases including hypertension, and there have been several reports on the mechanism of action related to the cardiovascular disease of primed stem cells (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 Article ID 685383).

In addition, the present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease or an immune disease, comprising as an active ingredient a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture medium thereof.

On the other hand, when stem cells are activated through priming, they are involved in immune regulation and inflammatory reactions, and can be applied to various inflammatory diseases and immune diseases, and there are various reports on mechanisms related to immune regulation and inflammatory responses of primed stem cells. (J Orthop Res. 2016 Apr 6, 1-12; Stem Cell Rev and Rep (2014) 10: 351-375; Stem Cells International 2016 Article ID 9364213).

Preferably, the histone deacetylation inhibitor may be valproic acid (VPA), sodium butyrate (NaB), nicotinamide (NAD), or sirtinol, but is not limited thereto. It does not work.

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

In the present invention, the "culture medium" includes a medium capable of supporting stem cell growth and survival in vitro, a secretion of cultured stem cells contained in the medium, and the like. The medium used for cultivation includes all common mediums used in the art suitable for culturing stem cells. Media and culture conditions can be selected according to the cell type. The medium used for the culture is preferably a cell culture minimum medium (CCMM), and generally contains a carbon source, a nitrogen source, and a trace element component. Such cell culture minimal media include, for example, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI1640, F-10, F-12, αMEM (α Minimal Essential Medium), GMEM (Glasgow's Minimal essential Medium), Iscove's Modified Dulbecco's Medium, etc., but are not limited to this.

In addition, the medium may include antibiotics such as penicillin, streptomycin, and gentamicin.

On the other hand, the present invention, the stem cells, their secretions, forms containing all of the media components, forms containing only the secretions and media components, separating only the secretions or used alone or in combination with stem cells, or by administering only stem cells It is also possible to use it in a form that produces secretions in the body.

The stem cells can usually be obtained using any method known in the art.

Stem cells treated with the histone deacetylation inhibitor and priming factor of the present invention can be used as a cell therapy for the treatment of certain diseases, and the treatment can be direct treatment or pre-treatment of the molecules. .

The "cell therapy agent" refers to an autologous, allogenic, or xenogenic cell that is grown in vitro to restore the functions of cells and tissues, or to increase or select the cell's biological properties by other methods. It means a medicine used for the purpose of treatment, diagnosis, and prevention through a series of actions.

The cell therapeutic agent can be administered to the human body through any 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; Pulmonary arterial hypertension associated with collagen vascular disease, congenital lung short circuit, portal hypertension, HIV infection, drugs or toxins; Pulmonary arterial hypertension associated with thyroid disorder, glycogen storage disease, Gaucher disease, hereditary hemorrhagic capillary dilatation, hemochromatosis, myeloproliferative disorder or splenectomy; Pulmonary arterial hypertension associated with pulmonary capillary angiomatosis; Persistent pulmonary hypertension in newborns; Pulmonary hypertension associated with chronic obstructive pulmonary disease, interstitial pulmonary disease, hypoxia-induced alveolar hypoventilation disorder, hypoxia-induced sleep disorder breathing or chronic exposure to high altitudes; Pulmonary hypertension associated with developmental abnormalities; And pulmonary hypertension caused by thromboembolic obstruction of the distal pulmonary artery, more preferably idiopathic pulmonary arterial hypertension, familial pulmonary arterial hypertension or chronic obstructive pulmonary disease, 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, cystic fibrosis (Cystic Fibrosis), Post transplantation late and chronic solid organ rejection, Graft-versus-host disease, Graft-versus-host disease, Multiple sclerosis, Systemic lupus erythematosus Systemic lupus erythematosus, Sjogren syndrome, Hashimoto thyroiditis, polymyositis, scleroderma, Addison disease, vitiligo, pernicious anemia , Glomerulonephritis and pulmonary fibrosis, Inflammatory Bowel Diesese, Crohns disease, Autoimmune Diabetes, Diabetes Diabetic retinopathy, Rhinitis, Ischemia-reperfusion injury, Post-angioplasty restenosis, Chronic obstructive pulmonary disease (COPD), Graves' disease (Graves disease), gastrointestinal allergy, conjunctivitis, atherosclerosis, coronary artery disease, angina, cancer metastasis, arterial disease or mitochondrial disease However, it is not limited thereto.

The pharmaceutical composition of the present invention may be prepared by using a pharmaceutically acceptable and physiologically acceptable adjuvant in addition to the active ingredient, and as an adjuvant, an excipient, a disintegrant, a sweetener, a binder, a coating agent, an expanding agent, a lubricant, a lubricant Alternatively, a solubilizing agent such as a flavoring agent can be used. The pharmaceutical composition of the present invention can be preferably formulated into a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. Acceptable pharmaceutical carriers in compositions formulated as liquid solutions include, as sterile and biocompatible, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets.

Pharmaceutical formulation forms of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions and sustained release formulations of active compounds, etc. You can. The pharmaceutical composition of the present invention is a conventional manner through intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. Can be administered. 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. Thus, the type of disease, the severity of the disease, the type and content of active and other ingredients contained in the composition, the type of formulation and the patient's age, weight, general health status, gender and diet, time of administration, route of administration and composition It can be adjusted according to various factors, including the rate of secretion, the duration of treatment, and drugs used simultaneously.

Hereinafter, examples will be described in detail to help understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Experimental example >

The following experimental examples are intended to provide an experimental example commonly applied to each embodiment according to the present invention.

1. From human umbilical cord MSCs  culture

Human UC was obtained from healthy normal full-term newborns with parental written consent in accordance with guidelines approved by the Asan Hospital Bioethics Review Board. Consent was obtained from all mothers prior to UC collection. UC-derived MSCs (UC-MSCs) include 2-mM L-glutamine, 20-mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) (pH 7.3), minimal-essential medium ( minimum-essential 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 ( fetal bovine serum (FBS) (HyClone), 5-ng / mL human epithelial growth factor (Sigma-Aldrich, St. Louis, MO), 10-ng / ml basic fibroblast growth factor and 50-mg / ml long- R3 insulin-like growth factor -1 (ProSpec, Rehovot, Israel) are added to the low-glucose Dulbecco's modified Eagle's medium (DMEM) (HyClone, Pittsburgh, PA) 5% CO ₂ at atmospheric conditions, were incubated in 37 ℃. UC-MSCs that were passaged and expanded to 5 or less were used to maintain pluripotency. Expression of surface proteins was analyzed as previously reported (Stem Cells and Dev. 24 (2015) 1658-1671).

2. Cell migration analysis

The 8-μm polycarbonate membrane was coated with 50 μl of 1.0% gelatin (Sigma) for 1 hour. UC-MSCs were primed for 1 day by mixing with VPA (0.5-mM; Sigma-Aldrich) or 5-Aza (1-μM; Sigma-Aldrich) alone or with 50-nM S1P. UC-MSCs were separated with TrypLE solution (Thermo Scientific, Pittsburgh PA), washed and resuspended in DMEM containing 0.5% bovine serum albumin (BSA), transwell inserts at a density of 3 X 10 ⁴ cells / well ( Transwell inserts) (Corning Costar, Pittsburgh, PA). 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. Cells remaining in the upper chamber were scraped off with cotton wool, and the migrated cells were fixed with a 4% paraformaldehyde (PFA) solution dissolved in phosphate-buffered saline (PBS), and then 0.5% 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. MSCs  In vitro characterization

Cell proliferation of MSCs, fibroblast colony-forming unit-fibroblast (CFU-F), pluripotency (ex vivo differentiation into chondrocyte, osteoblast or adipocyte lineage) and in vitro anti-inflammatory assays As reported in (Stem Cells and Dev. 24 (2015) 1658-1671).

4. Western Blot  And real-time quantitative PCR (real-time quantitative PCR ; RQ - PCR )

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

5. Statistical Analysis

To identify statistical significance differences, data were analyzed using a non-parametric Mann Whitney test or a one-way ANOVA with the Bonferroni post-hoc test. The present inventors performed all the analyzes using GraphPad Prism 6.0 software (GraphPad Software, La Jolla, CA), and statistical significance was defined as p <0.05, 0.01, or 0.001.

6. Experimental design

It is an object of the present invention to treat interstitial cystitis / bladder pain syndrome (IC / BPS) in a rat model, umbilical cord-derived MSCs derived from human umbilical cord primed with V1P + S1P (UC) -In addition to measuring the effectiveness of MSCs, the in vivo cellular properties of transplanted cells were also extensively observed. Control or V1P + S1P primed UC-MSCs were administered to the bladder-damaged mice, and the effects on bladder urination function, urinary tract epithelial detachment, mast cell infiltration, tissue fibrosis, apoptosis, and tumor formation were affected. It was measured. In all experiments, 2 independent experiments were performed on 5 independent animals per group. These were treated by random distribution to the injured group, cell transplantation or vehicle administration group, and cystometry group. The investigators involved in the surgical process were not informed of the type and dose of the cells administered. All bladder pressure measurements, histology, and gene expression measurements were performed by researchers who do not know information about treatment groups. Animals that died unexpectedly due to bladder damage or catheterization were excluded from all analyzes. All animal experiments were conducted in accordance with the guidelines and regulations of the Animal Experiment Ethics Committee of the University of Ulsan Medical School (IACUC-2014-14-167).

7. Animal model and UC - MSCs  transplantation

The HCl injection IC / BPS rat model was previously reported (Stem Cells and Development 24, 2015, 1648-1657). One week after HCl damage, the lower abdomen was dissected and 2.5 × 10 ⁵ (250 K) UC-MSCs or PBS were directly administered to the outer wall of the bladder and the ceiling using a 500 μm syringe and a 26-gauge needle. One day prior to stem cell administration, indomethacin (PMG Pharm Co., Ltd. Ansan, Korea; every 12 h at 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. Non-anesthesia ( Unanesthetized ) And Non-inhibition (unrestrained) Bladder pressure measurement chart  Collection (bladder pressure measurement under non-anesthesia)

Cystometrograms were performed in non-anesthesia and non-inhibited mice in metabolic cages. For intravesical pressure (IVP) and intra-abdominal pressure (IAP) recording measurements, a catheter was inserted simultaneously 3 days prior to intravesical pressure measurement. Briefly, after induction of anesthesia, a cuffed polyethylene catheter (PE-50; Becton-Dickinson, Parsippany, NJ, USA) was inserted into the bladder ceiling through an intraperitoneal incision. To record the IAP, a peritoneal balloon (Latex; Daewoo Medical, Incheon, Korea) near the cuff of the catheter tip was placed proximal to the bladder and tied to another catheter with a silk thread. The polyethylene catheter (PE-50) was heated in warm water, the tip of the insert was increased ~ 1.5 times the original length, and filled with heparinized saline (100 IU / mL). When the bladder catheter was implanted, the stretched catheter was inserted into the femoral vein. Thereafter, the catheters penetrated through the subcutaneous space, escaped through the animal's back, and attached to the skin of the back. After surgery, each rat was bred individually and maintained in the same way.

For awake cystometric analysis, a T-tube connected to a pressure transducer (Research Grade Blood Pressure Transducer; Harvard Apparatus, Holliston, MA, USA) and a microinjection pump (PHD22 / 2000 pump; Harvard Apparatus) was used. Through a double valve, an indwelling catheter was connected to the bladder. To record the IAP, another indwelling catheter with a body fluid filled abdominal balloon was connected to a different pressure transducer. Sterilized saline was injected into the bladder at a rate of 0.4 mL / min, and urine volume was continuously recorded through a body fluid collector connected with a force displacement transducer (Research Grade Isometric Transducer; Harvard Apparatus). IVP, IAP and urination were continuously recorded using a MP150 data acquisition system (Biopac Systems, Goleta, CA, USA) equipped with Acq Knowledge 3.8.1 software at a sampling rate of 50 Hz. All urination cycle values measured for 8 minutes from each animal were used for evaluation.

When IVP increased beyond 15 cmH ₂ O from baseline without urine discharge, it was measured by non-voiding contraction (NVC). BP means the lowest bladder pressure while the bladder is filled, MP means the highest bladder pressure during the urination cycle, MV means the urine volume during urine discharge, and RV means the residual urine volume after urine discharge. BC is defined as MV + RV, MI refers to the interval between urination contractions.

9. Pulmonary artery hypertension; PAH ) Animal model

A monocrotaline (MCT) induced PAH rat model was established according to previous reports (Stem Cells and Development 24, 2015, 1658-1671). Male specific-pathogen-free Lewis rats (8 weeks of age, 250-280 g) were bred without restriction of food and water, under control of breeding temperature and light (12 hour contrast cycle). PAH was induced by subcutaneous administration of MCT (60 mg / kg, Sigma). Control rats received the same volume of PBS. Two weeks after administration of MCT or PBS, UC-MSCs pretreated with non-priming-induced UC-MSCs or VPA + S1P were administered through the tail vein at a density of 2.5 × 10 ⁵ cells per 200 μl PBS. In a vehicle-treated control group, 200 µl PBS was administered without cells. Before 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. Systolic Right ventricular pressure (right ventricular systolic pressure; RVSP ) And right ventricular hypertrophy (RVH) measurement

 4 weeks after administration of MCT or PBS, patient maintained under breath through a ventilator (Harvard Apparatus, Holliston, MA) and under anesthesia with zoletil (40 mg / kg) and rumpun (rompun; 10 mg / kg) Systolic right ventricular pressure was measured by direct puncture of the diaphragm using a 26G needle connected to a monitor (MDE Escort II patient monitor, Arleta). Right ventricular pressure (RVP) maintained breathing through the ventilator. To measure RVH, the right ventricle of the heart was separated from the interventricular septum, and the weight of the left ventricle (LV + S), including the right ventricle (RV) and the ventricular septum, was measured.

11. Histology and gene expression analysis

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

For gene expression analysis, whole RNA was prepared using RNeasy Mini Kit (Qiagen Inc., Valencia, CA), reverse transcription was performed using TaqMan Reverse Transcription Reagents (Applied Biosystems), and PikoReal Real-Time PCR System ( Thermo Scientific) and iQ SYBR Green PCR Master Mix (Bio-Rad, Hercules, CA) were used to perform real-time quantitative PCR (RQ-PCR). For 5 independent animals per treatment group, three randomly selected regions (n = 15) from each slide were used to quantify digital images. Similarly, RQ-PCR was repeated twice (n = 10) from 5 randomly selected animals per group to obtain gene expression data.

< Example  1> On S1P  By human UC - MSCs For priming  There is 5- Aza's  effect

Low concentration treatment of 5-Aza and VPA (1-μM and 0.5-mM) greatly increases the expression of CXCR4 in UC-MSCs (Figure 1A). Next, the inventors confirmed the effect of the epigenetic modulator on the basic characteristics of UC-MSCs. Independent of the priming of 50-nM S1P, 5-Aza and VPA both did not significantly affect the expression of surface marker proteins (CD29, CD73 and CD90 positive, CD34 and CD45 negative) (FIGS. 1B and 2A). In addition, S1P priming treated with 5-Aza (5-Aza + S1P) or VPA (VPA + S1P) had little effect on the multi-line differentiation ability, and multi-line differentiation ability was glycosaminoglycans; Based on in vitro differentiation analysis of chondrocytes, osteoblasts and adipocyte lineages by measuring the increase in the level of mineral deposition (mineral deposition; alizarin red S) and lipid accumulation (oil red O staining), respectively. (Fig. 1C and Fig. 2B).

In contradiction to the up-regulation of CXCR4 (FIG. 1A), 5-Aza + S1P primed UC-MSCs in transwell migration assay showed a decrease in migration activity for the SDF-1 response by approximately 30% compared to unprimed cells. (Fig. 1E). In addition, 5-Aza + S1P priming severely impaired the activity of clonogenic CFU-F, which indicates the frequency of self-renewal with clonogenic progenitor cells (FIG. 1F). Cell proliferation of MSCs was little changed by priming of 5-Aza + S1P (FIG. 1D).

< Example  2> Low concentration S1P  Upon processing UC - MSCs Priming  Strengthening VPA

Next, the present inventors investigated the effect of VPA on priming of UC-MSCs when treating low concentration S1P. To this end, we have applied 50-nM S1P four times less than the optimal dose (200-nM) used for priming of fat- and UCB-derived MSCs. Unlike VPA alone and S1P alone, priming UC-MSCs during VPA + S1P treatment increased chemotactic activity against SDF-1 by 2 to 3 times higher than unprimed cells (Figure 3A and Figure 3B). The response to SDF-1 enhanced by VPA + S1P priming was completely different from that of 5-Aza + S1P priming. Next, the inventors confirmed the state of the signaling pathways associated with the movement of HSPCs. Consistent with chemotactic analysis, UC-MSCs exposed to VPA + S1P increased phosphorylation of MAPK ^{p42 / 44} and AKT proteins, indicating activation of the signaling pathway (Figure 3C). These results show that unlike 5-Aza, VPA can promote the priming of UC-MSCs by activating the MAPK ^{p42 / 44} and AKT signaling pathways even at low concentrations of S1P.

< Example  3> VPA + With S1P Primed UC - MSCs  Improving treatment ability

We tested the effect of VPA + S1P priming on other cell activity related to the therapeutic effect of MSCs. Unlike 5-Aza + S1P (Fig. 1), VPA + S1P primed UC-MSCs improved both cell proliferation (Fig. 4A) and clonogenic CFU-F activity (Fig. 4B). However, cell proliferation capacity and CFU-F activity were not observed to be increased in UC-MSCs treated with VPA alone or S1P alone (FIGS. 4A and 4B). Since MSCs affect anti-inflammatory and immunomodulatory abilities, we tested whether VPA + S1P priming affects the anti-inflammatory effects of UC-MSCs. Accordingly, the present inventors prepared condition media collected from unprimed UC-MSCs or VPA alone or VPA + S1P primed UC-MSCs, and the condition media was stimulated with lipopolysaccharide (LPS) alveolar macrophages ( It was tested whether the suppression of tumor necrosis factor-α (TNF-α) secretion from alveolar macrophages cells (MH-S cells) was possible. The conditioned medium collected from UC-MSCs was effective in reducing the secretion of TNF-α from LPS stimulated MH-S cells (FIG. 4C). In particular, conditioned medium collected from VPA + S1P primed UC-MSCs further inhibited TNF-α secretion than unprimed UC-MSCs or VPA alone primed UC-MSCs. The inventors used CM collected with human lung fibroblasts, IMR90, as a control, which was shown to further increase TNF-α secretion in vitro anti-inflammatory analysis.

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

< Example  3> Various VPA  And S1P  According to the concentration SDF Chemotaxis analysis for -1a

The present inventors tried to determine the lowest concentrations of VPA and S1P by performing chemotaxis analysis for SDF-1a according to various VPA and S1P concentrations.

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

< Example  4> In IC / BPS treatment, VPA + S1P Primed UC - MSC  Confirmation of the possibility of treatment in vivo

Next, we confirmed the in vivo effect of UC-MSCs using an IC / BPS animal model established with HCl injection. As a result of bladder function analysis using a non-anesthesia bladder pressure measurement (awake cystometry), HCl injection induced IC / BPS mice (HCl-IC group) showed irregular urination compared to control (sham-operated; sham) mice, and urination In addition to reducing the micturition interval (MI), it was found that the micturition volume (MV), maximum pressure, micturition pressure (MP) and bladder capacity (BC) were also lowered (FIG. 7A and FIG. 7b). Single transplantation of 2.5 × 10 ⁵ cell number of naive UC-MSCs (UC-MSC 250K) or VPA + S1P primed cells (UC-MSC 250K + VPA + S1P) improved impaired urination parameters. Importantly, VPA + S1P primed UC-MSCs showed better effect than control cells (FIGS. 7A and 7B). In particular, in the HC-IC group animals, the frequency of contraction was increased during the non-voiding periods (NVC), which was significantly improved in VPA + S1P primed UC-MSCs, but the control UC -Not much improvement in MSCs (Figures 7A and 7B). The results of awake cystometry under non-anesthesia were also consistent with the effect of improving the function, severe urinary urothelium denudation in rat models due to administration of control or VPA + S1P primed 2.5 × 10 ⁵ UC-MSCs, Abnormal histological phenomena such as mast cell infiltration, fibrosis, and cell death have been shown to be restored (FIGS. 8A and 8B). These abnormal histological phenomena are also characteristic of human IC / BPS bladder. In particular, recovery of histological transformation showed a better improvement effect in bladder tissue implanted with VPA + S1P primed UC-MSC than control cells. Taken together, the results indicated that for IC bladder treatment, VPA + S1P priming can significantly improve the therapeutic potential of UC-MSCs.

< Example  5> PAH  In animal model UC - MSC VPA + S1P -Improved the possibility of mediating

Next, the present inventors confirmed the effect of VPA + S1P priming under in vivo conditions using an MCT-derived PAH animal model. Similar to the previous report, RVSP increased significantly after 4 weeks of MCT administration. Unlike the control UC-MSC (PAH + MSC group), administration of UC-MSC primed with VPA + S1P (PAH + VPA + S1P-MSC group) significantly reduced the elevation of MCT-induced RVSP (FIG. 9a). In addition, after administration of MCT, RV / (LV + S) was also increased, VPA + S1P-MSC significantly reduced RV hypertrophy, and unprimed UC-MSC had little effect (FIG. 9A). In addition, administration of VPA + S1P-MSC reduced lung tissue inflammation induced by MCT, membrane thickness index and increase in α-SMA ⁺ smooth muscle cells (FIG. 9B). Taken together, the results indicated that priming of UC-MSC by VPA + S1P promotes angiogenesis and can induce a microenvironment that suppresses inflammatory responses.

Since the specific parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that this specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereby. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (20)

A composition for promoting umbilical cord mesenchymal stem cell (UC-MSC) activity comprising valproic acid (VPA) and sphingosine-1-phosphate (S1P) as active ingredients. delete delete According to claim 1, wherein the VPA is a composition for promoting umbilical cord mesenchymal stem cell (UC-MSC) activity, characterized in that contained in a concentration of 0.1 to 1.0 mM. The composition for promoting umbilical cord mesenchymal stem cell (UC-MSC) activity according to claim 1, wherein the S1P is contained at a concentration of 10 to 50 nM. delete According to any one of claims 1, 4 and 5, wherein the umbilical cord-derived mesenchymal stem cell (UC-MSC) activity is cell mobility, colony-forming ability and anti-inflammatory activity, characterized in that the umbilical cord-derived intermediate Composition for promoting mesenchymal stem cell (UC-MSC) activity. Umbilical cord-derived mesenchymal stem comprising the step of treating the isolated umbilical cord-derived mesenchymal stem cells (UC-MSC) with valproic acid (VPA) and sphingosine-1-phosphate (S1P). Method for promoting stem cell (UC-MSC) activity. delete delete 9. The method of claim 8, wherein the VPA is treated at a concentration of 0.1 to 1.0 mM, umbilical cord-derived mesenchymal stem cell (UC-MSC) activity. 9. The method of claim 8, wherein the S1P is treated at a concentration of 10 to 50 nM, and umbilical cord-derived mesenchymal stem cell (UC-MSC) activity is promoted. Prevention of hypertension including umbilical cord mesenchymal stem cells (UC-MSC) treated with valproic acid (VPA) and sphingosine-1-phosphate (S1P) or a culture medium thereof as an active ingredient Or therapeutic pharmaceutical compositions. delete delete 14. The method of claim 13, wherein the hypertension is idiopathic pulmonary arterial hypertension; Familial pulmonary arterial hypertension; Pulmonary arterial hypertension associated with collagen vascular disease, congenital lung short circuit, portal hypertension, HIV infection, drugs or toxins; Pulmonary arterial hypertension associated with thyroid disorder, glycogen storage disease, Gaucher disease, hereditary hemorrhagic capillary dilatation, hemochromatosis, myeloproliferative disorder or splenectomy; Pulmonary arterial hypertension associated with pulmonary capillary angiomatosis; Persistent pulmonary hypertension in newborns; Pulmonary hypertension associated with chronic obstructive pulmonary disease, interstitial pulmonary disease, hypoxia-induced alveolar hypoventilation disorder, hypoxia-induced sleep disorder breathing or chronic exposure to high altitudes; Pulmonary hypertension associated with developmental abnormalities; And pulmonary hypertension due to thromboembolic obstruction of the distal pulmonary artery, a pharmaceutical composition for preventing or treating hypertension. Inflammatory disease comprising umbilical cord mesenchymal stem cells (UC-MSC) treated with valproic acid (VPA) and sphingosine-1-phosphate (S1P) or a culture medium thereof as an active ingredient Or pharmaceutical composition for the prevention or treatment of immune disease. delete delete The method of claim 17, wherein the inflammatory disease or immune disease is osteoarthritis, rheumatoid arthritis, cystitis, interstitial cystitis, asthma, dermatitis, atopy, psoriasis, cystic fibrosis. ), Post transplantation late and chronic solid organ rejection, graft-versus-host disease, transplant rejection disease, multiple sclerosis, systemic lupus erythematosus (systemic) lupus erythematosus, Sjogren syndrome, Hashimoto thyroiditis, polymyositis, scleroderma, Addison disease, vitiligo, pernicious anemia, glomerulonephritis (glomerulonephritis) and pulmonary fibrosis, Inflammatory Bowel Diesese, Crohns disease, Autoimmune Diabetes, Diabetic retinopathy ), Rhinitis, Ischemia-reperfusion injury, Post-angioplasty restenosis, Chronic obstructive pulmonary disease (COPD), Graves disease , Gastrointestinal allergy, conjunctivitis, atherosclerosis, coronary artery disease, angina, cancer metastasis, small arterial disease and mitochondrial disease It characterized in that the inflammatory disease or immune disease prevention or treatment pharmaceutical composition.

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