Composition comprising melatonin for promoting stem cell activity, and method for priming stem cells by using same

Abstract

The present invention demonstrates the functional enhancement of melatonin-primed stem cells through ten indicators including self-renewal ability, engraftment rate, anti-inflammatory ability, and immunosuppressive ability in a cell culture model. It has been identified that in the primed stem cells according to the present invention, the activity of glutathione (GSH) is increased, through signaling involving cyclic adenosine monophosphate (cAMP) response element-binding protein-1 (CREB1) and nuclear factor (erythroid-derived-2) like-2 (NRF2) as a molecular mechanism and, simultaneously, the expression of PD-L1 protein is increased by JAK1-STAT3 signaling. The stem cells primed according to the present invention have improved stemness, and thus can be effectively used in the treatment of inflammatory diseases, immune diseases and the like.

Description

Composition for promoting stem cell activity containing melatonin and stem cell priming technique using the same

[0001] The present invention was completed by confirming that the functionality of stem cells is enhanced when melatonin is used as a stem cell priming factor. The present invention developed a priming technology that enhances stem cell functionality while simultaneously ensuring safety through a low-concentration mixture of melatonin and valproic acid (MT+VPA).

[0002] This invention was carried out with the support of the Ministry of Science and ICT's inter-ministerial regenerative medicine technology development project, "Development of a platform technology for stem cell treatment targeting interstitial cystitis using real-time analysis of stem cell functionality indicators, high-sensitivity titer measurement technology, single-cell analysis, and immune monitoring analysis" (Project ID: 1711179439, Project No.: 00040242).

[0003] Stem cell therapy based on adult mesenchymal stem cells (MSCs) is being used as an important source for the development of treatments for various intractable diseases due to its many advantages, including significantly reduced immune response and cancer incidence after transplantation, preferential migration to damaged areas, functional recovery of damaged cells, and immunomodulatory functions.

[0004] In particular, stem cell therapy, which was mainly used for the regeneration of the nervous system, blood system, and musculoskeletal system, is gradually expanding its use to treat severe and intractable diseases (asthma, atopic dermatitis, urinary disorders, acute kidney injury, diabetic complications, bronchopulmonary dysplasia, leukemia, etc.) and as an anticancer treatment. In particular, the effectiveness of MSCs in immune-related chronic inflammatory diseases such as diabetes, rheumatoid arthritis, and SLE, as well as in transplant rejection, is being suggested based on their ability to promote immune regulation and tissue regeneration.

[0005] The therapeutic efficacy of stem cell therapy is known to stem from the paracrine effects of specific factors secreted by the transplanted stem cells, known as the secretome, rather than from the direct differentiation and regeneration of the transplanted stem cells themselves. As the various secretome factors secreted after transplantation have been revealed to ultimately exert therapeutic effects by regulating the regeneration of damaged tissue and the activity of immune cells, active research is being conducted to analyze the types and functions of MSC secretome proteins and to efficiently manipulate and utilize them to further enhance therapeutic efficacy.

[0006] However, the low survival rate of stem cells due to immune response after transplantation and the loss of stem cell capacity (stemness) resulting in inconsistent and low therapeutic efficacy are problems that must be overcome. In addition, from the perspective of practical application, there are technical limitations such as the lack of a standardized stem cell treatment production protocol and limited culture capacity and culture technology. Therefore, basic research and technology development that can solve these problems are urgently needed.

[0007] Accordingly, the inventors of the present invention have developed a technology to secure a high-functionality fusion stem cell treatment that can be rapidly introduced into clinical trials, and have secured evidence of the Mode of Action (MoA) for an advanced stem cell treatment technology based on a single-cell analysis method, thereby resolving the high unmet medical needs of severe intractable asthma and developing a next-generation stem cell treatment that can secure global market entry and reliability.

[0008] The present invention has been conceived to solve the above problems and meet the above needs, and the purpose of the present invention is to provide a priming technique capable of promoting the activity (functionality) of stem cells.

[0009] According to the present invention, the purpose is to provide a combination of compounds and a combination ratio thereof that can improve the functionality of stem cells.

[0010] The present invention also aims to provide a stem cell therapeutic agent and pharmaceutical composition for treating inflammatory diseases and immune diseases using stem cells with improved functionality.

[0011] To solve the above-described problem, a composition for promoting stem cell activity comprising melatonin and a histone deacetylation inhibitor as active ingredients is provided.

[0012] In the present invention, the histone deacetylation inhibitor includes valproic acid (VPA), sodium butyrate (NaB), nicotinamide (NAD), and sirtinol, but most preferably valproic acid (VPA).

[0013] In the present invention, melatonin and a histone deacetylase inhibitor (preferably valproic acid) may be mixed at a concentration ratio of 2:2 to 5. According to a preferred embodiment of the present invention, melatonin and valproic acid are mixed at a concentration ratio of 2:5.

[0014] In the present invention, the stem cells include, but are not limited to, neural stem cells, liver 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 stem cells.

[0015] In the present invention, the stem cell activity includes cell motility, colony forming ability and anti-inflammatory activity.

[0016] The present invention also provides stem cells whose activity has been stimulated by treatment (priming) with the above-described stem cell activity-stimulating composition. Stem cells whose activity has been stimulated according to the present invention can be used as a cell therapeutic agent.

[0017] The present invention also provides a method for promoting stem cell activity, comprising a step of treating isolated stem cells with melatonin and a histone deacetylase inhibitor.

[0018] In another embodiment of the present invention, a pharmaceutical composition for preventing or treating an inflammatory disease or an immune disease, comprising stem cells or a culture medium thereof treated with melatonin and a histone deacetylase inhibitor as active ingredients; a use of the pharmaceutical composition for treating an inflammatory disease or an immune disease; and a method for preventing or treating an inflammatory disease or an immune disease using the pharmaceutical composition are provided.

[0019] In the present invention, the inflammatory disease or immune disease is osteoarthritis, rheumatoid arthritis, cystitis, interstitial cystitis, asthma, dermititis, atopy, psoriasis, cystic fibrosis, post transplantation late and chronic solid organ rejection, graft-versus-host disease, transplant rejection disease, multiple sclerosis, systemic lupus erythematosus, Sjogren syndrome, Hashimoto thyroiditis, polymyositis, scleroderma, Addison disease, vitiligo, pernicious anemia, Glomerulonephritis and pulmonary fibrosis, Inflammatory Bowel Disease, Crohn's 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 artery disease, and mitochondrial disease, but are not limited thereto.

[0020] The present invention confirms that the functionality of stem cells is promoted when treated with melatonin, and provides a technique for priming (or promoting) stem cells using melatonin.

[0021] The present invention also provides a combination of melatonin and valproic acid that can enhance the promotion of stem cell functionality.

[0022] Melatonin and valproic acid of the present invention enhance the self-renewal and cell migration capabilities of stem cells. Stem cells primed according to the present invention can be administered to a subject to alleviate inflammation and mitigate immune responses, making them useful as stem cell therapeutics.

[0023] Figures 1a and 1b illustrate the results of analysis after treating umbilical cord-derived MSCs (WJ-MSCs) with melatonin or a combination of melatonin and valproic acid at different concentrations, respectively, according to one embodiment of the present invention. The upper images of Figures 1a and 1b illustrate the results of confirming colony forming units, and the lower images illustrate the results of analyzing chemotaxis (cell migration ability).

[0024] FIG. 2a and FIG. 2b are the results of measuring self-renewal ability (CFU-F, FIG. 2b) and cell migration ability (Chemotaxis, FIG. 2a) after treatment with VPA fixed at 0.5 mM and MT concentrations adjusted from 100 μM to 500 μM according to one embodiment of the present invention.

[0025] Figures 3a to 3d show the results of analyzing the self-renewal capacity (Colony-Forming Unit-Fibroblast; CFU-F) of stem cells from each of the four types of WJ-MSC (#195, #140, #183, #202) after treatment with MT+VPA.

[0026] Figures 4a to 4d show the results of analyzing the migratory ability (Chemotaxis) of stem cells after treating each of the four types of WJ-MSC (#195, #140, #183, #202) with MT+VPA.

[0027] Figure 5 shows the results of examining the versatility of the melatonin + valproic acid-primed MSC manufacturing method established from human umbilical cord-derived MSCs, after treating 2 lots of adipose-derived MSCs (AD-MSC#202, #195) with melatonin and valproic acid together, and confirming the self-renewal ability and cell migration ability of the stem cells.

[0028] Figure 6 shows the results of analyzing the antioxidant capacity of MSCs primed with melatonin + valproic acid using the Glutathione Recovery Capacity Assay to verify the enhanced stem cell properties of MSCs primed with melatonin + valproic acid.

[0029] Figures 7a to 7c are the results of confirming the expression of genes and proteins involved in various functions, such as the CREB1-NRF2 associated gene pathway, in MSCs primed with melatonin + valproic acid according to one embodiment of the present invention.

[0030] Figure 8a shows the results of confirming the secretion of inflammatory cytokines TNFa and IL6 in MSCs primed with melatonin + valproic acid, and Figure 8b shows the results of confirming the expression of inflammatory genes in MSCs primed with melatonin + valproic acid.

[0031] Figure 9 shows the results of a T cell stimulation assay performed after stimulating PBMC (Pheripheral Blood mononuclear cell) with PHA (phytohemagglutinin) to induce an immune response in order to confirm the immunosuppressive capacity of MSC primed with melatonin + valproic acid.

[0032] Figure 10 shows the results of confirming the pro-angiogenesis of MSCs primed with melatonin + valproic acid according to one embodiment of the present invention.

[0033] Figure 11a is a schematic diagram of an experimental process for evaluating the anti-asthmatic treatment efficacy using MSCs with enhanced antioxidant capacity and engraftment rate according to one embodiment of the present invention. Figures 11b to 11e show the results of analyzing the lung tissue inflammatory response and changes in cytokine expression levels (Figure 11c), and the number of inflammatory cells and changes in cytokines in bronchoalveolar lavage fluid (BALF) (Figures 11b, 11d, and 11e) 7 days after transplanting MSCs cultured normally (NT) and MSCs primed with melatonin + valproic acid into a poly IC-induced asthma animal model.

[0034] Figures 12aa, 12ab, and 12b show the results of confirming the preclinical efficacy of stem cells through bladder function tests (awake cystometry) after producing an interstitial cystitis model according to one embodiment of the present invention.

[0035] Figure 13 shows the results of Hematoxylin & Eosin (H&E) staining, Masson-trichrome staining, and TB staining performed on bladder tissue to evaluate the degree of progression of interstitial cystitis following administration of MSCs primed with melatonin + valproic acid according to one embodiment of the present invention.

[0036] Figures 14a, 14b, 14c, and 14d show the results of confirming the efficacy of MT-VPA treatment on bladder voiding function. 5.0Х10 in STZ-DUA rats 5 The results of awake cystometry were measured one week after injection of naive hUC-MSCs or MT+VPA MSCs, and intravesical pressure (IVP), intraabdominal pressure (IAP), and residual volume (RV) were measured.

[0037] Figures 15a, 15b, 15c and 15d show the results of quantitative analysis of bladder voiding function factors. Experimental control group (PBS) or Naive hUC-MSC and MT+VPA MSC (5.0Х10 5 ) were injected into sham-operated and STZ-DUA rats. Awake cystometry was performed to quantitatively analyze voiding function factors. Data were analyzed by one-way analysis of variance (ANOVA) and Bonferroni post hoc comparison (**p<0.01, ***p<0.001, compared with the PBS group; ##p<0.01, ###p<0.001).

[0038] Figure 16 shows the results of MT-VPA treatment on tissue damage repair. One week after cell injection in the designated groups, bladder sections were stained with hematoxylin-eosin (upper panel; magnification, 100X; scale bar, 200 μm), Masson's trichome staining (middle panel; magnification, 100X; scale bar, 200 μm), and toluidine blue staining (lower panel; magnification, 400X; scale bar, 100 μm). Nuclei were stained with Mayer's hematoxylin.

[0039] Figure 17 shows the results of quantitative analysis of tissue staining of 5 animals per group in the quantitative analysis of awake-state cystoscopy. Data were analyzed by one-way analysis of variance (ANOVA) and Bonferroni post hoc comparison (**p<0.01, ***p<0.001, compared to PBS group; ##p<0.01, ###p<0.001).

[0040] Hereinafter, the present invention will be described in detail. The advantages and features of the present invention, as well as the embodiments that achieve them, will become clearer with reference to the embodiments described below. However, the present invention is not limited to the embodiments disclosed below and may be implemented in various different forms. These embodiments are provided solely to ensure the complete disclosure of the present invention and to fully inform those skilled in the art of the scope of the invention. The present invention is defined solely by the scope of the claims. Like reference numerals designate like elements throughout the specification.

[0041] Unless otherwise defined, all terms (including technical and scientific terms) used herein may be used in a sense commonly understood by those of ordinary skill in the art to which the present invention pertains. Furthermore, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless explicitly and specifically defined otherwise. The terminology used herein is for the purpose of describing embodiments and is not intended to limit the present invention. In this specification, singular forms also include plural forms, unless specifically stated otherwise.

[0042] The present invention provides a method for treating stem cells with melatonin and a histone deacetylase inhibitor as a priming technique for enhancing their functionality. According to the present invention, treatment with melatonin and a histone deacetylase inhibitor enhances the self-renewal and cell migration abilities of stem cells.

[0043] The stem cells of the present invention may be treated with melatonin, a histone deacetylase inhibitor, or a composition containing them before, during, or after culturing.

[0044] The term 'stem cell' used in this specification refers to a cell that has the ability to self-replicate and differentiate into two or more cells, and can be classified into totipotent stem cells, pluripotent stem cells, and multipotent stem cells.

[0045] The stem cells of the present invention may be appropriately selected without limitation depending on the purpose, and may be derived from adult cells of any known tissue, cell, or other organ derived from a mammal, including a human, preferably a human. For example, they may be derived from bone marrow, umbilical cord blood, placenta (or placental tissue cells), fat (or adipose tissue cells), etc.

[0046] For example, the stem cells may be stem cells obtained without limitation 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, and may be stem cells derived from a fetus, postnatally, or adult.

[0047] In a preferred embodiment of the present invention, the stem cells are selected from the group consisting of neural stem cells, liver 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 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 are not limited thereto.

[0048] In the present invention, the 'histone deacetylation inhibitor' may be, but is not limited to, valproic acid (VPA), sodium butyrate (NaB), nicotinamide (NAD), or sirtinol, and is most preferably valproic acid (hereinafter also referred to as VPA).

[0049] In the present invention, "melatonin (hereinafter referred to as MT)" is a biological hormone secreted by the pineal gland in the brain and is medically used to treat insomnia. Melatonin is synthesized in the body by detecting photoperiods, such as the length of day and night, and is known to regulate the human sleep-wake rhythm and daily and seasonal circadian rhythms, inducing natural sleep.

[0050] The term 'priming' used in this specification refers to a phenomenon in which the reactivity (activity) is enhanced to enhance the therapeutic efficacy of stem cells, and in the present invention, the activity of the stem cells is promoted using a histone deacetylation inhibitor and a priming factor that induce the priming.

[0051] In the present invention, 'stem cell activity' means cell mobility, cell proliferation ability, pluripotency (in vitro differentiation into chondrocyte, osteocyte or adipocyte lineage), colony formation ability or anti-inflammatory activity of stem cells.

[0052] The present invention provides a pharmaceutical composition for preventing or treating inflammatory or immune diseases, comprising stem cells or a culture solution thereof treated with melatonin and a histone deacetylase inhibitor as an active ingredient.

[0053] In the present invention, the 'culture medium' includes a medium capable of supporting stem cell growth and survival in vitro, secretions of cultured stem cells contained in the medium, etc. The medium used for culture includes all conventional media used in the art suitable for culturing stem cells. The medium and culture conditions can be selected depending on the type of cell. The medium used for culture is preferably a cell culture minimum medium (CCMM), which generally contains carbon source, nitrogen source, and trace element components. Examples of such cell culture minimum media include, but are not limited to, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, αMEM (α Minimal essential Medium), GMEM (Glasgow's Minimal essential Medium), Iscove's Modified Dulbecco's Medium, etc.

[0054] Additionally, the medium may contain antibiotics such as penicillin, streptomycin, and gentamicin.

[0055] In the present invention, the 'pharmaceutical composition' may contain a pharmaceutically acceptable carrier, and the carrier may include ion exchange resin, alumina, aluminum stearate, lecithin, serum protein, buffer substance, water, salt, electrolyte, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substrate, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol, and wool fat.

[0056] In the present invention, the 'pharmaceutical composition' may be characterized by being formulated for intravenous, intraperitoneal, intramuscular, intraarterial, oral, intracardiac, intramedullary, intrathecal, transdermal, enteral, subcutaneous, sublingual or topical administration, and may additionally contain auxiliary agents such as buffers, antimicrobial preservatives, surfactants, antioxidants, tonicity regulators, preservatives, thickeners or viscosity modifiers. The pharmaceutical composition of the present invention may be prepared in the form of a solution, suspension, emulsion, gel or powder.

[0057] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the severity of symptoms, the patient's weight, age, sex, administration method, and administration time, and a skilled physician can easily determine an effective dosage for the desired treatment or prevention.

[0058] In the present invention, the term 'patient' generally includes not only humans, but may also include other animals, such as other primates, rodents, dogs, cats, horses, sheep, pigs, etc. The term 'patient' in the present invention includes subjects other than humans who are diagnosed with or suspected of having solid cancer.

[0059] Meanwhile, the present invention can be used in a form including all of the stem cells, their secretions, and medium components, a form including only the secretions and medium components, a form in which only the secretions are separated and used alone or together with stem cells, or a form in which only stem cells are administered to produce secretions in the body.

[0060] The above stem cells can be obtained using any method commonly known in the art.

[0061] Stem cells treated with the histone deacetylase inhibitor and priming factor of the present invention can be used as a cell therapy agent for treating specific diseases, and the treatment can be direct treatment or pre-treatment with the molecules.

[0062] The above 'cell therapy product' refers to a medicine used for treatment, diagnosis, and prevention purposes through a series of actions such as proliferating and selecting living autologous, allogenic, and xenogenic cells in vitro or changing the biological characteristics of cells through other methods to restore the functions of cells and tissues.

[0063] The above cell therapy agent can be administered to the human body through any common route as long as it can reach the target tissue.

[0064] In the present invention, the inflammatory disease or immune disease is hypoactive bladder, osteoarthritis, rheumatoid arthritis, cystitis, interstitial cystitis, asthma, dermititis, atopy, psoriasis, cystic fibrosis, post transplantation late and chronic solid organ rejection, graft-versus-host disease, transplant rejection disease, multiple sclerosis, systemic lupus erythematosus, Sjogren syndrome, Hashimoto thyroiditis, polymyositis, scleroderma, Addison disease, vitiligo, pernicious anemia, Glomerulonephritis and pulmonary fibrosis, Inflammatory Bowel Disease, Crohn's 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 artery disease, or mitochondrial disease, but are not limited thereto.

[0065] The pharmaceutical composition for the prevention or treatment of inflammatory or immune diseases, comprising stem cells or a culture thereof treated with the melatonin and histone deacetylase inhibitor of the present invention as active ingredients, may be particularly effective in treating underactive bladder. The underactive bladder (UAB) of the present invention includes cases in which activity is reduced due to diabetes or other conditions.

[0066] Hereinafter, in order to help understand the present invention, examples will be given in detail.

[0067] [Example 1]

[0068] Experimental method

[0069] 1-1. Research Approval

[0070] All animal experiments were approved by the Institutional Animal Care and Use Committee of Ulsan University College of Medicine (IACUC-2020-12-141). Human umbilical cord (UC) and fat samples were obtained from healthy, normal, full-term infants after obtaining written parental consent in accordance with guidelines approved by the Human Experimentation Ethics Committee of Seoul Asan Medical Center (IRB#: 2015-0303). Informed consent was obtained from all pregnant women prior to UC collection.

[0071]

[0072] 1-2. Cell culture and in vitro characterization of MSCs

[0073] Human UC MSCs (UC-MSCs) were cultured in low-glucose DMEM containing 10% heat-inactivated fetal bovine serum (FBS), 5 ng / ml human epidermal growth factor (Sigma-Aldrich), 10 ng / ml basic FGF, and 50 ng / ml long-R3 insulin-like growth factor-1 (ProSpec, Rehovot, Israel). Human adipose tissue-derived MSCs (AD-MSCs) were established as previously reported and maintained using the same process as UC-MSCs. All MSCs used in the present invention were expanded for no more than 7 passages to ensure functionality and maintained in a humidified atmosphere with 5% CO2 at 37°C. In vitro MSCs cell activity assays, including cell proliferation, colony forming unit (CFU) for self-renewal-fibroblasts, trans-well migration in response to platelet-derived growth factor (10 ng / mL PDGF-AA, R&D Systems, Minneapolis, MN, USA), angiogenic potential via Matrigel tube-formation assay, in vitro anti-inflammatory, and suppressive properties in response to allogeneic stimulation via immunomodulatory assay, were performed as described previously. Key functions of MSCs were quantified by digital image analysis using Image Pro 5.0 software (Media-Cybernetics, Rockville, MD, USA). FACS data analysis was performed using FlowJo software 7.6.5 (FlowJo, LLC, Ashland, OR, USA).

[0074]

[0075] 1-3. Real-time observation of GRC in living MSCs

[0076] To track GSH changes in living single cells in real time under various culture conditions, we performed GRC analysis using the Operetta High-Content Imaging System (HH12000000; PerkinElmer, Waltham, MA, USA) with 200 or 400 magnification. The GRC analysis was performed as a non-destructive, integrated, and image-based high-throughput analysis for qualitative and quantitative analysis of GSH dynamics in living MSCs. The GRC analysis was performed based on the unique properties of FreSHtracer (Fluorescent real-time thiol tracer; Cell2in, Inc., Seoul, Korea), a reversible chemical probe for GSH. In the reaction with GSH, FreSHtracer exhibited a λmax spectral shift of its UV-visible absorbance from 520 nm to 430 nm, which resulted in a decrease in the fluorescence emission intensity at 580 nm (F580, λex 520 nm) and an increase in the fluorescence intensity at 510 nm (F510, λex 430 nm). To obtain the fluorescence ratio (FR) value of FreSHtrace, the fluorescence emissions excited at 430 and 520 nm were detected at 510 and 580 nm, respectively. The fluorescence signals of FreSHtrace were analyzed using Harmony High-Content Imaging and Analysis Software 3.1 (PerkinElmer) with a confocal mode.

[0077]

[0078] 1-4. Gene expression and Western blotting analysis

[0079] Quantitative measurements of transcripts of the indicated genes were performed using 50 ng of total RNA as previously reported. Relative expression levels of the genes were measured using the 2 -ΔΔCt method, and GAPDH was used as an endogenous control gene.

[0080] For Western blot analysis, cell extracts (30 μg) were prepared with RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and separated on SDS-PAGE gels. The expression levels of the indicated proteins were measured using specific antibodies as probes. The signal densities for the indicated proteins were measured and quantified using NIH Image J software.

[0081]

[0082] 1-5. Statistical Analysis

[0083] Data were statistically analyzed using the nonparametric Mann-Whitney test or one- or two-way ANOVA with Bonferroni post-hoc testing. All analyses were performed using GraphPad Prism 7.0 software (GraphPad Software, La Jolla, CA, USA). A p < 0.05 was considered statistically significant.

[0084]

[0085] 1-6. Lipopolysaccharide (LPS)-induced interstitial cystitis (IC) animal model and administration of MT+VPA MSCs

[0086] All animal experiments were approved by the Institutional Animal Care and Use Committee of Ulsan University College of Medicine (IACUC-2019-12-129). LPS injection into the LPS-induced IC rat model was performed as previously described. To establish the LPS-IC animal model, 8-week-old female Sprague-Dawley rats (OrientBio, Gapyong, Gyeonggi-do, Korea) were administered PS+LPS once a week for 5 weeks. Using this disease model that induced IC, the therapeutic efficacy of M-MSC stem cells was investigated. Five weeks after PS+LPS administration, an abdominal incision was made, and PBS vehicle or MT+VPA-primed MSCs were directly injected into the anterior wall and submucosal layer of the bladder dome using a 500 μm syringe and a 26-gauge needle, as previously reported. One week after MSCs administration, bladder voiding function and tissue damage were measured using non-anesthetized and unrestrained cystometry (awake cystometry) and histological staining, respectively.

[0087]

[0088] 1-7. Cystometrogram by awake cystometry

[0089] Cystomanometry was performed in nonanesthetized, unrestrained rats housed in metabolic cages. Simultaneous catheterization was performed 3 days before cystometry for recording intravesical pressure (IVP) and intraabdominal pressure (IAP), as previously reported. The bladder was accessed using an inflatable PE-50 catheter (Clay Adams, Parsippany, NJ) connected to a pressure transducer (Research Grade Blood Pressure Transducer; Harvard Apparatus, Holliston, MA, USA) and a microinjection pump (PHD22 / 2000 pump; Harvard Apparatus). Micturition volume (MV) was continuously recorded by a collecting tube connected to a physical displacement transducer (Research Grade Isometric Transducer; Harvard Apparatus), while sterile saline was infused into the bladder at approximately 0.4 ml / min. IVP, IAP, and MV were continuously recorded using Acq Knowledge 3.8.1 software and an MP150 data acquisition system (Biopac Systems, Goleta, CA, USA) at a sampling rate of 50 Hz. The average values ​​were calculated from three repeated voids, with five animals in each group (n = 5). Non-voiding contraction (NVC) was calculated when the increase in IVP exceeded 2 cmH2O from the baseline without voiding.BP (bladder basal pressure) is the lowest bladder pressure during bladder filling, MP (micturition pressure) is the maximum bladder pressure during the voiding cycle, MV (micturition volume) is the volume of urine voided, and RV (residual volume) is the volume of urine remaining after voiding. BC (bladder capacity) is the sum of MV and RV, and MI (micturition interval) refers to the interval between voiding contractions.

[0090]

[0091] 1-8. Thelper (Th) type 2 response-induced asthma animal model and administration of MT+VPA MSCs

[0092] All animal experiments were approved by the Institutional Animal Care and Use Committee of Ulsan University College of Medicine (IACUC-2015-12-061). Six-week-old BALB / c mice (Orient Bio, Gapyeong, Gyeonggi-do, Korea) were used to create an asthma mouse model.

[0093] On days 0 and 7, mice were sensitized by intraperitoneal administration of 2 mg Alum (Thermo, #77161, USA), and challenged by intranasal administration of 50 μg OVA (Sigma, #A5503, USA) after anesthesia on days 14, 15, 16, 21, 22, and 23 to induce impairment. The mice were intravenously injected with 1.0 X 10 5Navie MSCs and MT+VPA MSCs were injected into the lungs. BALF, lymph node, and lung tissues were obtained from mice 24 hours after the final immunization. The number of monocytes, basophils, neutrophils, and lymphocytes, as well as changes in cytokine expression in BALF and lung tissues, were measured as previously described. For histopathological evaluation, the lungs were perfused with 5 ml of PBS through the right ventricle and inflated with 1 ml of PBS through the trachea. The inflated lungs were fixed by immersion in 10% neutral buffered formalin for 24 hours. The fixed lung tissues were embedded in paraffin and sectioned at 4 μm thickness. The extent of inflammation in the bronchial and perivascular areas was examined by hematoxylin and eosin staining. Engraftment of injected MSCs was determined by immunofluorescence analysis of human β2-microglobulin (ab15976; Abcam, USA) and visualized using FITC-labeled secondary antibodies. Nuclei were relatively stained with 4'-6-diamino-2-phenylindole (DAPI; Sigam, USA).

[0094]

[0095] [Example 2]

[0096] Establishment of core technologies for enhancing stem cell function and evaluation of in vitro and in vivo efficacy

[0097] 2-1. Discovery of low-molecular compounds to enhance MSC antioxidant capacity and survival rate.

[0098] The present inventors, among the various priming factors for enhancing MSC function, conducted an efficacy evaluation after treating with a combination of melatonin (MT) and previously discovered valproic acid (VPA).

[0099] To establish the optimal mixed composition of MT and VPA for securing multifunctional MSCs in a single process, the conditions for treating umbilical cord-derived MSCs (WJ-MSCs) with various concentrations of a culture medium composition (MT) for enhancing antioxidant activity were analyzed. As a result, when the MT+VPA combination was treated, the colony forming unit (self-renewal activity) increased in a dose-dependent manner compared to when the cells were treated with MT alone, and chemotaxis (cell migration activity) increased when MT was treated at 50 μM rather than 25 μM. Therefore, it can be seen that the treatment with the MT+VPA combination is a candidate substance for enhancing the function of stem cells. (See Figs. 1a and 1b)

[0100] Since it is important to secure optimal reagent treatment conditions (time, concentration, etc.) in order to develop it as a therapeutic agent, the following was conducted to evaluate the function of stem cells by treating MSCs with compounds separately or together.

[0101]

[0102] 2-2. Confirmation of conditions for combined treatment with melatonin (MT) and valproic acid (VPA).

[0103] In order to find the conditions that maximize the effect when treating with a combination of MT and VPA, VPA was fixed at 0.5 mM, and the concentration of MT was adjusted from 100 μM to 500 μM, and then self-renewal ability (CFU-F) and cell migration ability (Chemotaxis) were measured (Fig. 2a and Fig. 2b).

[0104] Referring to Figures 2a and 2b, it can be confirmed that the optimal MT+VPA combination treatment conditions are 200 μM MT and 0.5 mM VPA. In other words, it can be confirmed that MT and VPA must be treated at a ratio of approximately 2:5 to most effectively increase cell activity.

[0105]

[0106] 2-3. Confirmation of stem cell self-renewal and cell migration ability following combined treatment with melatonin (MT) and valproic acid (VPA).

[0107] In this example, the self-renewal ability and cell migration ability were confirmed after MT+VPA treatment according to the stem cell type. Referring to FIGS. 3A to 3D and 4A to 4D, it can be confirmed that the self-renewal ability (Colony-Forming Unit-Fibroblast; CFU-F) and cell migration ability (Chemotaxis) significantly increased due to MT+VPA priming in all four types of WJ-MSC (#195, #140, #183, #202).

[0108] In addition, in order to study the universality of the MT+VPA-MSC manufacturing method established from human umbilical cord-derived MSC, 2 lots (AD-MSC#202, #195) of adipose-derived MSCs at clinical research level were provided from the Stem Cell Center of Seoul Asan Medical Center. When MT and VPA were treated together, it was confirmed that the self-renewal ability and cell migration ability of stem cells increased in AD-MSCs as well, confirming that the development of MT+VPA MSCs is not limited to a specific cell group and can be utilized universally (see Fig. 5).

[0109]

[0110] 2-3. Confirmation of enhanced stem cell function following combined treatment with melatonin (MT) and valproic acid (VPA).

[0111] To verify the enhanced stem cell properties of MSCs following MT+VPA combination treatment, experiments that serve as important indicators for maintaining MSC function were performed.

[0112] First, we conducted the Glutathione Recovery Capacity Assay, which can measure in real time the recovery capacity of glutathione (GSH), a representative antioxidant indicator indicating resistance to ROS. The results are shown in Fig. 6, and referring to Fig. 6, it can be confirmed that MT+VPA-treated MSCs have a higher antioxidant capacity than naive MSCs (NT in Fig. 6).

[0113] Furthermore, comparative analysis of the expression of genes and proteins involved in various functions, including the existing CREB1-NRF2 associated gene pathway (Figures 7a to 7c), revealed that most gene and protein expression levels increased in MT+VPA-treated MSCs. Therefore, it can be expected that MT+VPA MSCs are stem cells with superior therapeutic potential compared to naïve MSCs.

[0114] That is, according to the present example, it can be seen that the MT+VPA combination can improve stemness, such as improving resistance to ROS in stem cells.

[0115]

[0116] 2-4. Confirmation of the anti-inflammatory response of stem cells following combined treatment with melatonin (MT) and valproic acid (VPA).

[0117] As a result of measuring the anti-inflammation response of MT+VPA MSCs, it was confirmed that the secretion of representative inflammation-related cytokines, TNFa and IL6, was reduced (Fig. 8a), and the expression of genes such as mTNF, mCCL2, mCCL7, mIL1-β, mIL6, and mIL18 was also reduced (Fig. 8b).

[0118]

[0119] 2-5. Confirmation of the immunosuppressive effect of stem cells treated with a combination of melatonin (MT) and valproic acid (VPA).

[0120] In this example, to confirm the immunosuppressive capacity of MSCs treated with MT+VPA, PBMCs (Pheripheral Blood mononuclear cells) were stimulated with PHA (phytohemagglutinin) to induce an immune response, and then a T cell stimulation assay was performed to evaluate the suppressive capacity of MSCs. Referring to Figure 9, it can be seen that treatment with MT+VPA can induce excellent immunosuppressive capacity in stem cells.

[0121] In addition, referring to Figure 10, it can be confirmed that the pro-angiogenesis of MT+VPA treated MSCs is also stably enhanced.

[0122]

[0123] [Example 3]

[0124] Evaluation of the anti-asthmatic treatment efficacy using MSCs with enhanced antioxidant capacity and engraftment rate.

[0125] In this example, to evaluate the in vivo effectiveness of MT+VPA priming MSC functional enhancement, conventionally cultured MSCs (NT) and MT+VPA primed MSCs were transplanted into a poly IC-induced asthma animal model (OVA-induced), and 7 days later, the lung tissue inflammatory response and changes in cytokine expression levels, the number of inflammatory cells in bronchoalveolar lavage fluid (BALF), and changes in cytokines were investigated (Figs. 11a to 11e).

[0126] Referring to Figures 11b to 11e, it can be confirmed that the inflammatory response of lung tissue, the expression of inflammatory cytokines, and the degree of inflammatory cell infiltration in BALF were effectively suppressed in the group injected with MT+VPA-primed MSCs.

[0127] As a result of studying the mechanism of the enhanced anti-asthmatic therapeutic effect of MT+VPA MSCs, it was confirmed that the group transplanted with MT+VPA primed MSCs had a higher engraftment rate in the lung tissue compared to the group transplanted with NT MSCs, and the engrafted cells (human b2 microglobin; hB2M+) existed as type II alveolar (SFPTC+) peripheral cells (SPFTC-).

[0128]

[0129] [Example 4]

[0130] Evaluation of the efficacy of MSCs in the treatment of LPS-induced interstitial cystitis by enhancing antioxidant capacity and engraftment rate.

[0131] The present inventors also created an interstitial cystitis rat model representing the clinical phenotype by injecting protamine sulfate (PS) and lipopolysaccharidated (LPS) intravesically once a week for 5 weeks, and confirmed the preclinical efficacy of stem cells through bladder function tests (awake cystometry).

[0132] Referring to Figures 12a and 12b, the results of this experiment show that the LPS-administered group showed a significant decrease in micturition interval (MI), residual volume (RV), and bladder capacity (BC), and an increase in micturition press (MP). By confirming that a single administration of MT+VPA-primed MSCs improved bladder dysfunction in the LPS model, the preclinical efficacy of stem cells for interstitial cystitis disease models can be confirmed.

[0133] In addition, in this experiment, in order to evaluate the degree of progression of interstitial cystitis, Hematoxylin & Eosin (H&E) staining, Masson-trichrome staining, and TB staining were performed on bladder tissue. As a result, as shown in Figure 13, it was confirmed that severe inflammation and mast cells were increased in the LPS-induced interstitial cystitis model, and that these pathological findings were effectively suppressed by stem cell administration.

[0134]

[0135] [Example 5]

[0136] Confirmed effectiveness in treating diabetic cystopathy

[0137] 5-1. Experimental method

[0138] In this study, 8-week-old female Sprague-Dawley rats were used. Type 1 diabetes was induced using STZ (streptozotocin; Sigma Chemical Company, St. Louis, MO, USA). After overnight fasting, STZ (50 mg / kg) dissolved in 0.1 M citrate buffer, pH 4.5, was injected intraperitoneally. The non-diabetic control group received an equal volume of citrate buffer. After 72 hours, blood glucose levels were assessed through tail blood samples, and rats with blood glucose levels exceeding 200 mg / dL (16.7 mmol / L) were confirmed as diabetic and included in the subsequent study. Three weeks after diabetes induction, diabetic rats were anesthetized using 0.2 mL of Zoletil1 (Virbac Laboratories, Carros, France), and the therapeutic efficacy of MSCs (WJ-MSCs) was evaluated. Human MSCs were resuspended in 200 μL of phosphate-buffered saline (PBS) and injected directly into the outer layer (serosa) of the anterior bladder wall. In non-diabetic and untreated diabetic groups, PBS was injected instead of stem cells. Treatment outcomes were assessed 1 week (short-term) or 2 and 4 weeks (long-term) after MSC injection using awake cystometry and histological analysis.

[0139]

[0140] 5-2. Confirmation of the therapeutic effect of combination priming using melatonin and valproic acid on diabetic cystopathy.

[0141] Figures 14a, 14b, 14c and 14d are the results of confirming the therapeutic efficacy of MT-VPA treatment on bladder voiding function according to Example 5-1, and Figures 15a, 15b, 15c and 15d are the results of quantitative analysis of bladder voiding function factors.

[0142] Considering the beneficial effects of melatonin (MT) and valproic acid (VPA)-primed mesenchymal stem cells (MSCs) on various intractable diseases, we evaluated the therapeutic efficacy of MT+VPA-primed human umbilical cord-derived MSCs (hUC-MSCs) in a rat model of streptozotocin (STZ)-induced diabetic detrusor underactivity (DUA). Naïve cultured hUC-MSCs or MT+VPA MSCs (5.0х10 5 ) transplanted into diabetic DUA, impaired bladder function was improved, with increases in maximum pressure (MP) and bladder emptying efficiency (BVE) and decreases in voiding interval (MI), voided volume (MV), bladder capacity (BC), and residual volume (RV) (see Figs. 14a to 14d and 15a to 15d). In particular, the results of quantitative analysis of bladder function through awake cystometry in animals injected with MT+VPA MSCs compared to naïve-cultured hUC-MSCs showed significant improvements in the above-mentioned bladder function indices, verifying the therapeutic efficacy of MT+VPA MSCs in improving hypoactive bladder function.

[0143] Consistent with the results of voiding function assessment using awake cystometry analysis, MT-VPA MSCs showed superior efficacy in improving tissue damage compared to naïve hUC-MSCs, resulting in expansion of the muscle layer, elevated expression of the muscle marker α-smooth muscle actin (a-SMA), and reduced mast cell infiltration within the detrusor muscle (Figs. 16 and 17). Collectively, these results demonstrated the enhanced therapeutic efficacy of MT+VPA priming for the pathogenesis of diabetic DUA and the mechanism of action of MSC treatment in vivo.

[0144]

[0145] In summary, the combination of melatonin (MT) and valproic acid (VPA) enhanced stem cell activity, increasing stem cell engraftment rates and exhibiting anti-inflammatory and antioxidant effects. Therefore, melatonin (MT) and valproic acid (VPA) can be used as a priming technique to enhance stem cell functionality while simultaneously ensuring safety.

[0146]

[0147] The above examples are merely illustrative of the present invention, and the scope of the present invention is not limited to these examples. These examples are provided to more fully explain the present invention to those of ordinary skill in the art.

Claims

1. A composition for promoting stem cell activity containing melatonin and a histone deacetylation inhibitor as active ingredients.

2. A composition for promoting stem cell activity in claim 1, wherein the histone deacetylation inhibitor is at least one selected from the group consisting of valproic acid (VPA), sodium butyrate (NaB), nicotinamide (NAD), and sirtinol.

3. A composition for promoting stem cell activity in claim 1, wherein the histone deacetylation inhibitor is valproic acid (VPA).

4. In paragraph 1, A composition for promoting stem cell activity, wherein melatonin and a histone deacetylation inhibitor are mixed at a concentration ratio of 2:2 to 5.

5. In paragraph 1, A composition for promoting stem cell activity, wherein the stem cell is at least one cell selected from the group consisting of neural stem cells, hepatic stem cells, hematopoietic stem cells, umbilical cord stem cells, epidermal stem cells, gastrointestinal stem cells, endothelial stem cells, muscle stem cells, mesenchymal stem cells, and pancreatic stem cells.

6. A composition for promoting stem cell activity in claim 1, wherein the stem cell activity is cell mobility, colony forming ability and anti-inflammatory activity.

7. Stem cells whose activity is promoted by being treated with a composition according to any one of claims 1 to 6.

8. A method for promoting stem cell activity, comprising a step of treating separated stem cells with melatonin and a histone deacetylation inhibitor.

9. In paragraph 8, A method for promoting stem cell activity, wherein the histone deacetylase inhibitor is at least one selected from the group consisting of valproic acid (VPA), sodium butyrate (NaB), nicotinamide (NAD), and sirtinol.

10. In paragraph 8, A method for promoting stem cell activity, wherein melatonin and a histone deacetylation inhibitor are mixed at a concentration ratio of 2:2 to 5.

11. A pharmaceutical composition for preventing or treating inflammatory or immune diseases, comprising stem cells or a culture solution thereof treated with melatonin and a histone deacetylase inhibitor as an active ingredient.

12. In paragraph 11, A pharmaceutical composition for preventing or treating an inflammatory disease or an immune disease, wherein the histone deacetylase inhibitor is at least one selected from the group consisting of valproic acid (VPA), sodium butyrate (NaB), nicotinamide (NAD), and sirtinol.

13. In paragraph 11, The above inflammatory diseases or immune diseases include underactive bladder, osteoarthritis, rheumatoid arthritis, cystitis, interstitial cystitis, asthma, dermititis, atopy, psoriasis, cystic fibrosis, post transplantation late and chronic solid organ rejection, graft-versus-host disease, transplant rejection disease, multiple sclerosis, systemic lupus erythematosus, Sjogren syndrome, Hashimoto thyroiditis, polymyositis, scleroderma, Addison disease, vitiligo, pernicious anemia, Glomerulonephritis and pulmonary fibrosis, Inflammatory Bowel Disease, Crohn's disease, Autoimmune Diabetes, Diabetic retinopathy, Rhinitis, Ischemia-reperfusion injury, Post-angioplasty restenosis, Chronic obstructive pulmonary disease;A pharmaceutical composition for preventing or treating an inflammatory disease or an immune disease, wherein the disease is at least one disease selected from the group consisting of COPD, Graves disease, gastrointestinal allergy, conjunctivitis, atherosclerosis, coronary artery disease, angina, cancer metastasis, small artery disease, and mitochondrial disease.;

Images