Stem cell priming composition

Abstract

The present invention relates to a priming composition that can enhance the immunomodulatory or regenerative activity of stem cells.

Description

Technical Field

[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 that supported the present invention]

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

[0004] [Ministry Name] Small and Medium Venture Business Department

[0005] [Research Management Specialized Institution] Korea Technology and Information Promotion Agency for SMEs

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

[0007] [Research Subject Title] Development of dual-target therapeutic agents for refractory inflammatory bowel disease utilizing stem cell secretions

[0008] [Leading Institution] Jongjin Bioscience Company Limited

[0009] [Research Period] 2024.07.01~2024.09.23

Background Art

[0010] Stem cells are undifferentiated cells in the pre-differentiation stage obtained from various tissues of embryos and adults, and have characteristics such as self-renewal, differentiation potential, and immortality. Stem cells are divided into embryonic stem cells and adult stem cells according to their tissue of origin. While therapeutic experiments using embryonic stem cells are difficult due to ethical concerns and the potential for tumor formation, adult stem cells have the advantage of being easily obtained from various tissues, and active research is underway to apply them to the treatment of various diseases.

[0011] Among stem cells, pluripotent mesenchymal stem cells (MSCs) are components of the tissue matrix of all adult organs, generally located in the perivascular region, and play a crucial role in tissue homeostasis, surveillance, repair, and remodeling. A typical immunophenotypic profile of mesenchymal stem cells includes the expression of surface markers CD44, CD73, CD90, and CD105, and deficiencies in CD34, CD45, CD14, and HLA-DR. Mesenchymal stem cells have the potential for multi-system differentiation into osteocytes, adipocytes, and chondrocytes.

[0012] Mesenchymal stem cells are known to suppress inflammation, induce the generation of regulatory T cells (Tregs), and induce the death of immune cells involved in cell death, and the development of a variety of therapeutic agents using them is actively underway.

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

[0014] As a result, recent research has been actively conducted to improve the phylogenetic differentiation and function of mesenchymal stem cells by modulating biological, biochemical, and / or biophysical factors in order to enhance 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 media, (b) MSC priming using hypoxia, (c) MSC priming using pharmacological drugs and chemicals, (d) MSC priming using biomaterials and other culture conditions, and (e) techniques for priming MSCs with other molecules. [Overview of the Initiative] [Problems that the invention aims to solve]

[0015] Modes for carrying out the invention technical challenges One object of the present invention is to provide a priming composition that can enhance the immunomodulatory or regenerative activity of stem cells.

[0016] Another object of the present invention is to provide stem cells or culture media derived from the stem cells in which immunomodulatory activity or regenerative activity is enhanced by treatment with the composition.

[0017] Another object of the present invention is to provide diverse applications for stem cells or culture media derived from such stem cells, whose immunomodulatory or regenerative activity has been enhanced by treatment with the composition.

[0018] However, the technical problems that this invention aims to solve are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those with ordinary skill in the art from the following description. [Means for solving the problem]

[0019] solution Hereinafter, various specific examples of the invention described herein are described with reference to the drawings. In the following description, various specific details, such as specific forms, compositions, and processes, are described for a complete understanding of the invention. However, certain specific examples can be carried out without one or more of these specific details, or in conjunction with other known methods and forms. In other examples, known processes and manufacturing techniques are not described as specific details so as not to unnecessarily obscure the invention. Any reference throughout this specification to “one specific example” or “specific example” means that the special features, forms, compositions, or properties described in association with the specific example are included in one or more specific examples of the invention. For this reason, the situations in which “one specific example” or “specific example” is expressed in various places throughout this specification do not necessarily refer to the same specific example of the invention. In addition, special features, forms, compositions, or properties may be combined in any suitable way in one or more specific examples.

[0020] In this invention, unless otherwise defined, all scientific and technical terms used herein have the same meanings as those commonly understood by those skilled in the art in which this invention pertains.

[0021] (blank)

[0022] One embodiment of the present invention relates to a composition for enhancing the immunomodulatory or regenerative activity of stem cells, comprising agmatine or a pharmaceutically acceptable salt thereof; punicalagin, its stereoisomer or a pharmaceutically acceptable salt thereof; or a mixture thereof.

[0023] In the present invention, the "stem cell" is an undifferentiated cell in the pre-differentiation stage obtained from embryonic and adult tissues, and has characteristics such as self-renewal, differentiation ability, and immortality. Stem cells are divided into pluripotent, multipotent and unipotent stem cells according to their differentiation potential. Adult stem cells are characterized by multipotency, while embryonic stem cells are characterized by pluripotency. In the present invention, the stem cells targeted for function enhancement are not limited to embryonic stem cells, adult stem cells, or induced pluripotent stem cells (iPS), and may include all of the above.

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

[0025] In the present invention, the mesenchymal stem cell may be a mesenchymal stem cell derived from umbilical cord, umbilical cord blood, Wharton's Jelly, bone marrow, adipose, muscle, nerve, skin, amnion, placenta, amniotic fluid, tonsil, dental pulp, periodontal ligament, dental follicle, or the like, but is 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 naturally produced from arginine. The chemical formula is C5H 14 N4, with a molecular weight of 130.195 g / mol, and a CAS number of 306-60-5. Agmatine is known to exert regulatory effects particularly on various molecular targets. It provides a basis for additional research into potential application fields through effects on the neurotransmitter system, ion channels, nitric oxide (NO) synthesis, and polyamine metabolism.

[0027] [Chemical formula 1]

[0028] [ka]

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

[0030] [Chemical formula 2]

[0031] [ka]

[0032] In the composition of the present invention, the agmatine or a pharmaceutically acceptable salt thereof may be present in concentrations of 10-1000 μM, 100-800 μM, 200-600 μM, or 200-400 μM, but is not limited thereto.

[0033] In the compositions of the present invention, punicalagin, its stereoisomers, or pharmaceutically acceptable salts may be present in concentrations of 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, but are not limited thereto.

[0034] In the present invention, even if described as agmatine or punicalagin, it further includes pharmaceutically acceptable salts of the said compound. Such pharmaceutically acceptable salts are those suitable for medical application and generally considered by those skilled in the art (for example, because such salts are not harmful to the subjects that can be treated with the said salt), or salts that produce acceptable side effects within their respective therapeutic applications. Generally, such pharmaceutically acceptable salts are those 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). However, the present invention, in principle, further includes, for example, intermediates in the production of the compound according to the present invention or its physiologically functional derivatives, or intermediates in the production of pharmaceutically acceptable salts of the compound according to the present invention or its physiologically functional derivatives, which are pharmaceutically unacceptable in themselves. Such salts include water-insoluble salts, and in particular, water-soluble salts.

[0035] In each case, a person skilled in the art can easily determine whether a particular compound or physiologically functional derivative of the present invention can form a salt, that is, whether the compound or physiologically functional derivative of the present invention has a group that can carry an electric charge, such as an amino group or a carboxyl group.

[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 pharmaceuticals, which are water-insoluble or especially water-soluble acid addition salts. Depending on the substituents of the compounds of the present invention, salts with bases may be even more preferred. Acid addition salts may be formed, for example, by mixing a solution of the compounds 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 may 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).Exemplary examples of pharmaceutically acceptable salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitertate, borate, bromide, butyrate, calcium edetate, camphorate, camphor sulfonate, camylate, carbonate, chloride, citrate, digluconate, dihydrochloride, dodecyl sulfate, edetate, edicilate, ethanesulfonate, formate, fumarate, galactate, galacturonate, gluconate, glutamate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrobromide, hydrochloride, hydroiodide, and 2-hydrochloride. This includes, but is not limited to, oxyethanesulfonates, hydroxynaphthoates, iodides, isobutyrates, isothioates, lactates, laurates, lauryl sulfates, malates, maleates, malonates, mandelates, methanesulfonates (mesylates), methyl sulfates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pantothenates, pectinates, persulfates, 3-phenylpropionates, phosphates / diphosphates, phthalates, piclates, pivalates, polygalacturonates, propionates, salicylates, stearates, sulfates, subelates, succinates, tannates, tartrates, tosylates, undecanoates, valerates, and others.

[0037] Salts that are pharmaceutically unacceptable, for example, obtained as process products during the production of compounds according to the present invention on an industrial scale, are further included in the present invention and, if desired, can be converted to pharmaceutically acceptable salts by methods known to those skilled in the art.

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

[0039] [Chemical formula 3]

[0040] [ka]

[0041] On the other hand, in the present invention, even if "punicalagin" is mentioned, it includes stereoisomers of the aforementioned compound. Since the compounds according to the present invention may have an asymmetric carbon center, they may exist as R or S isomers or racemic compounds, and all of these optical isomers and mixtures may be included in the scope of the present invention.

[0042] Furthermore, not only the compounds of the present invention but also their salts may contain various amounts of solvent when separated into crystalline form, for example. Therefore, the scope of the present invention may include not only solvates of the compounds of the present invention, particularly hydrates, but also solvates of salts of the compounds of the present invention, particularly hydrates. In particular, the present invention may include hydrates of the compounds, salts, and / or physiologically functional derivatives according to the present invention that contain 1, 2, or 1 / 2 water molecules relative to the stoichiometric ratio.

[0043] For 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] [ka]

[0046] In the aforementioned chemical formula 4,

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

[0048] If R1 is a hydroxyl group and R2 is hydrogen, then β-punicalagin may also be used.

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

[0050] In this specification, unless otherwise specified, punicalagin may mean a racemic mixture of α-punicalagin and β-punicalagin.

[0051] When using the composition of the present invention, among the diverse functions of stem cells, immunomodulatory activity in particular, such as immunosuppressive or anti-inflammatory activity, can be enhanced. Specifically, when stem cells are treated with the composition of the present invention, 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), or mRNA encoding these proteins, which are factors related to the immunosuppressive or anti-inflammatory activity of stem cells, increases, while the expression level of IL-6 (Interleukin 6), a factor related to inflammation, or mRNA encoding this protein, decreases.

[0052] When using the composition of the present invention, the regenerative activity, in particular, can be enhanced among the diverse functions of stem cells. Specifically, when stem cells are treated with the composition of the present invention, it can be said 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), or mRNA encoding these proteins, has increased.

[0053] In the present invention, changes in the mRNA expression level can be measured by methods such as PCR, qPCR, qRT-PCR, semi-quantitative RT-PCR, digital PCR, Northern blotting, mRNA-SEQ, and microarrays, but are not limited thereto. Any method known in the industry for measuring gene expression levels can be used without limitation. Furthermore, in the present invention, changes in the protein expression level can be measured by methods such as antibody-based assays, enzyme-linked immunosorbent assays (ELISA), immunoassay or Western blotting, flow cytometry, and mass spectrometry, but are not limited thereto. Any method known in the industry for measuring protein expression levels may be included without limitation.

[0054] (blank)

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

[0056] In the present invention, the treatment may be carried out by adding agmatine or a pharmaceutically acceptable salt thereof; punicalagin, its stereoisomer or a pharmaceutically acceptable salt thereof; or a mixture thereof to the culture medium during stem cell culture.

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

[0058] In the present invention, the culture of stem cells may be carried out in a culture medium containing components necessary for stem cell culture that are widely known in the art, or in a culture medium that additionally contains components that can promote the proliferation of stem cells. Here, the culture medium may be a concept that includes all natural culture media, synthetic culture media, and selective culture media.

[0059] In the present invention, the culture 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 culture medium can be selected depending on the cell type and culture conditions, but is not limited to, for example, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, MEM alpha (Minimal essential Medium alpha), GMEM (Glasgow's Minimal essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), and CDM (Chemically Defined Medium).

[0060] In the present invention, other culture conditions such as temperature and culture time required for the culture can be the same as those for normal stem cell culture.

[0061] As an example, the method of the present invention may include the step of culturing stem cells in a serum medium containing agmatine or a pharmaceutically acceptable salt thereof; punicalagin, its stereoisomer 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 5-15 volume% fetal bovine serum (FBS) or human platelet lysate (hPL), or a chemically defined medium from which serum / plasma and animal / human-derived extracts / lysates have been excluded. Furthermore, the culture conditions may be, but are not limited to, culturing stem cells for 1-6 days or 1-4 days at a temperature of 35-40°C and 5 volume% CO2.

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

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

[0064] (blank)

[0065] A further embodiment of the present invention relates to stem cells treated with the composition provided in the present invention, thereby enhancing their immunomodulatory or regenerative activity.

[0066] In the present invention, stem cells treated with the composition of the present invention may have increased expression levels 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 immunosuppressive ability or anti-inflammatory activity, or mRNA encoding these proteins, compared to stem cells not treated with the composition. Conversely, the expression level of IL-6 (Interleukin 6), a factor related to inflammation, or mRNA encoding this protein may be decreased, thus enhancing immunosuppressive ability or anti-inflammatory activity.

[0067] Stem cells treated with the composition of the present invention may have increased expression levels of one or more proteins selected from the group consisting of regeneration-related factors such as 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), or mRNA encoding these proteins, compared to stem cells not treated with the composition, and may exhibit enhanced regenerative activity.

[0068] (blank)

[0069] According to yet another embodiment of the present invention, the present invention relates to a stem cell culture medium or a concentrate, purified product or fraction derived from the culture medium that has been treated with the composition provided in the present invention to enhance immunomodulatory or regenerative activity.

[0070] In the present invention, the stem cell culture medium may include a culture medium obtained after culturing stem cells for enhancing their immunomodulatory or regenerative activity by the method of the present invention.

[0071] The present invention provides a prepared culture medium from which cells have been removed from the culture medium of the stem cells.

[0072] In the present invention, the "conditioned medium" refers to a medium from which stem cells have been cultured and then the cells have been removed, and whose function has been modified by containing active ingredients produced by the cells during the culture of stem cells or factors that are useful for the maintenance and survival of the cells.

[0073] In the present invention, the prepared medium may be the supernatant obtained by centrifuging the culture medium obtained after culturing stem cells for enhancing their immunomodulatory or regenerative activity using the method of the present invention at 500 to 1,500 x g.

[0074] Furthermore, in the present invention, the prepared 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] Furthermore, the present invention can provide a secretome derived from the stem cell culture medium.

[0076] In the present invention, the term "secretome" refers to a composition comprising one or more substances secreted by a cell, and is also called a "cyclitome," "secretome," or "cellular secretory." 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 not, and may additionally contain one or more substances not secreted by a cell (e.g., culture medium, additives, nutrients, etc.) as needed.

[0077] In the present invention, the secretome may be obtained by filtering a culture medium obtained after culturing stem cells by the method of the present invention to enhance their immunomodulatory or regenerative activity, so that it contains molecules with a molecular weight cut-off value of 1 to 10 kDa or more.

[0078] In the present invention, the secretome may be obtained by, if necessary, centrifuging the culture medium obtained after culturing stem cells by the method of the present invention to enhance their immunomodulatory or regenerative activity, collecting the supernatant, filtering it through a 0.1 to 0.3 μm filter, preferably a 0.2 or 0.22 μm filter, and then filtering it to a constant volume using a tangential flow filtration (TFF) apparatus to remove molecules with a molecular weight cutoff of 1 to 10 kDa or less, so that it contains molecules with a molecular weight of 1 to 10 kDa or more.

[0079] In the present invention, during constant-volume filtration, the supernatant can be concentrated at 0-25°C while replacing the supernatant with sterile water for injection using a peristatic tubing pump or a diaphragm pump.

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

[0081] The stem cell culture medium, or the adjusted medium from which cells have been removed, or the secretome fraction provided in the present invention may have increased levels 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 mRNA encoding these proteins, and decreased levels of the inflammatory factor IL-6 (Interleukin 6) or mRNA encoding it.

[0082] (blank)

[0083] According to yet another embodiment of the present invention, the present invention relates to a cell therapy agent comprising stem cells treated with a composition provided in the present invention, thereby enhancing immunomodulatory or regenerative activity.

[0084] In the present invention, the term "cell therapy agent" refers to a pharmaceutical product used for therapeutic, diagnostic, or preventive purposes by a series of actions, such as growing and selecting living autologous, allogenic, or xenogenic cells outside the body, or altering the biological properties of cells by other means, in order to repair the function of cells and tissues.

[0085] In the present invention, the cell therapy 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), and a mixture of one or more of these components, and other common additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed.

[0086] In the present invention, depending on the dosage form, the cell therapy agent may be appropriately supplemented with suspending agents, solubilizers, stabilizers, isotonic agents, preservatives, anti-adsorption agents, surfactants, diluents, excipients, pH adjusters, analgesics, buffers, sulfur-containing reducing agents, antioxidants, etc., as needed. Examples of the suspending agents include, but are not limited to, methylcellulose, polysorbate 80, hydroxyethylcellulose, gum arabic, tragacanth powder, sodium carboxymethylcellulose, and polyoxyethylene sorbitan monolaurate.

[0087] In the present invention, examples of the solubilizer include polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, macrogol, and castor oil fatty acid ethyl ester. Examples of stabilizers include, but are not limited to, dextran 40, methylcellulose, gelatin, sodium sulfite, and sodium metasulfate.

[0088] In the present invention, the isotonic agent may include, but is not limited to, D-mannitol and sorbitol.

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

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

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

[0092] In the present invention, the antioxidants include, but are not limited to, 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 gallate, propyl gallate, or chelating agents such as ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, and sodium metaphosphate.

[0093] In the present invention, the cell therapy agent may be administered in doses of approximately 1,000 to 10,000 cells / dose, 1,000 to 100,000 cells / dose, 1,000 to 1,000,000 cells / dose, 1,000 to 10,000,000 cells / dose, 1,000 to 100,000,000 cells / dose, 1,000 to 1,000,000,000 cells / dose, or 1,000 to 10,000,000,000 cells / dose, based on an adult patient weighing 70 kg, either once or several times per day at regular time intervals.

[0094] The injectable product according to the present invention can be manufactured in the form of a filled injection, taking the amount commonly known in the industry depending on the patient's constitution and type of defect. [Effects of the Invention]

[0095] When stem cells are treated with the composition according to the present invention, the immunomodulatory activity of the stem cells is enhanced, and their regenerative activity is also increased, making it usable as a cell therapy agent for the treatment of various diseases. [Brief explanation of the drawing]

[0096] [Figure 1] In Experiment Example 1, the graph shows the results of qRT-PCR analysis of changes in IL-10 mRNA expression levels in stem cell-derived RNA compared to the control group, when the stem cell culture medium was treated with agmatine at a concentration of 200 μM (A200), when it was treated with punicalagin at a concentration of 5 nM (P5), or when it was treated with agmatine at a concentration of 400 μM and punicalagin at a concentration of 10 nM (A400+P10).

[0097] [Figure 2] In Experiment Example 1, the graph shows the results of qRT-PCR analysis of changes in IL-1RA mRNA expression levels in stem cell-derived RNA compared to the control group, when the stem cell culture medium was treated with agmatine at a concentration of 200 μM (A200), when it was treated with punicalagin at a concentration of 5 nM (P5), or when it was treated with agmatine at a concentration of 400 μM and punicalagin at a concentration of 10 nM (A400+P10).

[0098] [Figure 3]In Experiment Example 1, the graph shows the results of qRT-PCR analysis of changes in Galectin-1 mRNA expression levels in stem cell-derived RNA compared to the control group, when the stem cell culture medium was treated with agmatine at a concentration of 200 μM (A200), when it was treated with punicalagin at a concentration of 5 nM (P5), or when it was treated with agmatine at a concentration of 400 μM and punicalagin at a concentration of 10 nM (A400+P10).

[0099] [Figure 4] In Experiment Example 1, the graph shows the results of qRT-PCR analysis of changes in Galectin-3 mRNA expression levels in stem cell-derived RNA compared to the control group, when the stem cell culture medium was treated with agmatine at a concentration of 200 μM (A200), when it was treated with punicalagin at a concentration of 10 nM (P10), or when it was treated with both agmatine at a concentration of 400 μM and punicalagin at a concentration of 10 nM (A400+P10).

[0100] [Figure 5] In Experimental Example 1, the graph shows the results of qRT-PCR analysis of changes in TSG-6 mRNA expression levels in stem cell-derived RNA compared to the control group, when the stem cell culture medium was treated with agmatine at a concentration of 200 μM (A200), when it was treated with punicalagin at a concentration of 10 nM (P10), or when it was treated with both agmatine at a concentration of 400 μM and punicalagin at a concentration of 10 nM (A400+P10).

[0101] [Figure 6]In Experiment Example 1, the graph shows the results of qRT-PCR analysis of changes in VEGF-A mRNA expression levels in stem cell-derived RNA compared to the control group, when the stem cell culture medium was treated with agmatine at a concentration of 200 μM (A200), when it was treated with punicalagin at a concentration of 5 nM (P5), or when it was treated with agmatine at a concentration of 400 μM and punicalagin at a concentration of 10 nM (A400+P10).

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

[0103] [Figure 8] In Experimental Example 2, the graph shows the results of ELISA analysis of changes in FGF-2 cytokine expression levels in stem cell culture medium when the culture medium was treated with agmatine to a concentration of 0-400 μM (A0-400) and punicalagin to a concentration of 0-10 nM (P0-10) compared to the control group. However, in Figure 8, P0A0 represents the result of treatment with the solvent DMSO. [Modes for carrying out the invention]

[0104] Best mode for carrying out the invention One embodiment of the present invention relates to a composition for enhancing the immunomodulatory or regenerative activity of stem cells, comprising agmatine or a pharmaceutically acceptable salt thereof; punicalagin, its stereoisomer or a pharmaceutically acceptable salt thereof; or a mixture thereof.

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

[0106] The aforementioned stem cells may be mesenchymal stem cells derived from the umbilical cord, umbilical cord blood, Wharton's jelly, bone marrow, fat, muscle, nerve, skin, amniotic membrane, placenta, amniotic fluid, tonsils, dental pulp, periodontal ligament, or dental sac.

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

[0108] The aforementioned punicalagin, its stereoisomers, or pharmaceutically acceptable salts may be included at concentrations 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), or the mRNA encoding them, or induce a decrease in the expression level of IL-6 (Interleukin 6) or the mRNA encoding it.

[0110] Modes for carrying out the invention The present invention will be described in more detail below through the examples. These examples are merely for the purpose of illustrating the present invention in more detail, and it will be obvious to those with ordinary skill in the art that the scope of the present invention is not limited by these examples, as is the essence of the invention.

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[0112] Examples

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[0114] [Preparation Example 1] Culture of Mesenchymal Stem Cells

[0115] Umbilical cords were obtained from healthy fetuses of healthy mothers. The umbilical cords were washed twice with DPBS, then once with 83% ethanol, and then twice again with PBS. After removing all blood vessels from the umbilical cord tissue, the umbilical cord tissue was cut into 1-2 mm fragments. Then, Liberase (Roche) was added to the fragmented umbilical cord tissue samples at 37°C for 90 minutes according to the manufacturer's protocol to prepare tissue lysates. The samples were then treated with MEM alpha (Gibco) nutrient medium containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin (P / S) (Hyclone, Cytiva) antibiotics to inactivate Liberase. The tissue lysates were filtered through a 100 μm cell strainer (pluriSelect), and the filtrate was centrifuged at 1,000 g for 5 minutes. Next, the supernatant was removed after the cell pellet was taken out, and the nutrient medium was added to the cell pellet and resuspended by pipetting. The viability of the cells in the cell suspension was measured using Trypan blue solution. The cells were inoculated into a culture vessel, the nutrient medium was added, and the cells were cultured at 5% CO2 and 37°C. Thereafter, the culture medium was changed every 3 days, and primary culture was carried out for 10 days. After 10 days, the morphology of the cultured cells was observed using a light microscope, and the morphology of mesenchymal stem cells was observed. Flow cytometry was performed using the Stemflow Human MSC Analysis Kit (BD Bioscience) according to the manufacturer's protocol, and it was confirmed that primary culture of umbilical cord-derived mesenchymal stem cells was complete. After washing the confirmed cells with DPBS, TrypLE Express (no phenol red) (Gibco) solution was added, and the cells were reacted in an incubator at 37°C for 5 minutes before being removed from the culture vessel. The TrypLE solution was neutralized by adding the aforementioned nutrient medium to the sample, and then the sample was centrifuged at 300g for 5 minutes. Subsequently, the supernatant was removed, the cell pellet was decontaminated, 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 trypan blue solution.The cells were inoculated into a new culture vessel, nutrient medium was added, and then cultured at 5% CO2 and 37°C, with subculturing performed at 3-day intervals.

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

[0118] The umbilical cord-derived mesenchymal stem cells prepared in Preparation Example 1 were cultured in a medium containing agmatine. The agmatine was prepared by adding a solution of agmatine sulfate (Sigma-Aldrich) dissolved in distilled water to the medium so that the final concentration of agmatine in the medium was 100, 200, or 400 μM. For primary culture aimed at promoting cell activity, 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) was used. For secondary culture aimed at obtaining cell culture medium, 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) was used. However, for the negative control group, the same amount of distilled water was added to the medium.

[0119] Specifically, 5 ml of primary culture medium to which agmatine has been added as described above is dispensed into a T25 flask, and the umbilical cord-derived mesenchymal stem cells prepared in Preparation Example 1 are subcultured to cell passage 5-6, then cultured at 2,500-3,500 cells / cm³. 2The culture medium was inoculated into the flask at the specified density and cultured for 2 days under conditions of 5% CO2 and 37°C. The culture medium was collected from each cultured stem cell into a 15 ml tube and centrifuged at 850 g for 10 minutes. The stem cells from which the culture medium had been collected were washed once with 3 mL of DPBS (Dulbecco's Phosphate Buffered Saline), and then 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. Subsequently, the culture medium was collected from each cultured stem cell into a 15 ml tube and centrifuged at 850 g for 10 minutes. The supernatant was obtained by removing the precipitated material from each centrifuged tube and stored at -80°C until analysis. Total RNA was extracted from each cell of the stem cells from which the culture medium had been collected using the GeneJET RNA Purification Kit (Thermo Scientific) according to the manufacturer's protocol and stored at -80°C until analysis.

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

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

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

[0125] The umbilical cord-derived mesenchymal stem cells prepared in Preparation Example 1 were cultured in a medium containing both agmatine and punicalagin. The experiment was carried out 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 and secondary culture media so that the final concentration of agmatine in each medium was 100, 200, or 400 μM, and the final concentration of punicalagin was 1, 5, or 10 nM.

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

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

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

[0130] Based on the measured RNA concentration values, 1 μg of each RNA sample was sampled. Then, cDNA for each sample was synthesized using the Maxime RT PreMix Kit (Intron) and Alpha Cycler 1 (PCR max) according to the manufacturer's protocol. Next, qRT-PCR was performed on the cDNA samples prepared 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 were purchased from Cosmo Genetech (KR), and 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 after each treatment were normalized by GAPDH expression levels, and the relative mRNA expression levels are shown graphically in Figures 1-6. The statistical significance levels for each sample group, compared to the control group (mean mRNA expression levels in the DW-treated and DMSO-treated groups), are shown in each figure, based on an unparied two-tailed t-test. (*p≦0.05;**p≦0.01;***p≦0.001)

[0131] [Table 1]

[0132] As shown in Figures 1-6, 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-inflammatory effects, increased significantly compared to a control group treated with the solvent alone. The expression level of VEGF-A mRNA, a representative factor related to regeneration, also increased significantly compared to the control group treated with the solvent alone. Furthermore, when mesenchymal stem cells were treated with both agmatine and punicalagin, the expression levels of the aforementioned factors increased even further.

[0133] This revealed that agmatine, punicalagin, or mixtures thereof according to the present invention have a priming effect that enhances the immunosuppressive and regenerative activity of stem cells.

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[0135] [Experimental Example 2] Evaluation of the effect of enhancing the function of immunomodulatory activity in stem cells

[0136] In Example 3, to confirm whether the anti-inflammatory activity-related functions of mesenchymal stem cells were enhanced by treatment with agmatine and / or punicalagin, 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. TMUsing a GO Microplate Spectrophotometer (Thermo Scientific), the changes in IL-6 and FGF-2 cytokine content in the cell culture media obtained in Examples 1, 2, and 3, according to the manufacturer's protocol, were analyzed, and the results are shown in Figures 7 and 8. Unparied two-tailed t-tests were used to determine the statistical significance levels for each sample group compared to the control group (mean value of IL-6 content in the DW-treated and DMSO-treated groups), and these levels are indicated in the figures. (*p≦0.05;**p≦0.01;***p≦0.001)

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

[0138] Furthermore, as shown in Figure 8, when mesenchymal stem cells were treated with agmatine and punicalagin according to the present invention, we were able to confirm that the expression level of FGF-2, a representative regeneration factor, increased significantly compared to the control group treated with the solvent alone.

[0139] This revealed that agmatine, punicalagin, or mixtures thereof according to the present invention have a priming effect that enhances the anti-inflammatory function of stem cells.

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[0141] Through the experiments described above, it was found that priming stem cells with agmatine, punicalagin, or a mixture thereof according to the present invention activates the immunomodulatory and regenerative activities of the stem cells themselves, thereby enhancing the efficacy of the stem cells as a cell therapy agent. Furthermore, it was found that the priming treatment increases the expression levels of immunosuppressive and anti-inflammatory factors and regenerative factors contained in the stem cell culture medium, allowing the stem cell culture medium, or the adjusted medium from which cells have been removed, or the secretome, to be used as a therapeutic agent for various diseases.

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[0143] The above description of the present invention is illustrative, and a person with ordinary skill in the art to which the present invention pertains will understand that it can be easily modified into other specific forms without altering the technical idea or essential features of the present invention. Therefore, the embodiments described above should be understood to be illustrative and not limiting in all respects. [Industrial applicability]

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

Claims

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

2. The composition for enhancing the immunomodulatory or regenerative activity of stem cells according to claim 1, wherein the stem cells are embryonic stem cells, adult stem cells, or induced pluripotent stem cells (IPS).

3. The stem cell immunomodulatory or regenerative activity enhancing composition according to claim 1, 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, tonsils, dental pulp, periodontal ligament, or dental sac.

4. The composition for enhancing the immunomodulatory or regenerative activity of stem cells according to claim 1, comprising agmatine or a pharmaceutically acceptable salt thereof at a concentration of 10 to 1000 μM.

5. The composition for enhancing the immunomodulatory or regenerative activity of stem cells according to claim 1, comprising punicalagin, its stereoisomer, or a pharmaceutically acceptable salt thereof at a concentration of 0.1 to 100 nM.

6. The composition contains stem cell IL-1RA (Interleukin 1 receptor antagonist), IL-10 (Interleukin 10), TGF-β (transforming growth factor beta), PDGF (platelet derived growth factor), VEGF-A (vascular endothermal growth factor A), FGF-2 (fibroblast growth factor 2), EGF (epidermal growth factor), and HGF (hepatocyte growth factor). The stem cell immunomodulatory or regenerative activity enhancing composition according to claim 1, which induces an increase in the expression level of one or more proteins selected from the group consisting of factors, or mRNA encoding them, or induces a decrease in the expression level of IL-6 (Interleukin 6) or mRNA encoding it.

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

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