Stem cell priming composition

Abstract

The present invention relates to a priming composition capable of enhancing the immunomodulatory activity or regenerative activity of stem cells.

Description

Stem cell priming composition

[0001] The present invention relates to a priming composition capable of enhancing the immunomodulatory activity or regenerative activity of stem cells.

[0002] [Research and development project supporting this invention]

[0003] [Project Number] RS-2024-00460565

[0004] [Ministry Name] Ministry of SMEs and Startups

[0005] [Research Management Specialist Agency] Small and Medium Business Technology Information Promotion Agency

[0006] [Research Project Name] Startup Growth Technology Development Project (TIPS)

[0007] [Research Project Name] Development of a Dual-Target Treatment for Intractable Inflammatory Bowel Disease Using Stem Cell Secretomes

[0008] [Organizer] Jeongjin Bioscience Co., Ltd.

[0009] Research Period: July 1, 2024 - September 23, 2024

[0010] Stem cells are undifferentiated cells that can be obtained from various tissues of embryos and adults, and have characteristics such as self-renewal, differentiation, and immortality. Stem cells can be divided into embryonic stem cells and adult stem cells depending on the tissue of origin. While therapeutic experiments using embryonic stem cells are difficult due to ethical issues and the possibility of tumor formation, adult stem cells have the advantage of being easily obtained from various tissues, and active research is being conducted to apply them to the treatment of various diseases.

[0011] Among stem cells, multipotent mesenchymal stem cells (MSCs) are a component of the tissue matrix of all adult organs, likely located in perivascular regions, where they play a crucial role in tissue homeostasis, surveillance, repair, and remodeling. Typical immunophenotypic profiles of MSCs include the expression of the surface markers CD44, CD73, CD90, and CD105, and the absence of CD34, CD45, CD14, and HLA-DR. MSCs possess multilineage differentiation potential into osteocytes, adipocytes, and chondrocytes.

[0012] Mesenchymal stem cells are known to suppress inflammation, induce the production of regulatory T cells (Treg), or induce the death of immune cells involved in apoptosis, and various therapeutic agents using them are being actively developed.

[0013] The properties of mesenchymal stem cells are influenced by biological, biochemical, and biophysical factors both in vivo and in vitro, driven by interactions between cells, the extracellular matrix (ECM), and soluble bioactive factors. Mesenchymal stem cells interact with surrounding tissues and cells in a three-dimensional space, modulating the ECM to promote angiogenesis, produce anti-inflammatory molecules, prevent cell death (anti-apoptotic effects), and modulate the immune system.

[0014] Accordingly, recent research has been actively conducted to enhance the lineage differentiation and function of mesenchymal stem cells by controlling biological, biochemical, and / or biophysical factors to improve the therapeutic potential of mesenchymal stem cells. One known method is the technique of priming stem cells using proinflammatory mediators. Representative priming techniques include (a) MSC priming using inflammatory cytokines or mediators, (b) MSC priming using hypoxia, (c) MSC priming using pharmacological drugs and chemicals, (d) MSC priming using biomaterials and other culture conditions, and (e) MSC priming with other molecules.

[0015] One object of the present invention is to provide a priming composition capable of enhancing the immunomodulatory activity or regenerative activity of stem cells.

[0016] Another object of the present invention is to provide stem cells or a culture solution derived from the stem cells with enhanced immunomodulatory activity or regenerative activity through treatment with the above-described composition.

[0017] Another object of the present invention is to provide various applications of stem cells or stem cell-derived culture fluids having enhanced immunomodulatory activity or regenerative activity through treatment with the above-described composition.

[0018] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0019] Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, various specific details, such as specific configurations, compositions, and processes, are set forth to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and configurations. In other instances, well-known processes and manufacturing techniques have not been described in specific detail so as not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the appearances of "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0020] Unless otherwise specifically defined herein, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0021]

[0022] According to one embodiment of the present invention, the present invention relates to a composition for enhancing the immune-regulating activity or regenerative activity of stem cells, comprising agmatine or a pharmaceutically acceptable salt thereof; punicalagin, a stereoisomer thereof or a pharmaceutically acceptable salt thereof; or a mixture thereof.

[0023] In the present invention, the above "stem cell" is an undifferentiated cell in the pre-differentiation stage that can be obtained from each tissue of an embryo or adult, and has characteristics such as self-renewing, differentiation, and immortality. Stem cells can be divided into pluripotency, multipotency, and unipotency stem cells according to their differentiation ability, and adult stem cells are characterized by multipotency, and embryonic stem cells are characterized by pluripotent differentiation ability. In the present invention, the stem cell that is the target of the above function enhancement may include all of them, without limitation, whether it is an embryonic stem cell, an adult stem cell, or an induced pluripotent stem cell (IPS).

[0024] In the present invention, the adult stem cell may be a mesenchymal stem cell, a mesenchymal stromal cell derived from mammalian tissue including a human, a mesenchymal stem cell derived from mammalian tissue including a human, a multipotent stem cell, or an amniotic epithelial cell, and is preferably a mesenchymal stem cell, but is not limited thereto.

[0025] In the present invention, the mesenchymal stem cells may be mesenchymal stem cells derived from umbilical cord, umbilical cord blood, Wharton's Jelly, bone marrow, fat, muscle, nerve, skin, amniotic membrane, placenta, amniotic fluid, tonsil, dental pulp, periodontal ligament, and dental follicle, but are not limited thereto.

[0026] In the present invention, the "agmatine" is a 1-(4-aminobutyl)guanidine compound represented by the following chemical formula 1, which is a compound naturally produced from arginine. The chemical formula is C5H 14N4, molecular weight 130.195 g / mol, CAS number 306-60-5. Agmatine is known to exert modulatory effects on several molecular targets, particularly neurotransmitter systems, ion channels, nitric oxide (NO) synthesis, and polyamine metabolism, providing a basis for further research into potential applications.

[0027] [Chemical Formula 1]

[0028]

[0029] In the present invention, the "punicalagin" is a type of phenol compound, ellagitannin, and is a 2,3-(S)-hexahydroxydiphenoyl-4,6-(S,S)-galazyl-D-glucose compound represented by the following chemical formula 2. Punicalagin is found as alpha and beta isomers in pomegranates (Punica granatum) and the like. The chemical formula of punicalagin is C 48 H 28 O 30 , the molecular weight is 1084.71 g / mol and the CAS number is 65995-63-3.

[0030] [Chemical Formula 2]

[0031]

[0032] In the composition of the present invention, the agmatine or a pharmaceutically acceptable salt thereof may be included at a concentration of, but not limited to, 10 to 1000 μM, 100 to 800 μM, 200 to 600 μM, or 200 to 400 μM.

[0033] In the composition of the present invention, the punicalagin, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof may be included at a concentration of, but not limited to, 0.1 to 100 nM, 1 to 80 nM, 1 to 20 nM, 5 to 20 nM, 1 to 10 nM, or 5 to 10 nM.

[0034] Even if agmatine or punicalagin is described in the present invention, it also includes pharmaceutically acceptable salts of said compounds. Such pharmaceutically acceptable salts are salts generally considered by those skilled in the art to be suitable for medical applications (e.g., because such salts are not harmful to a subject to be treated with said salt), or salts that cause acceptable side effects within the respective treatment. Typically, such pharmaceutically acceptable salts are salts considered acceptable by regulatory authorities such as the U.S. Food and Drug Administration (FDA), the European Medicines Agency (EMA), or the Pharmaceuticals and Medical Devices Agency (PMDA) of the Ministry of Health, Labour and Welfare of Japan. However, the present invention also includes salts of the compounds of the present invention that are not pharmaceutically acceptable in themselves, for example, as intermediates in the preparation of the compounds of the present invention or physiologically functional derivatives thereof, or as intermediates in the preparation of pharmaceutically acceptable salts of the compounds of the present invention or physiologically functional derivatives thereof. Such salts include water-insoluble salts, and in particular, water-soluble salts.

[0035] In each case, a person skilled in the art can readily determine whether a particular compound according to the invention or a physiologically functional derivative thereof is capable of forming a salt, i.e. whether the compound according to the invention or a physiologically functional derivative thereof has a group capable of carrying a charge, such as, for example, an amino group, a carboxyl group, etc.

[0036] Exemplary salts of the compounds of the present invention are acid addition salts or salts with bases, particularly pharmaceutically acceptable inorganic and organic acid addition salts and salts with bases commonly used in pharmacy, which are water-insoluble or particularly water-soluble acid addition salts. Depending on the substituents of the compounds of the present invention, salts with bases may also be suitable. Acid addition salts can be formed, for example, by mixing a solution of a compound of the present invention with a solution of a pharmaceutically acceptable acid, such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. Similarly, pharmaceutically acceptable base addition salts include alkali metal salts (e.g., sodium or potassium salts); alkaline earth metal salts (e.g., calcium or magnesium salts); and salts formed with suitable organic ligands (e.g., ammonium, quaternary ammonium and amine cations formed using counter anions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyl sulfonates and aryl sulfonates).Illustrative examples of pharmaceutically acceptable salts include acetate, adipate, alginate, arginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphorate, camphorsulfonate, camsylate, carbonate, chloride, citrate, digluconate, dihydrochloride, dodecylsulfate, edetate, edisylate, ethanesulfonate, formate, fumarate, galactate, galacturonate, gluconate, glutamate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrobromide, hydrochloride, hydroiodide, Including but not limited to 2-hydroxy-ethanesulfonate, hydroxynaphthoate, iodide, isobutyrate, isothionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, methanesulfonate (mesylate), methyl sulfate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate / diphosphate, phthalate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, sulfate, suberate, succinate, tannate, tartrate, tosylate, undecanoate, valerate, etc.

[0037] Salts which are not pharmaceutically acceptable and which may be obtained, for example, as process products during the preparation of the compounds according to the invention on an industrial scale, are also encompassed by the present invention and, if desired, can be converted into pharmaceutically acceptable salts by methods known to those skilled in the art.

[0038] As an example, in the present invention, the agmatine may be used in the form of a sulfate represented by the following chemical formula 3, but is not limited thereto.

[0039] [Chemical Formula 3]

[0040]

[0041] Meanwhile, even if the compound is described as punicalagin in the present invention, it includes stereoisomers of the compound. Since the compound according to the present invention may have an asymmetric carbon center, it may exist as an R or S isomer or a racemic compound, and all of these optical isomers and mixtures may be included within the scope of the present invention.

[0042] In addition, the compounds of the present invention, as well as their salts, may contain varying amounts of solvent, for example when isolated in crystalline form. Accordingly, solvates, particularly hydrates, of the compounds of the present invention, as well as solvates, particularly hydrates, of salts of the compounds of the present invention, may be included within the scope of the present invention. More particularly, the present invention may include hydrates of the compounds, salts, and / or physiologically functional derivatives according to the present invention, which contain one, two, or half water molecules with respect to the stoichiometry.

[0043] As an example, in the present invention, the punicalagin may be α-punicalagin or β-punicalagin represented by the following chemical formula 4, but is not limited thereto.

[0044] [Chemical Formula 4]

[0045]

[0046] In the above chemical formula 4,

[0047] If R1 is hydrogen and R2 is a hydroxyl group, it is α-punicalagin,

[0048] If R1 is a hydroxy group and R2 is hydrogen, it may be β-punicalazine.

[0049] As an example, in the present invention, the punicalagin may be used in a racemic form in which α-punicalagin and β-punicalagin are mixed, but is not limited thereto.

[0050] Unless specifically stated herein, the punicalagin may refer to a racemic mixture of α-punicalagin and β-punicalagin.

[0051] When using the composition of the present invention, among the various functions of stem cells, immunomodulatory activity, particularly immunosuppressive activity or anti-inflammatory activity, can be enhanced. Specifically, when treating stem cells with the composition of the present invention, it can mean that the expression level of one or more proteins selected from the group consisting of IL-1RA (Interleukin 1 receptor antagonist), IL-10 (Interleukin 10), and TGF-β (transforming growth factor beta), which are factors related to the immunosuppressive activity or anti-inflammation of stem cells, or mRNA encoding the same, is increased, and the expression level of IL-6 (Interleukin 6), which is a factor related to inflammation, or mRNA encoding the same, is decreased.

[0052] When using the composition of the present invention, among the various functions of stem cells, particularly regenerative activity can be enhanced. Specifically, when treating stem cells with the composition of the present invention, it may mean that the expression level of one or more proteins selected from the group consisting of VEGF-A (vascular endothelial growth factor A), FGF-2 (fibroblast growth factor 2), EGF (epidermal growth factor), TGF-β (transforming growth factor beta), PDGF (platelet derived growth factor), and HGF (hepatocyte growth factor), which are factors related to stem cell regeneration, or mRNA encoding the same, is increased.

[0053] In the present invention, the change in the expression level of the mRNA can be measured by, but is not limited to, PCR; qPCR; qRT-PCR; semi-quantitative RT-PCR; digital PCR; Northern blot; mRNA-SEQ; microarray, etc., and any method known in the art for measuring the expression level of a gene can be used without limitation. In addition, in the present invention, the change in the expression level of the protein can be measured by, but is not limited to, antibody-based assay; enzyme-linked immunosorbent assay (ELISA); immuno or Western blot; flow cytometry, mass spectrometry, etc., and any method known in the art for measuring the expression level of a protein can be included without limitation.

[0054]

[0055] According to another embodiment of the present invention, there is provided a method for enhancing the immunomodulatory activity or regenerative activity of stem cells, comprising a step of treating stem cells with agmatine or a pharmaceutically acceptable salt thereof; punicalagin, a stereoisomer thereof or a pharmaceutically acceptable salt thereof; or a mixture thereof.

[0056] In the present invention, the treatment can be performed by adding agmatine or a pharmaceutically acceptable salt thereof; punicalagin, a stereoisomer thereof or a pharmaceutically acceptable salt thereof; or a mixture thereof to the culture medium during the culture of stem cells.

[0057] In the present invention, the above treatment may be performed in vitro or ex vivo.

[0058] In the present invention, the culture of the stem cells may be performed in a medium containing components necessary for stem cell culture widely known in the art, or in a medium additionally containing components capable of promoting stem cell proliferation. Here, the medium may be a concept encompassing a natural medium, a synthetic medium, or a selective medium.

[0059] In the present invention, the medium may be a cell culture minimum medium (CCMM) containing a carbon source, a nitrogen source, and trace element components, and may additionally contain antibiotics such as penicillin, streptomycin, and gentamicin. In the present invention, the medium may be selected according to the type of cell and culture conditions, and for example, Dulbecco's Modified Eagle's Medium (DMEM), Minimal essential Medium (MEM), Basal Medium Eagle (BME), RPMI1640, F-10, F-12, Minimal essential Medium alpha (MEM alpha), Glasgow's Minimal essential Medium (GMEM), Iscove's Modified Dulbecco's Medium (IMDM), and Chemically Defined Medium (CDM) may be used, but is not limited thereto.

[0060] In the present invention, other culture conditions such as temperature and culture time required for the culture may follow the culture conditions of conventional stem cells.

[0061] As an example, the method of the present invention may include a step of culturing stem cells in a serum medium containing agmatine or a pharmaceutically acceptable salt thereof; punicalagin, a stereoisomer thereof or a pharmaceutically acceptable salt thereof; or a mixture thereof. Here, the serum medium may be, but is not limited to, MEM alpha (Minimal essential Medium alpha) medium supplemented with 5 to 15% by volume of fetal bovine serum (FBS) or human platelet lysate (hPL), or a chemically defined medium excluding serum / plasma and animal / human-derived extracts / lysates. In addition, the culture conditions may be, but are not limited to, culturing the stem cells at a temperature of 35 to 40°C and 5% by volume of CO2 for 1 to 6 days or 1 to 4 days.

[0062] As an example, the method of the present invention may include a step of culturing stem cells in a serum-free medium containing agmatine or a pharmaceutically acceptable salt thereof; punicalagin, a stereoisomer thereof or a pharmaceutically acceptable salt thereof; or a mixture thereof. Here, the serum-free medium may be Dulbecco's Modified Eagle's Medium (DMEM). If necessary, L-glutamine may be additionally added to the serum-free medium, and for example, L-glutamine may be added at a concentration of more than 0 and less than or equal to 10 mM, 1 to 5 mM, or 1 to 3 mM, but is not limited thereto. In addition, the culture conditions may be, but are not limited to, culturing the stem cells at a temperature of 35 to 40°C and under conditions of 5% by volume of CO2 for 1 to 6 days, or 1 to 4 days.

[0063] As an example, the method of the present invention may include a step of first culturing stem cells in a serum medium containing agmatine or a pharmaceutically acceptable salt thereof; punicalagin, a stereoisomer thereof or a pharmaceutically acceptable salt thereof; or a mixture thereof; and a step of second culturing stem cells in a serum-free medium containing agmatine or a pharmaceutically acceptable salt thereof; punicalagin, a stereoisomer thereof or a pharmaceutically acceptable salt thereof; or a mixture thereof. Here, the serum medium may be a MEM alpha (Minimal essential Medium alpha) medium supplemented with 5 to 15 volume% of fetal bovine serum (FBS). In addition, the serum-free medium may be DMEM (Dulbecco's Modified Eagle's Medium), and may have L-glutamine added thereto if necessary. For example, L-glutamine may be added at a concentration of more than 0 and less than 10 mM, 1 to 5 mM, or 1 to 3 mM, but is not limited thereto. In addition, the primary and secondary culture conditions may be, but are not limited to, culturing stem cells for 1 to 6 days and 1 to 4 days, respectively, under conditions of a temperature of 35 to 40°C and 5% by volume of CO2.

[0064]

[0065] According to another embodiment of the present invention, the present invention relates to stem cells having enhanced immunomodulatory activity or regenerative activity by being treated with a composition provided by the present invention.

[0066] In the present invention, stem cells treated with the composition of the present invention may have an increased expression level of one or more proteins selected from the group consisting of IL-1RA (Interleukin 1 receptor antagonist), IL-10 (Interleukin 10), galectin-1, galectin-3, TSG-6 (Tumor Necrosis Factor (TNF)-α-Stimulated Gene 6), and TGF-β (transforming growth factor beta), which are factors related to immunosuppression or anti-inflammation, or mRNA encoding the same, and a decreased expression level of IL-6 (Interleukin 6), which is a factor related to inflammation, or mRNA encoding the same, thereby enhancing immunosuppression or anti-inflammation activity.

[0067] Stem cells treated with the composition of the present invention may have increased regenerative activity, as compared to stem cells not treated with the composition, as shown by an increased expression level of one or more proteins selected from the group consisting of VEGF-A (vascular endothelial growth factor A), FGF-2 (fibroblast growth factor 2), EGF (epidermal growth factor), TGF-β (transforming growth factor beta), PDGF (platelet derived growth factor), and HGF (hepatocyte growth factor), which are regeneration-related factors, or mRNA encoding the same.

[0068]

[0069] According to another embodiment of the present invention, the present invention relates to a stem cell culture solution or a concentrate, purified product or fraction derived from the culture solution, wherein the immunomodulatory activity or regenerative activity is enhanced by being treated with the composition provided by the present invention.

[0070] In the present invention, the stem cell culture solution described above may include a culture solution obtained after culturing to enhance the immune regulation activity or regenerative activity of stem cells according to the method of the present invention.

[0071] The present invention can provide a conditioned medium from which cells have been removed from the culture medium of the above-mentioned stem cells.

[0072] In the present invention, the "conditioned medium" refers to a medium in which stem cells have been cultured and then the cells have been removed, and which has a changed function by containing effective ingredients produced by the cells or factors that help in the maintenance and survival of the cells.

[0073] In the present invention, the conditioned medium may be a supernatant recovered by centrifuging the culture solution obtained after culturing to enhance the immune regulation activity or regenerative activity of stem cells according to the method of the present invention at 500 to 1,500 xg.

[0074] In addition, in the present invention, the conditioned medium may be obtained by filtering the culture solution obtained as described above or the supernatant recovered by centrifugation as described above through a 0.1 to 0.3 μM filter, preferably a 0.2 or 0.22 μM filter.

[0075] In addition, the present invention can provide a secretome derived from the above-mentioned stem cell culture solution.

[0076] In the present invention, the "secretome" refers to a composition containing one or more substances secreted from cells, and is also called "secretomes," "secretomes," or "cell secretomes." The secretome may contain one or more cytokines, one or more exosomes, and / or one or more microvesicles. In the present invention, the secretome may be purified or unpurified, and may additionally contain one or more substances (e.g., culture medium, additives, nutrients, etc.) that are not secreted from cells, if necessary.

[0077] In the present invention, the secretome may be filtered from a culture solution obtained after culturing stem cells to enhance their immune-regulatory activity or regenerative activity according to the method of the present invention so as to include molecules having a molecular weight cut-off value of 1 to 10 kDa or more.

[0078] In the present invention, the secretome may be obtained by centrifuging the culture solution obtained after culturing for enhancing the immune regulation activity or regenerative activity of stem cells according to the method of the present invention at 500 to 1,500 xg, if necessary, to recover the supernatant, and then filtering it with a 0.1 to 0.3 μM filter, preferably a 0.2 or 0.22 μM filter, and then using a tangential flow filtration (TFF) device to filter out molecules having a molecular weight cut-off value of 1 to 10 kDa or less, so that molecules having a molecular weight of 1 to 10 kDa or more are included.

[0079] In the present invention, the supernatant can be diluted with water for injection using a peristatic tubing pump or a diaphragm pump during the above-described filtration, and the concentration can be performed at 0 to 25°C.

[0080] The present invention may further include a step of freeze-drying the concentrate obtained as described above for 6 to 10 hours, if necessary. In the present invention, the freeze-drying can be used to obtain a powdered preparation of secretome or its concentrate.

[0081] The stem cell culture medium provided by the present invention, or the conditioned medium from which cells have been removed, or the secretome as a fraction, may have an increased level of one or more proteins selected from the group consisting of IL-1RA (Interleukin 1 receptor antagonist), IL-10 (Interleukin 10), TGF-β (transforming growth factor beta), PDGF (platelet derived growth factor), VEGF-A (vascular endothelial growth factor A), FGF-2 (fibroblast growth factor 2), EGF (epidermal growth factor), and HGF (hepatocyte growth factor) or an mRNA encoding the same, compared to the stem cell culture medium from which the composition of the present invention has not been treated, or the conditioned medium from which cells have been removed, or the secretome as a fraction, and may have a decreased level of the inflammatory factor IL-6 (Interleukin 6) or an mRNA encoding the same.

[0082]

[0083] According to another embodiment of the present invention, there is provided a cell therapeutic agent comprising stem cells having enhanced immunomodulatory activity or regenerative activity treated with a composition provided by the present invention.

[0084] In the present invention, the above-mentioned "cell therapeutic agent" means a medicine used for the purpose of treatment, diagnosis, and prevention 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 by other methods to restore the functions of cells and tissues.

[0085] In the present invention, the cell therapeutic agent may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be, for example, saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, HSA (human serum albumin), or a mixture of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added.

[0086] In the present invention, the cell therapeutic agent may appropriately add a suspending agent, a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an anti-adsorption agent, a surfactant, a diluent, an excipient, a pH adjuster, a soothing agent, a buffer, a sulfur-containing reducing agent, an antioxidant, etc., depending on the formulation. Examples of the suspending agent include, but are not limited to, methylcellulose, polysorbate 80, hydroxyethylcellulose, gum arabic, mal traganthus, sodium carboxymethylcellulose, and polyoxyethylene sorbitan monolaurate.

[0087] In the present invention, the solution auxiliary agent may include polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, macrogol, castor oil fatty acid ethyl ester, etc. The stabilizer may include, but is not limited to, dextran 40, methylcellulose, gelatin, sodium sulfite, sodium metasulfate, etc.

[0088] In the present invention, the above-mentioned tonic agent may include, but is not limited to, D-mannitol, sorbitol, etc.

[0089] In the present invention, examples of the preservative include, but are not limited to, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, sorbic acid, phenol, cresol, chlorocresol, etc.

[0090] In the present invention, examples of the anti-adsorption agent include, but are not limited to, human serum albumin, lecithin, dextran, ethylene oxide-propylene oxide copolymer, hydroxypropyl cellulose, methyl cellulose, polyoxyethylene hydrogenated castor oil, polyethylene glycol, etc.

[0091] In the present invention, examples of the sulfur-containing reducing agent include, but are not limited to, those having a sulfhydryl group such as N-acetylcysteine, N-acetylhomocysteine, thioctoic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, glutathione, and thioalkanoic acid having 1 to 7 carbon atoms.

[0092] In the present invention, examples of the antioxidant include, but are not limited to, chelating agents such as erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbic acid palmitate, L-ascorbic acid stearate, sodium bisulfite, sodium sulfite, triamyl gallic acid, propyl gallic acid, or sodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, and sodium metaphosphate.

[0093] In the present invention, the cell therapeutic agent may be administered in divided doses once or several times a day at regular intervals, or may be administered multiple times at regular intervals, for example, at a dose of about 1,000 to 10,000 cells / time, 1,000 to 100,000 cells / time, 1,000 to 1,000,000 cells / time, 1,000 to 10,000,000, 1,000 to 100,000,000 cells / time, 1,000 to 1,000,000,000 cells / time, 1,000 to 10,000,000,000 cells / time.

[0094] The injection product according to the present invention can be manufactured in the form of a filled injection by taking an amount commonly known in the art according to the patient's constitution and type of defect.

[0095] When the composition according to the present invention is treated on stem cells, the immunomodulatory activity of the stem cells is enhanced and the regenerative activity is also promoted, so that the composition can be used as a cell therapy agent for treating various diseases.

[0096] Figure 1 is a graph showing the results of analyzing the change in the expression level of IL-10 mRNA in stem cell-derived RNA by qRT-PCR when the culture medium of stem cells was treated with a concentration of 200 μM agmatine (A200), a concentration of 5 nM punicalagin (P5), or a concentration of 400 μM agmatine and a concentration of 10 nM punicalagin (A400+P10) compared to the control group in Experimental Example 1.

[0097] Figure 2 is a graph showing the results of analyzing the change in the expression level of IL-1RA mRNA in stem cell-derived RNA by qRT-PCR when the culture medium of stem cells was treated with a concentration of 200 μM agmatine (A200), a concentration of 5 nM punicalagin (P5), or a combination of agmatine at a concentration of 400 μM and punicalagin at a concentration of 10 nM (A400+P10) compared to the control group in Experimental Example 1.

[0098] Figure 3 is a graph showing the results of analyzing the change in the expression level of Galectin-1 mRNA in stem cell-derived RNA by qRT-PCR when the culture medium of stem cells was treated with a concentration of 200 μM agmatine (A200), a concentration of 5 nM punicalagin (P5), or a concentration of 400 μM agmatine and a concentration of 10 nM punicalagin (A400+P10) compared to the control group in Experimental Example 1.

[0099] Figure 4 is a graph showing the results of analyzing the change in the expression level of Galectin-3 mRNA in stem cell-derived RNA by qRT-PCR in the culture medium of stem cells treated with a concentration of 200 μM agmatine (A200), a concentration of 10 nM punicalagin (P10), or a combination of agmatine at a concentration of 400 μM and punicalagin at a concentration of 10 nM (A400+P10) compared to the control group in Experimental Example 1.

[0100] Figure 5 is a graph showing the results of analyzing the change in the expression level of TSG-6 mRNA in stem cell-derived RNA by qRT-PCR when the culture medium of stem cells was treated with a concentration of 200 μM agmatine (A200), a concentration of 10 nM punicalagin (P10), or a concentration of 400 μM agmatine and a concentration of 10 nM punicalagin (A400+P10) compared to the control group in Experimental Example 1.

[0101] Figure 6 is a graph showing the results of analyzing the change in the expression level of VEGF-A mRNA in stem cell-derived RNA by qRT-PCR when the culture medium of stem cells was treated with agmatine at a concentration of 200 μM (A200), with punicalagin at a concentration of 5 nM (P5), or with agmatine at a concentration of 400 μM and punicalagin at a concentration of 10 nM (A400+P10) compared to the control group in Experimental Example 1.

[0102] Figure 7 is a graph showing the results of ELISA analysis of changes in the expression level of IL-6 cytokines in stem cell culture media when agmatine was treated in a concentration of 0 to 400 μM (A0 to 400) and punicalagin was treated in a concentration of 0 to 10 nM (P0 to 10) in the stem cell culture medium compared to the control group in Experimental Example 2. However, in Figure 7, P0A0 is the result of treatment with the solvent DMSO.

[0103] Figure 8 is a graph showing the results of ELISA analysis of changes in the expression level of FGF-2 cytokines in stem cell culture media when agmatine was treated in a concentration of 0 to 400 μM (A0 to 400) and punicalagin was treated in a concentration of 0 to 10 nM (P0 to 10) in the culture medium of stem cells compared to the control group in Experimental Example 2. However, in Figure 8, P0A0 is the result of treatment with the solvent DMSO.

[0104] According to one embodiment of the present invention, the present invention relates to a composition for enhancing the immune-regulating activity or regenerative activity of stem cells, comprising agmatine or a pharmaceutically acceptable salt thereof; punicalagin, a stereoisomer thereof or a pharmaceutically acceptable salt thereof; or a mixture thereof.

[0105] The above stem cells may be embryonic stem cells, adult stem cells, or induced pluripotent stem cells (IPS).

[0106] The above stem cells may be mesenchymal stem cells derived from umbilical cord, umbilical cord blood, Wharton's Jelly, bone marrow, fat, muscle, nerve, skin, amniotic membrane, placenta, amniotic fluid, tonsil, dental pulp, periodontal ligament or dental follicle.

[0107] The agmatine or a pharmaceutically acceptable salt thereof may be included at a concentration of 10 to 1000 μM.

[0108] The above punicalagin, a stereoisomer thereof or a pharmaceutically acceptable salt thereof may be included at a concentration of 0.1 to 100 nM.

[0109] The composition may induce an increase in the expression level of one or more proteins selected from the group consisting of IL-1RA (Interleukin 1 receptor antagonist), IL-10 (Interleukin 10), TGF-β (transforming growth factor beta), PDGF (platelet derived growth factor), VEGF-A (vascular endothelial growth factor A), FGF-2 (fibroblast growth factor 2), EGF (epidermal growth factor), and HGF (hepatocyte growth factor) of stem cells or an mRNA encoding the same, or may induce a decrease in the expression level of IL-6 (Interleukin 6) or an mRNA encoding the same.

[0110] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0111]

[0112] Example

[0113]

[0114] [Preparation Example 1] Culturing of mesenchymal stem cells

[0115] Umbilical cords were obtained from healthy fetuses of healthy mothers. The umbilical cords were washed twice with DPBS, once with 83% ethanol, and then twice with PBS. After removing all blood vessels within the umbilical cord tissue, the cord tissue was minced into 1–2 mm fragments. Liberase (Roche) was added to the fragmented umbilical cord tissue samples for 90 min at 37°C according to the manufacturer's protocol to prepare tissue lysates. Liberase was then inactivated by treating the samples with MEM alpha (Gibco) nutrient medium containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin (P / S) (Hyclone, Cytiva). The above tissue lysate was filtered through a 100 μm Cell Strainer (pluriSelect), and the filtrate was centrifuged at 1,000 g for 5 minutes. The supernatant except for the cell pellet was then removed, and the nutrient medium was added to the cell pellet and resuspended by pipetting. The viability of the cells contained in the cell suspension was measured using a Trypan blue solution. The cells were seeded in a culture vessel, the nutrient medium was added, and the cells were cultured at 5% CO2 and 37 °C. Thereafter, the medium was replaced every 3 days, and primary culture was performed for 10 days. After 10 days, the morphology of the cultured cells was observed through an optical microscope, and the morphology of mesenchymal stem cells was observed. Flow cytometry analysis was performed using the Stemflow Human MSC Analysis Kit (BD Bioscience) according to the manufacturer's protocol, confirming that the primary culture of umbilical cord-derived mesenchymal stem cells was completed. After washing the identified cells with DPBS, TrypLE Express (no phenol red) (Gibco) solution was added and incubated in an incubator at 37°C for 5 minutes to detach the cells from the culture vessel.The TrypLE solution was neutralized by adding the above nutrient medium to the sample, and centrifuged at 300 g for 5 minutes. The supernatant excluding the cell pellet was then removed, and the cell pellet was resuspended by pipetting with the nutrient medium. The viability of the cells contained in the cell suspension was measured using a trypan blue solution. The cells were inoculated into a new culture dish, nutrient medium was added, and the cells were cultured at 5% CO2 and 37°C, and subcultures were performed at 3-day intervals.

[0116]

[0117] [Example 1] Priming of mesenchymal stem cells using agmatine

[0118] The umbilical cord-derived mesenchymal stem cells prepared in the above Preparation Example 1 were cultured in a medium containing agmatine. The agmatine was prepared by dissolving agmatine sulfate (Sigma-Aldrich) in distilled water and adding it to the medium so that the final concentration of agmatine in the medium was 100, 200, or 400 μM. The medium used for primary culture aimed at promoting cell activity was MEM alpha (Gibco) medium supplemented with 10% (v / v) FBS (Gibco) and 1% (v / v) penicillin / streptomycin (Hyclone, Cytiva) or 0.5% (v / v) gentamicin (Gibco). The medium used for secondary culture aimed at obtaining cell culture fluid was DMEM 1X (low-glucose, no phenol red) (Gibco) and MEM alpha (Gibco) medium supplemented with 2 mM L-glutamine (Gibco) and 1% (v / v) penicillin / streptomycin (Hyclone, Cytiva) or 0.5% (v / v) gentamicin (Gibco). However, as a negative control, an equal volume of distilled water was added to the medium.

[0119] Specifically, 5 ml of the primary culture medium containing agmatine as described above was dispensed into a T25 flask, and the umbilical cord-derived mesenchymal stem cells prepared in Preparation Example 1 were subcultured to passage number 5 to 6 (Cell passage 5 to 6) to 2,500 to 3,500 cells / cm 2 The stem cells were seeded into the flask at a density of 10 and cultured for 2 days under conditions of 5% CO2 and 37°C. Each medium was collected from the cultured stem cells in a 15 ml tube and centrifuged at 850 g for 10 minutes. The stem cells from which the medium was collected were washed once with 3 mL DPBS (Dulbecco's Phosphate Buffered Saline), and 5 mL of secondary culture medium was added to the cells and cultured for an additional 2 days under conditions of 5% CO2 and 37°C. Thereafter, each medium was collected from the cultured stem cells in a 15 ml tube and centrifuged at 850 g for 10 minutes. The supernatant excluding the precipitated material was obtained from each centrifuged tube and stored at -80°C until analysis. Total RNA from each cell was extracted from the stem cells from which the medium was collected using the GeneJET RNA Purification Kit (Thermo Scientific) according to the manufacturer's protocol, and stored at -80°C until analysis.

[0120]

[0121] [Example 2] Priming of mesenchymal stem cells using punicalagin

[0122] The umbilical cord-derived mesenchymal stem cells prepared in the above Preparation Example 1 were cultured in a medium containing punicalagin. The punicalagin was prepared by dissolving punicalagin (Sigma-Aldrich) in 0.25% (v / v) dimethyl sulfoxide (DMSO) and adding the solution to the medium so that the final concentration of punicalagin in the medium was 1, 5, or 10 nM. The experiment was performed in the same manner as in Example 1, but a solution containing punicalagin was added instead of agmatine to the primary culture medium and the secondary culture medium. However, as a negative control, an equal amount of 0.25% (v / v) DMSO was added to the medium.

[0123]

[0124] [Example 3] Priming of mesenchymal stem cells using agmatine and punicalagin

[0125] The umbilical cord-derived mesenchymal stem cells prepared in the above Preparation Example 1 were cultured in a medium containing both agmatine and punicalagin. The experiment was performed in the same manner as in Example 1, but a solution of agmatine sulfate (Sigma-Aldrich) dissolved in distilled water and a solution of punicalagin (Sigma-Aldrich) dissolved in 0.25% (v / v) DMSO (dimethyl sulfoxide) were added to the primary culture medium and the secondary culture medium, respectively, so that the final concentration of agmatine contained in each medium was 100, 200, or 400 μM, and the final concentration of punicalagin was 1, 5, or 10 nM.

[0126]

[0127] [Experimental Example 1] Evaluation of the functional enhancement effect of stem cells on immune-regulatory activity and regenerative activity.

[0128] In order to confirm whether the functions related to the immunoregulatory activity and regenerative activity of mesenchymal stem cells were enhanced by treatment with agmatine and / or punicalagin in Examples 1, 2 and 3 above, changes in the expression levels of factors related to immunosuppression, anti-inflammation and regeneration were measured from RNA derived from stem cells.

[0129] Specifically, after thawing the RNA samples prepared in Examples 1, 2, and 3, the RNA concentration of each sample was measured using a spectrophotometer (NanoDrop One, Thermo fisher scientific), an Epoch Microplate Spectrophotometer (BioTek), and a Take3 Micro-volume plate (BioTek) according to the manufacturer's protocol.

[0130] Based on the measured RNA concentration value, 1 μg of each RNA sample was sampled, and cDNA of each sample was synthesized according to the manufacturer's protocol using Maxime RT PreMix Kit (Intron) and Alpha Cycler 1 (PCR max). Next, qRT-PCR was performed on the prepared cDNA samples according to the manufacturer's protocol using 2X qPCRBIO SyGreen Blue Mix Lo-ROX (PCR Biosystems), LightCycler 96 System (Roche), and QuantStudio6 Flex (Thermo fisher scientific). The primers used here were purchased from Cosmogenetech (KR), and the information on the primers for each gene is shown in Table 1 below. The qRT-PCR results for IL-10, IL-1RA, Galectin-1, Galectin-3, TSG-6, and VEGF-A mRNA genes according to each treatment were normalized to the GAPDH expression level, and the relative mRNA expression levels were graphically presented in Figures 1 to 6. The statistical significance level for each sample group compared to the control group (the average value of mRNA expression levels in the DW treatment group and DMSO treatment group) was indicated in each figure using an unpaired two-tailed t-test. (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001)

[0131] Gene Sequence of forward primer (5' → 3') Sequence of reverse primer (5' → 3') IL-10GAG ATG CCT TCA GCA GAG TGA AGA (SEQ ID NO: 1) AGT TCA CAT GCG CCT TGA TGT C (SEQ ID NO: 2) IL-1raCCA GCA AGA TGC AAG CCT TCA GAA T (SEQ ID NO: 3) CCA GAC TTG ACA CAG GAC AGG C (SEQ ID NO: 4) Galectin-1GGT CTG GTC GCC AGC AAC CTG AAT (SEQ ID NO: 5) TGA GGC GGT TGG GGA ACT TG (SEQ ID NO: 6) Galectin-3CCA AAG AGG GAA TGA TGT TGC C (SEQ ID NO: 7) TGA TTG TAC TGC AAC AAG TGA GC (SEQ ID NO: 8) TSG-6GGT GTG TAC CAC AGA GAA GCA (SEQ ID NO: 9) GGG TTG TAG CAA TAG GCA TCC (SEQ ID NO: 10) VEGF-ACGG TGC TGG AAT TTG ATA TTC ATT G (SEQ ID NO: 11) CGA TTC AAG TGG GGA ATG GC (SEQ ID NO: 12) GAPDHGAG AAG TAT GAC AAC AGC CTC AAG ATC A (SEQ ID NO: 13) CCA CCA CTG ACA CGT TGG CA (SEQ ID NO: 14)

[0132] As shown in Figures 1 to 6, when mesenchymal stem cells were treated with agmatine or punicalagin according to the present invention, the expression levels of IL-10, IL-1RA, Galectin-1, Galectin-3, and TSG-6 mRNA, which are factors related to immunosuppression and anti-inflammation, were significantly increased compared to the control group treated with only the solvent, and the expression level of VEGF-A mRNA, a representative factor related to regeneration, was also significantly increased compared to the control group treated with only the solvent. In addition, when mesenchymal stem cells were treated with agmatine and punicalagin together, the expression levels of the above factors were confirmed to increase further.

[0133] Through this, it was found that agmatine, punicalagin, or a mixture thereof according to the present invention has a priming effect that enhances the immunosuppressive ability or regenerative activity of stem cells.

[0134]

[0135] [Experimental Example 2] Evaluation of the effect of enhancing the immune-regulatory activity of stem cells.

[0136] In order to confirm whether the function related to the anti-inflammatory activity of mesenchymal stem cells was enhanced by treatment with agmatine and / or punicalagin in the above Example 3, the supernatant obtained after secondary culture of stem cells was subjected to BD OptEIA Human IL-6 ELISA Set (Cat# 555220, BD Biosciences), R&D Human FGF basic DuoSet (Cat# DY233, R&D) and Epoch Microplate Spectrophotometer (BioTek), Multiskan TM The changes in the contents of IL-6 and FGF-2 cytokines in the cell cultures obtained in Examples 1, 2, and 3 were analyzed using a GO Microplate Spectrophotometer (Thermo Scientific) according to the manufacturer's protocol, and the results are shown in Figs. 7 and 8. The statistical significance level for each sample group compared to the control group (the average value of IL-6 contents in the DW treatment group and the DMSO treatment group) was indicated in the figure using an unpaired two-tailed t-test. (* p≤0.05; ** p≤0.01; *** p≤0.001)

[0137] As shown in Fig. 7, when agmatine and punicalagin according to the present invention were combined and treated with mesenchymal stem cells, it was confirmed that the expression level of IL-6, a representative inflammatory cytokine, was significantly reduced compared to the control group.

[0138] In addition, as shown in Fig. 8, when agmatine and punicalagin according to the present invention were treated to mesenchymal stem cells, it was confirmed that the expression level of FGF-2, a representative regenerative factor, was significantly increased compared to the control group treated with only the solvent.

[0139] Through this, it was found that agmatine, punicalagin, or a mixture thereof according to the present invention has a priming effect that enhances the anti-inflammatory function of stem cells.

[0140]

[0141] Through the above experiments, it was found that priming stem cells with agmatine, punicalagin, or a mixture thereof according to the present invention activates the immune-regulatory and regenerative activities of the stem cells themselves, thereby enhancing the utility of the stem cells as a cell therapeutic agent. In addition, it was found that the priming treatment increases the expression levels of immune-suppressive and anti-inflammatory factors and regenerative factors contained in the stem cell culture medium, and thus the stem cell culture medium, the conditioned medium from which cells have been removed, or the secretome can be utilized as a therapeutic agent for various diseases.

[0142]

[0143] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0144] The present invention relates to a priming composition capable of enhancing the immunomodulatory activity or regenerative activity of stem cells.

Claims

1. A composition for enhancing the immunomodulatory activity or regenerative activity of stem cells, comprising agmatine or a pharmaceutically acceptable salt thereof; punicalagin, a stereoisomer thereof or a pharmaceutically acceptable salt thereof; or a mixture thereof.

2. In paragraph 1, A composition for enhancing the immune regulation activity or regenerative activity of stem cells, wherein the stem cells are embryonic stem cells, adult stem cells or induced pluripotent stem cells (IPS).

3. In paragraph 1, A composition for enhancing the immune-regulatory activity or regenerative activity of stem cells, wherein the stem cells are mesenchymal stem cells derived from umbilical cord, umbilical cord blood, Wharton's Jelly, bone marrow, fat, muscle, nerve, skin, amniotic membrane, placenta, amniotic fluid, tonsil, dental pulp, periodontal ligament or dental follicle.

4. In paragraph 1, A composition for enhancing the immune-regulating activity or regenerative activity of stem cells, comprising the agmatine or a pharmaceutically acceptable salt thereof at a concentration of 10 to 1000 μM.

5. In paragraph 1, A composition for enhancing the immune-regulating activity or regenerative activity of stem cells, comprising the punicalagin, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof at a concentration of 0.1 to 100 nM.

6. In paragraph 1, The composition is a composition for enhancing the immune regulation activity or regenerative activity of stem cells, which induces an increase in the expression level of one or more proteins selected from the group consisting of IL-1RA (Interleukin 1 receptor antagonist), IL-10 (Interleukin 10), TGF-β (transforming growth factor beta), PDGF (platelet derived growth factor), VEGF-A (vascular endothelial growth factor A), FGF-2 (fibroblast growth factor 2), EGF (epidermal growth factor), and HGF (hepatocyte growth factor) of stem cells or mRNA encoding the same, or induces a decrease in the expression level of IL-6 (Interleukin 6) or mRNA encoding the same.

7. A method for enhancing the immunomodulatory activity or regenerative activity of stem cells, comprising a step of treating stem cells with agmatine or a pharmaceutically acceptable salt thereof; punicalagin, a stereoisomer thereof or a pharmaceutically acceptable salt thereof; or a mixture thereof.

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