Pretreatment composition for stem cells

Abstract

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

Description

Technical Field

[0001] This invention relates to a pretreatment composition capable of enhancing the immunomodulatory or regenerative activity of stem cells.

[0002] [Support the research and development project of this invention] [Project Number] RS-2024-00460565 [Responsible Authority] Ministry of Small and Medium Enterprises, South Korea [Research and Management Professional Organization] Korea Small and Medium Enterprise Technology and Information Promotion Agency [Research Project Title] Technology Incubation and Development Project (TIPS) [Research Topic Title] Development of a Dual-Target Therapy for Refractory Inflammatory Bowel Disease Utilizing Stem Cell Secretions [Supervisory Authority] JEONGJIN BIOSCIENCE Co., Ltd., South Korea [Research Period] July 1, 2024 ~ September 23, 2024 Background Technology

[0003] Stem cells are undifferentiated cells in the pre-differentiation stage that can be obtained from various tissues of embryos and adults. They possess characteristics such as self-renewal, differentiation, and immortality. Stem cells can be divided into embryonic stem cells and adult stem cells based on their tissue of origin. While using embryonic stem cells for therapeutic experiments presents ethical and tumorigenesis risks, adult stem cells have the advantage of being easily obtained from various tissues. Therefore, research aimed at applying them to the treatment of various diseases is actively underway.

[0004] Among stem cells, pluripotent mesenchymal stem cells (MSCs) are components of the stroma in all adult organs and tissues, often located in the perivascular region, and play a crucial role in tissue homeostasis, monitoring, repair, and remodeling. Typical immunophenotypes expressed by MSCs include the expression of surface markers CD44, CD73, CD90, and CD105, and the absence of CD34, CD45, CD14, and HLA-DR. MSCs possess the potential to differentiate into multiple lineages, including osteoblasts, adipocytes, and chondrocytes.

[0005] Mesenchymal stem cells are known to suppress inflammation, induce the generation of regulatory T cells (Tregs), or induce the death of immune cells involved in apoptosis. Therefore, research on developing various therapeutic agents using them is actively underway.

[0006] The characteristics of mesenchymal stem cells (MSCs) are influenced by in vivo and in vitro biological, biochemical, and biophysical factors through interactions between cells, the extracellular matrix (ECM), and soluble bioactive factors. MSCs interact with surrounding tissues and cells in three dimensions, promoting angiogenesis, producing anti-inflammatory molecules, inhibiting apoptosis (anti-apoptotic effects), and modulating the immune system by regulating the ECM.

[0007] Therefore, to enhance the therapeutic potential of mesenchymal stem cells (MSCs), recent research has been actively conducted on improving their lineage-specific differentiation and function by modulating biological, biochemical, and / or biophysical factors. One known approach is the priming of MSCs using proinflammatory mediators. Representative priming techniques include: (a) MSC priming using inflammatory cytokines or mediators; (b) MSC priming using a hypoxic environment; (c) MSC priming using pharmacological drugs and chemicals; (d) MSC priming using biological materials and other culture conditions; and (e) MSC priming using other molecules. Summary of the Invention

[0008] Technical issues One object of the present invention is to provide a pretreatment composition that can enhance the immunomodulatory or regenerative activity of stem cells.

[0009] Another object of the present invention is to provide stem cells or culture media derived from said stem cells that have been treated with the above composition to enhance their immunomodulatory or regenerative activity.

[0010] Another object of the present invention is to provide a variety of applications for stem cells or culture media derived therefrom that have been treated with the composition to enhance their immunomodulatory or regenerative activity.

[0011] However, the technical problems that this invention aims to solve are not limited to those mentioned above, and other technical problems not mentioned can be clearly understood by those skilled in the art through the following description.

[0012] Solution to the problem In the following description, various specific examples described herein will be illustrated with reference to the accompanying drawings. In this description, various specific details, such as specific forms, compositions, and processes, are described to provide a complete understanding of the invention. However, specific examples may be implemented without one or more of these specific details, or in combination with other known methods and forms. In other examples, known processes and manufacturing techniques are not described with specific details so as not to unnecessarily obscure the invention. Throughout this specification, references to “in one example” or “in a specific example” indicate that the specific feature, form, composition, or characteristic described in connection with that specific example is included in one or more examples of the invention. Therefore, the expressions “in one example” or “in a specific example” appearing throughout this specification do not necessarily refer to the same specific example of the invention. Furthermore, specific features, forms, compositions, or characteristics may be combined in any suitable manner in one or more examples.

[0013] Unless otherwise expressly defined in this invention, all scientific and technical terms used in this specification shall have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0014] According to one embodiment of the invention, a composition for enhancing the immunomodulatory or regenerative activity of stem cells is provided, comprising: agmatine or a pharmaceutically acceptable salt thereof; punicalagin, its stereoisomer or a pharmaceutically acceptable salt thereof; or a mixture thereof.

[0015] In this invention, "stem cell" refers to undifferentiated cells obtained from embryonic and adult tissues that are in the pre-differentiation stage and possess characteristics such as self-renewal, differentiation, and immortality. Based on their differentiation potential, stem cells can be classified into pluripotent, multipotent, and unipotent stem cells; adult stem cells are characterized by multipotency, while embryonic stem cells are characterized by pluripotency. In this invention, the stem cells used for functional enhancement are not limited; they can include embryonic stem cells, adult stem cells, or induced pluripotent stem cells (IPS).

[0016] In this invention, the adult stem cells may be mesenchymal stem cells, mesenchymal stromal cells derived from mammalian tissues including humans, mesenchymal stem cells derived from mammalian tissues including humans, multipotent stem cells, or amniotic epithelial cells, preferably mesenchymal stem cells, but not limited thereto.

[0017] In this 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, amnion, placenta, amniotic fluid, tonsils, dental pulp, periodontal ligament, and dental sac, but are not limited thereto.

[0018] In this invention, "agmatine" refers to a 1-(4-aminobutyl)guanidine compound represented by the following chemical formula 1, which is a naturally occurring compound derived from arginine. Its chemical formula is C5H 14 N4, with a molecular weight of 130.195 g / mol and CAS number 306-60-5, is known to play a regulatory role, particularly at multiple molecular targets. Its application in neurotransmitter systems, ion channels, nitric oxide (NO) synthesis, and polyamine metabolism pathways provides a foundation for further research into its potential applications.

[0019] [Chemical Formula 1]

[0020] In this invention, "punicalagin" is an ellagitannin belonging to the phenolic class of compounds, specifically a 2,3-(S)-hexahydroxybiphenyldicarboxyloyl-4,6-(S,S)-gallagyl-D-glucose compound represented by the following chemical formula 2. Punicalagin exists in plants such as pomegranate (Punica granatum) in α and β isomer forms. The chemical formula of punicalagin is C... 48 H 28 O 30 It has a molecular weight of 1084.71 g / mol and a CAS number of 65995-63-3.

[0021] [Chemical Formula 2]

[0022] In the compositions of the present invention, the concentration of guanidine or a pharmaceutically acceptable salt thereof may be from 10 μM to 1000 μM, 100 μM to 800 μM, 200 μM to 600 μM or 200 μM to 400 μM, but is not limited thereto.

[0023] In the compositions of the present invention, the concentration of punicin, its stereoisomers or pharmaceutically acceptable salts thereof may be 0.1 nM to 100 nM, 1 nM to 80 nM, 1 nM to 20 nM, 5 nM to 20 nM, 1 nM to 10 nM or 5 nM to 10 nM, but is not limited thereto.

[0024] In this invention, even when described as guanidine or punicalin, it includes a pharmaceutically acceptable salt of the compound. A pharmaceutically acceptable salt is one that is generally considered suitable for medical use by those skilled in the art (e.g., because such salt is harmless to the recipient) or that produces acceptable side effects in the respective treatment. Typically, a pharmaceutically acceptable salt is one permitted for use by regulatory agencies such as the U.S. Food and Drug Administration (FDA), the European Medicines Agency (EMA), or the Pharmaceuticals and Medical Devices Agency (PMDA) under the Japanese Ministry of Health, Labour and Welfare. However, this invention also includes, in principle, salts of compounds according to the invention that are not pharmaceutically acceptable themselves, for example, as intermediates in the preparation of compounds according to the invention or their physiologically active derivatives, or as intermediates in the preparation of pharmaceutically acceptable salts of compounds according to the invention or their physiologically active derivatives. The salts include water-insoluble salts, and particularly water-soluble salts.

[0025] In each case, those skilled in the art can readily determine whether a particular compound or its physiologically active derivative according to the present invention is capable of forming a salt, i.e., whether the compound or its physiologically active derivative according to the present invention has a charged group, such as an amino group, a carboxyl group, etc.

[0026] Exemplary salts of the compounds of the present invention are acid addition salts or salts formed with a base, particularly pharmaceutically acceptable inorganic and organic acid addition salts, and salts formed with pharmaceutically commonly used bases, which are water-insoluble or, in particular, water-soluble acid addition salts. Salts formed with a base may also be suitable according to the substituents of the compounds of the present invention. Acid addition salts can be formed, for example, by mixing a solution of the compounds of the present invention with a pharmaceutically acceptable acid solution (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 comprise: 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 salts, quaternary ammonium salts, and amine cations formed using halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyl sulfonates, and aryl sulfonates, etc., to counter anions). Examples of pharmaceutically acceptable salts include: acetates, adipates, alginates, arginine salts, ascorbic acid salts, aspartate salts, benzenesulfonates, benzoates, bicarbonates, bisulfates, tartrates, borates, bromides, butyrates, calcium EDTA, camphorates, camphor sulfonates, camphorane sulfonates, carbonates, chlorides, citrates, digluconates, dihydrochlorides, dodecyl sulfates, EDTA, ethanesulfonates, ethanesulfonates, formates, fumarates, galactobionates, galacturonic acid, gluconates, glutamates, glycerophosphates, hemisulfates, heptarates, hexanoates, hexylresorcinol salts, hydrobromide salts, hydrochloride salts, and hydroiodic acid. Salts, 2-hydroxyethanesulfonate, hydroxynaphthyl carboxylate, iodides, isobutyrate, hydroxyethyl sulfonate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, methanesulfonate (methanesulfonate), methyl sulfate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate / bisphosphate, phthalate, picrate, pentanoate, polygalacturonic acid ester, propionate, salicylate, stearate, sulfate, octanoate, succinate, tannate, tartrate, toluenesulfonate, undecanoate, valerate, etc., but not limited to these.

[0027] Even salts that are not pharmaceutically acceptable, such as those that can be obtained as processing products in the industrial-scale preparation of compounds according to the invention, are included in the invention and, if necessary, can be converted into pharmaceutically acceptable salts by methods known to those skilled in the art.

[0028] As an example, in this invention, the guanidine can be used in the sulfate form shown in Chemical Formula 3 below, but is not limited thereto.

[0029] [Chemical Formula 3]

[0030] On the other hand, in this invention, even when described as punicalin, it includes stereoisomers of the compound. Compounds according to the invention may have asymmetric carbon centers and therefore may exist as R or S isomers or racemic compounds; all such optical isomers and mixtures thereof are included within the scope of this invention.

[0031] Furthermore, the compounds and their salts of the present invention may contain varying amounts of solvent when separated, for example, in crystalline form. Therefore, solvates (especially hydrates) of the compounds of the present invention and solvates (especially hydrates) of the salts of the compounds of the present invention are both included within the scope of the present invention. More specifically, the present invention may include hydrates of compounds, salts, and / or physiologically active derivatives of the present invention that stoichiometrically contain one, two, or one / half a water molecule.

[0032] As an example, in this invention, the punicalin can be α-punicalin or β-punicalin as shown in Chemical Formula 4, but is not limited thereto.

[0033] [Chemical Formula 4]

[0034] In the chemical formula 4, When R1 is hydrogen and R2 is hydroxyl, it can be α-punicalin; When R1 is a hydroxyl group and R2 is hydrogen, it can be β-punicin.

[0035] As an example, in this invention, the punicalin can be used in a racemic form of a mixture of α-punicalin and β-punicalin, but is not limited thereto.

[0036] Unless otherwise specified in this specification, the term punicalin may refer to a racemic mixture of α-punicalin and β-punicalin.

[0037] When using the compositions of the present invention, immunosuppressive or anti-inflammatory activity can be enhanced in various functions of stem cells, particularly in their immunomodulatory activity. Specifically, this can refer to an increase in the expression levels of one or more proteins or their encoding mRNAs selected from the group consisting of interleukin-1 receptor antagonist (IL-1RA), interleukin-10 (IL-10), and transforming growth factor-β (TGF-β) when stem cells are treated with the compositions of the present invention.

[0038] When using the composition of the present invention, various functions of stem cells, particularly their regenerative activity, can be enhanced. Specifically, this can refer to an increase in the expression level of one or more proteins or mRNAs encoding factors related to stem cell regeneration, selected from the group consisting of vascular endothelial growth factor A (VEGF-A), fibroblast growth factor 2 (FGF-2), epidermal growth factor (EGF), transforming growth factor beta (TGF-β), platelet-derived growth factor (PDGF), and hepatocyte growth factor (HGF), when stem cells are treated with the composition of the present invention.

[0039] In this invention, changes in the expression level of mRNA can be measured using methods such as polymerase chain reaction (PCR), quantitative real-time polymerase chain reaction (qPCR), quantitative reverse transcription polymerase chain reaction (qRT-PCR), semi-quantitative reverse transcription polymerase chain reaction (semi-quantitative RT-PCR), digital PCR, Northern blot, messenger RNA sequencing (mRNA-SEQ), and microarrays, but are not limited to these methods. Any method known in the art for measuring gene expression levels can be used without limitation. Furthermore, in this invention, changes in the expression level of protein can be measured using methods such as antibody-based measurements, enzyme-linked immunosorbent assay (ELISA), Western blot or immunoblotting, flow cytometry, and mass spectrometry, but are not limited to these methods. Any method known in the art for measuring protein expression levels can be included without limitation.

[0040] According to another embodiment of the invention, a method for enhancing the immunomodulatory or regenerative activity of stem cells is provided, comprising the steps of treating the stem cells with guanidine or a pharmaceutically acceptable salt thereof; punicin, its stereoisomer or a pharmaceutically acceptable salt thereof; or a mixture thereof.

[0041] In this invention, the treatment can be carried out by adding guanidine or a pharmaceutically acceptable salt thereof, punicin, its stereoisomer or a pharmaceutically acceptable salt thereof, or a mixture thereof to the culture medium during stem cell culture.

[0042] In this invention, the treatment can be performed in vitro or ex vivo.

[0043] In this invention, the stem cell culture can be carried out in a culture medium containing components widely known in the art required for stem cell culture, or in a culture medium additionally containing components capable of promoting stem cell proliferation. Here, the culture medium can be any concept including natural culture media, synthetic culture media, or selective culture media.

[0044] In this invention, the culture medium may be a cell culture minimum medium (CCMM) containing carbon source, nitrogen source and trace element components, and may also contain antibiotics such as penicillin, streptomycin, and gentamicin. In this invention, the culture medium can be selected according to the cell type and culture conditions. For example, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, Ham F-10 Nutrient Mixture (F-10), Ham F-12 Nutrient Mixture (F-12), Minimal Essential Medium alpha (MEMalpha), Glasgow's Minimal Essential Medium (GMEM), Iscove's Modified Dulbecco's Medium (IMDM), Chemically Defined Medium / Serum-Free Chemically Defined Medium (CDM), etc., but not limited to these.

[0045] In this invention, the temperature and time required for culture, as well as other culture conditions, can follow conventional stem cell culture conditions.

[0046] As an example, the method of the present invention may include the step of culturing stem cells in a serum culture medium containing guanidine or a pharmaceutically acceptable salt thereof; punicein, its stereoisomer or a pharmaceutically acceptable salt thereof; or a mixture thereof. Here, the serum culture medium may be a minimum essential medium (MEM alpha) supplemented with 5% to 15% by volume fetal bovine serum (FBS) or human platelet lysate (hPL), or a chemically defined medium excluding serum / plasma and animal / human-derived extracts / lysates, but is not limited thereto. Furthermore, the culture conditions may be, but are not limited to, culturing stem cells at 35–40°C and 5% by volume CO2 for 1 to 6 days, or 1 to 4 days.

[0047] As an example, the method of the present invention may include the step of culturing stem cells in a serum-free medium containing guanidine or a pharmaceutically acceptable salt thereof; punicein, its stereoisomer or a pharmaceutically acceptable salt thereof; or a mixture thereof. Here, the serum-free medium may be DMEM medium. If necessary, L-glutamine may be additionally added to the serum-free medium, for example, at a concentration greater than 0 and less than or equal to 10 mM, 1 mM to 5 mM, or 1 mM to 3 mM, but not limited thereto. Furthermore, the culture conditions may be: culturing stem cells at a temperature of 35–40°C and 5% CO2 for 1 to 6 days, preferably 1 to 4 days, but not limited thereto.

[0048] As an example, the method of the present invention may include: a step of culturing stem cells in a serum medium containing guanidine or a pharmaceutically acceptable salt thereof; punicalin, its stereoisomer or a pharmaceutically acceptable salt thereof; or a mixture thereof; and a step of culturing stem cells in a serum-free medium containing guanidine or a pharmaceutically acceptable salt thereof; punicalin, its stereoisomer or a pharmaceutically acceptable salt thereof; or a mixture thereof. Here, the serum medium may be a minimum essential medium (MEM alpha) supplemented with 5% to 15% fetal bovine serum (FBS). Furthermore, the serum-free medium may be DMEM medium, and L-glutamine may be added as needed, for example, the concentration of L-glutamine added may be greater than 0 and less than or equal to 10 mM, 1 mM to 5 mM, or 1 mM to 3 mM, but is not limited thereto. Furthermore, the primary and secondary culture conditions can be, respectively, culturing stem cells for 1 to 6 days or 1 to 4 days at a temperature of 35 to 40°C and 5% CO2, but are not limited to these conditions.

[0049] According to another embodiment of the invention, there is a stem cell that is treated with the composition provided in the invention, thereby enhancing its immunomodulatory or regenerative activity.

[0050] In this invention, stem cells treated with the composition of this invention, compared with stem cells not treated with the composition, exhibit increased expression levels of one or more proteins or mRNAs encoding factors related to immunosuppressive capacity or anti-inflammatory effects, namely selected from the group consisting of interleukin-1 receptor antagonist (IL-1RA), interleukin-10 (IL-10), galectin-1, galectin-3, tumor necrosis factor (TNF)-α-Stimulated Gene 6 (TSG-6), and transforming growth factor beta (TGF-β), while the expression levels of inflammation-related factors, namely interleukin-6 (IL-6) or its encoding mRNA, are decreased, thereby exhibiting enhanced immunosuppressive capacity or anti-inflammatory activity.

[0051] Stem cells treated with the composition of the present invention, compared with stem cells not treated with the composition, exhibit increased expression levels of one or more proteins or mRNAs encoding regeneration-related factors selected from the group consisting of vascular endothelial growth factor A (VEGF-A), fibroblast growth factor 2 (FGF-2), epidermal growth factor (EGF), transforming growth factor beta (TGF-β), platelet-derived growth factor (PDGF), and hepatocyte growth factor (HGF), thereby exhibiting enhanced regenerative activity.

[0052] According to another embodiment of the present invention, it relates to a stem cell culture medium, or a concentrate, purified or fractionated product derived from the culture medium, which is treated with the composition provided by the present invention to enhance immunomodulatory or regenerative activity.

[0053] In this invention, the stem cell culture medium may include a culture medium obtained by culturing stem cells according to the method of this invention to enhance their immunomodulatory or regenerative activity.

[0054] In this invention, a conditioned medium obtained after removing cells from the culture medium of the said stem cells can be provided.

[0055] In this invention, the "conditioned medium" refers to the culture medium obtained by removing cells after culturing stem cells in a culture medium. It refers to a culture medium whose function changes by containing effective components produced by the cells due to stem cell culture or factors that help maintain and survive the cells.

[0056] In this invention, the conditional culture medium may be: a culture medium obtained by culturing stem cells according to the method of this invention to enhance their immunomodulatory or regenerative activity, and the supernatant recovered after centrifugation at 500xg to 1500xg.

[0057] Furthermore, in this invention, the conditional culture medium can be obtained by filtering the culture medium obtained as described above, or the supernatant recovered after centrifugation as described above, using a 0.1 μM to 0.3 μM filter (preferably a 0.2 μM or 0.22 μM filter).

[0058] Furthermore, in this invention, a secretome derived from the aforementioned stem cell culture medium can be provided.

[0059] In this invention, the term "secretome" refers to a composition containing one or more substances secreted from cells, also known as a "secretome," "secretosome," or "cellular secretosome." The secretome may contain one or more cytokines, one or more exosomes, and / or one or more microvesicles. In this invention, the secretome may be purified or unpurified, and may additionally contain one or more non-cellularly secreted substances (e.g., culture medium, additives, nutrients, etc.) as needed.

[0060] In this invention, the secretome can be: a product obtained by filtering from the culture medium obtained after culturing stem cells according to the method of this invention to enhance their immunomodulatory or regenerative activity, containing molecules with a molecular weight cut-off value of 1 kDa to 10 kDa or higher.

[0061] In this invention, the secretome can be: after culturing stem cells to enhance their immunomodulatory or regenerative activity according to the method of this invention, the cells can be centrifuged at 500xg to 1500xg as needed to recover the supernatant, then filtered through a 0.1μM to 0.3μM filter (preferably a 0.2μM or 0.22μM filter), and then dialyzed using a tangential flow filtration (TFF) device to remove molecules with a molecular weight cutoff of 1kDa to 10kDa or less, thereby obtaining a product containing molecules of 1kDa to 10kDa or more.

[0062] In this invention, the dialysis filtration can be performed using a peristaltic tubing pump or a diaphragm pump, by displacing and diluting the supernatant with water for injection, and by concentrating it at 0~25°C.

[0063] In this invention, if necessary, a step of freeze-drying the concentrate obtained as described above for 6 to 10 hours may also be included. In this invention, the freeze-drying process can convert the secretory group or its concentrate into a powder form.

[0064] In the stem cell culture medium, cell-free conditioned medium, or secretion group as a fractionation product provided by the present invention, compared with the stem cell culture medium, cell-free conditioned medium, or secretion group as a fractionation product untreated by the composition of the present invention, the levels of one or more proteins or their encoding mRNAs selected from the group consisting of interleukin-1 receptor antagonist (IL-1RA), interleukin-10 (IL-10), transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), vascular endothelial growth factor A (VEGF-A), fibroblast growth factor 2 (FGF-2), epidermal growth factor (EGF), and hepatocyte growth factor (HGF) are increased, while the levels of interleukin-6 (IL-6), an inflammatory factor, are also increased. The presence levels of IL-6 or its encoded mRNA are reduced.

[0065] According to another embodiment of the present invention, there is a cell therapy agent comprising stem cells treated with the composition provided by the present invention, thereby enhancing their immunomodulatory or regenerative activity.

[0066] In this invention, the "cell therapy agent" refers to a pharmaceutical product used for treatment, diagnosis, and prevention by a series of actions, such as proliferating and screening living autologous, allogenic, or xenogenic cells in vitro, or altering the biological characteristics of cells by other methods, in order to restore the function of cells and tissues.

[0067] In this invention, the cell therapy agent may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be, for example, physiological saline, sterile water, Ringer's solution, buffered physiological saline, glucose solution, maltodextrin solution, glycerol, ethanol, human serum albumin (HSA), and mixtures of one or more of these components. Other conventional additives such as antioxidants, buffer solutions, and antibacterial agents may also be added as needed.

[0068] In this invention, the cell therapy agent may, depending on its dosage form, 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. Examples of the suspending agents may include, but are not limited to, methylcellulose, polysorbate 80, hydroxyethylcellulose, gum arabic, tragacanth gum, sodium carboxymethylcellulose, polyoxyethylene sorbitan monolaurate, etc.

[0069] In this invention, examples of the co-solvent may include: polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, polyethylene glycol, castor oil fatty acid ethyl ester, etc. Stabilizers may include, but are not limited to, dextran 40, methylcellulose, gelatin, sodium sulfite, sodium metabisulfite, etc.

[0070] In this invention, the isotonic agent may include, for example, D-mannitol, sorbitol, etc., but is not limited thereto.

[0071] In this invention, the preservatives may include, for example, methylparaben, ethylparaben, sorbic acid, phenol, cresol, chlorocresol, etc., but are not limited thereto.

[0072] In this invention, the anti-adsorption agent may include, for example, human serum albumin, lecithin, dextran, ethylene oxide-propylene oxide copolymer, hydroxypropyl cellulose, methylcellulose, polyoxyethylene hydrogenated castor oil, polyethylene glycol, etc., but is not limited thereto.

[0073] In this invention, the sulfur-containing reducing agent may include, but is not limited to, compounds with thiol groups such as N-acetylcysteine, N-acetylhomocysteine, lipoic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, glutathione, and thioalkyl acids having 1 to 7 carbon atoms.

[0074] In this invention, the antioxidants may include, but are not limited to, isoascorbic acid, butylated hydroxytoluene, butylated hydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbate palmitate, L-ascorbate stearate, sodium bisulfite, sodium sulfite, tripentyl gallate, propyl gallate or sodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, sodium metaphosphate, and other chelating agents.

[0075] In this invention, the cell therapy agent, when used as a baseline for an adult patient weighing 70 kg, can be administered, for example, at doses of approximately 1,000-10,000 cells / dose, 1,000-1,000,000 cells / dose, 1,000-1,000,000 cells / dose, 1,000-1,000,000,000 cells / dose, 1,000-1,000,000,000 cells / dose, or 1,000-1,000,000,000,000 cells / dose, once or several times daily at fixed time intervals, or administered multiple times at prescribed time intervals.

[0076] The injectable product according to the present invention can be manufactured in a pre-filled injectable form using dosages conventionally known in the art, depending on the patient's physical condition and type of defect.

[0077] The effects of the invention When stem cells are treated with the composition of the present invention, their immunomodulatory activity is enhanced and their regenerative activity is improved, thus enabling them to be used as cell therapy agents for treating a variety of diseases. Attached Figure Description

[0078] Figure 1 The results of qRT-PCR analysis of IL-10 mRNA expression levels in stem cell-derived RNA in Experiment 1 are presented in graphical form, compared with the control group, when the stem cell culture medium was treated to a concentration of guanidine at 200 μM (A200), a concentration of punicaloside at 5 nM (P5), or a concentration of guanidine at 400 μM and a concentration of punicaloside at 10 nM (A400+P10).

[0079] Figure 2 The results of qRT-PCR analysis of IL-1RA mRNA expression levels in stem cell-derived RNA in Experiment 1 are presented in graphical form, compared with the control group, when the stem cell culture medium was treated to a concentration of guanidine at 200 μM (A200), a concentration of punicaloside at 5 nM (P5), or a concentration of guanidine at 400 μM and a concentration of punicaloside at 10 nM (A400+P10).

[0080] Figure 3 The results of qRT-PCR analysis of Galectin-1 mRNA expression levels in stem cell-derived RNA in Experiment 1 are presented in graphical form, compared with the control group, when the stem cell culture medium was treated to a concentration of 200 µM guanidine (A200), a concentration of 5 nM punicaloside (P5), or a combination of 400 µM guanidine and 10 nM punicaloside (A400+P10).

[0081] Figure 4 The results of qRT-PCR analysis of Galectin-3 mRNA expression levels in stem cell-derived RNA in Experiment 1 are presented in graphical form, compared with the control group, when the stem cell culture medium was treated to a concentration of guanidine at 200 µM (A200), a concentration of punicaloside at 10 nM (P10), or a combined treatment of guanidine at 400 µM and punicaloside at 10 nM (A400+P10).

[0082] Figure 5 The results of qRT-PCR analysis of TSG-6 mRNA expression levels in stem cell-derived RNA in Experiment 1 are presented in graphical form, compared with the control group, when the stem cell culture medium was treated to a concentration of guanidine at 200 µM (A200), a concentration of punicaloside at 10 nM (P10), or a combined treatment of guanidine at 400 µM and punicaloside at 10 nM (A400+P10).

[0083] Figure 6 The results of qRT-PCR analysis of VEGF-A mRNA expression levels in stem cell-derived RNA in Experiment 1 are presented in graphical form, compared with the control group, when the stem cell culture medium was treated to a concentration of 200 µM guanidine (A200), a concentration of 5 nM punicaloside (P5), or a combination of 400 µM guanidine and 10 nM punicaloside (A400+P10).

[0084] Figure 7 The results of Experiment 2, compared to the control group, show the effects of combined treatment in stem cell culture medium at concentrations of guanidine 0–400 µM (A0–400) and punicalin 0–10 nM (P0–10). It should be noted that… Figure 7 P0A0 in the figure represents the results of ELISA analysis of changes in the expression level of IL-6 cytokine in stem cell culture medium when treated with the solvent DMSO.

[0085] Figure 8 The results of ELISA analysis of FGF-2 cytokine expression levels in stem cell culture medium were presented in graphical form in Example 2, compared with the control group, when the cells were treated with a combination of guanidine at a concentration of 0–400 µM (A0–400) and punicein at a concentration of 0–10 nM (P0–10). It should be noted that… Figure 8 P0A0 in the figure represents the result of treating the solvent DMSO. Detailed Implementation

[0086] The best way to implement the invention According to one embodiment of the present invention, a composition for enhancing the immunomodulatory or regenerative activity of stem cells is provided, comprising: agmatine or a pharmaceutically acceptable salt thereof; punicalagin, its stereoisomer or a pharmaceutically acceptable salt thereof; or a mixture thereof.

[0087] The stem cells may be embryonic stem cells, adult stem cells, or induced pluripotent stem cells (IPS).

[0088] The stem cells may be mesenchymal stem cells derived from umbilical cord, umbilical cord blood, Wharton's jelly, bone marrow, fat, muscle, nerve, skin, amnion, placenta, amniotic fluid, tonsils, dental pulp, periodontal ligament, or dental sac.

[0089] The concentration of the guanidine or its pharmaceutically acceptable salt may be from 10 μM to 1000 μM.

[0090] The concentration of the stereoisomer or its pharmaceutically acceptable salt of the punicalin can be from 0.1 nM to 100 nM.

[0091] The composition can induce an increase in the expression level of one or more proteins or their encoding mRNA selected from the group consisting of stem cell-derived interleukin-1 receptor antagonist (IL-1RA), interleukin-10 (IL-10), transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), vascular endothelial growth factor A (VEGF-A), fibroblast growth factor 2 (FGF-2), epidermal growth factor (EGF), and hepatocyte growth factor (HGF), or induce a decrease in the expression level of interleukin-6 (IL-6) or its encoding mRNA.

[0092] Methods for implementing the invention The present invention will now be described in more detail through embodiments. These embodiments are only used to illustrate the invention more specifically, and it will be apparent to those skilled in the art that the scope of the invention is not limited to these embodiments, based on the spirit of the invention.

[0093] Example Preparation Example 1: Culturing of Mesenchymal Stem Cells Umbilical cords were obtained from healthy fetuses of healthy pregnant women. The cords were washed twice with DPBS, once with 83% ethanol, and then twice with PBS. After removing all blood vessels from the cord tissue, it was cut into 1-2 mm pieces. Liberase (Roche) was then added to the chopped cord tissue samples according to the manufacturer's operating procedures, and the mixture was reacted at 37°C for 90 minutes to prepare a tissue digest. 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 digest was filtered through a 100 μm cell strainer (pluriSelect), and the filtrate was centrifuged at 1000 g for 5 minutes. The supernatant, except for the cell pellet, was discarded, and the nutrient medium was added to the cell pellet, resuspending it by pipetting. Cell viability in the cell suspension was measured using trypan blue solution. The cells were seeded into culture vessels and cultured at 37°C with the nutrient medium added, then cultured for 10 days with 5% CO2. The medium was changed every 3 days. After 10 days, the morphology of the cultured cells was observed under an optical microscope, revealing the morphology of mesenchymal stem cells. Flow cytometry analysis was then performed using the Stemflow Human MSC Analysis Kit (BD Bioscience) according to the manufacturer's instructions, confirming the completion of the primary culture of umbilical cord-derived mesenchymal stem cells. After washing the confirmed cells with DPBS, TrypLE Express (no phenol red; Gibco) solution was added and the mixture was incubated at 37°C for 5 minutes to detach the cells from the culture vessel. The sample was then centrifuged at 300g for 5 minutes after adding the nutrient medium to neutralize the TrypLE solution. Subsequently, the supernatant, excluding the cell pellet, was discarded, and nutrient medium was added to the cell pellet, resuspending it by pipetting. Cell viability in the cell suspension was measured using trypan blue solution. The cells were seeded into new culture containers, nutrient medium was added, and the cells were cultured at 37°C with 5% CO2, and passaged every 3 days.

[0094] Example 1: Pretreatment of Mesenchymal Stem Cells with Agmatine The umbilical cord-derived mesenchymal stem cells prepared in Preparation Example 1 were cultured in a medium containing guanidine. The guanidine was added to the medium by dissolving guanidine sulfate (Sigma-Aldrich) in distilled water, so that the final concentration of guanidine in the medium reached 100, 200, or 400 μM. The primary culture medium used to promote cell viability 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 secondary culture medium used to obtain cell culture medium was DMEM 1X (low-glucose, no phenol red, Gibco) or MEM alpha (Gibco) medium supplemented with 2 mM L-glutamine (Gibco brand) and 1% (v / v) penicillin / streptomycin (P / S) (Hyclone; Cytiva) or 0.5% (v / v) gentamicin (Gibco). As a negative control, an equal volume of distilled water was added to the medium.

[0095] Specifically, 5 ml of the primary culture medium containing guanidine added as described above was dispensed into a T25 culture flask, and the umbilical cord-derived mesenchymal stem cells prepared in Preparation Example 1 were passaged to the 5th to 6th generation (Cell passage 5-6), and cultured at a density of 2500-3500 cells / cm². 2 The stem cells were seeded at a density of [insert density here] into the culture flasks and cultured for 2 days at 37°C with 5% CO2. The culture medium was collected from each cultured stem cell and centrifuged at 850g for 10 minutes in 15ml centrifuge tubes. After washing the collected stem cells once with 3mL of Durbecco's Phosphate Buffered Saline (DPBS), 5mL of secondary culture medium was added to the cells, and the cells were cultured for an additional 2 days at 37°C with 5% CO2. Subsequently, the culture medium from each cultured stem cell was collected from each cultured stem cell and centrifuged at 850g for 10 minutes. The supernatant (excluding precipitate) was obtained from each centrifuge tube and stored at -80°C until analysis. Total RNA was extracted from the collected stem cells using the GeneJET RNA Purification Kit (Thermo Scientific) following the manufacturer's instructions and stored at -80°C until analysis.

[0096] Example 2: Pretreatment of Mesenchymal Stem Cells with Punicalagin The umbilical cord-derived mesenchymal stem cells prepared in Preparation Example 1 were cultured in a medium containing punicalagin. Punicalagin was obtained by adding a solution of punicalagin (Sigma-Aldrich) dissolved in 0.25% (v / v) dimethyl sulfoxide (DMSO) to the medium, bringing the final concentration of punicalagin in the medium to 1 nM, 5 nM, or 10 nM. Experiments were conducted according to the same method as in Example 1, but a solution containing punicalagin was added to the primary and secondary culture media instead of guanidine. As a negative control, an equal volume of 0.25% (v / v) DMSO was added to the culture medium.

[0097] Example 3: Pretreatment of Mesenchymal Stem Cells with Agmatine and Punicalagin The umbilical cord-derived mesenchymal stem cells prepared in Preparation Example 1 were cultured in a medium containing both guanidine and punicaloside. The experiment was conducted according to the same method as in Example 1, but solutions of guanidine sulfate (Sigma-Aldrich) dissolved in distilled water and punicaloside (Sigma-Aldrich) dissolved in 0.25% (v / v) DMSO (dimethyl sulfoxide) were added to the primary and secondary culture media, respectively, to achieve a final concentration of guanidine at 100 μM, 200 μM, or 400 μM, and a final concentration of punicaloside at 1 nM, 5 nM, or 10 nM.

[0098] Experimental Example 1: Evaluation of the Immune Regulation Activity and the Function Enhancement Effect of the Regeneration Activity of Stem Cells To confirm whether treatment with guanidine and / or pungent glycoside in Examples 1, 2 and 3 enhanced the immunomodulatory and regenerative functions of mesenchymal stem cells, changes in the expression levels of factors related to immunosuppression, anti-inflammation and regeneration were measured from stem cell-derived RNA.

[0099] 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, Epoch Microplate Spectrophotometer; BioTek, and Take3 Micro-volume plate; BioTek) in accordance with the manufacturer's operating procedures.

[0100] Based on the measured RNA concentration, 1 μg of each RNA sample was taken and cDNA was synthesized using the Maxime RT PreMix Kit (Intron) and Alpha Cycler 1 (PCR max) according to the manufacturer's instructions. Subsequently, qRT-PCR analysis was performed on the prepared cDNA samples using 2X qPCRBIO SyGreen Blue Mix Lo-ROX (PCR Biosystems), LightCycler96 System (Roche), and QuantStudio6 Flex (Thermo Fisher Scientific) according to the manufacturer's instructions. Primers used were purchased from COSMOGENETECH (KR), and the primer information for each gene is shown in Table 1. Based on the qRT-PCR results of IL-10, IL-1RA, Galectin-1, Galectin-3, TSG-6, and VEGF-A mRNA genes in each treatment group, the expression levels were normalized using GAPDH levels, and the relative mRNA expression levels are presented graphically. Figures 1 to 6 In the figure, the statistical significance of each sample group relative to the control group (mean mRNA expression level in the DW treatment group and DMSO treatment group) was marked in each figure by unpaired two-tailed t-test (*p≤0.05; **p≤0.01; ***p≤0.001).

[0101] Table 1

[0102] like Figures 1 to 6 As shown, it can be confirmed that after treating mesenchymal stem cells with guanidine or pungent glycoside according to the present invention, compared with the control group treated with solvent only, the expression levels of immunosuppression and anti-inflammatory factors, namely IL-10, IL-1RA, Galectin-1, Galectin-3, and TSG-6 mRNA, were significantly increased, and the expression level of VEGF-A mRNA, a representative factor related to regeneration, was also significantly higher than that of the control group treated with solvent only. Furthermore, it can be confirmed that when mesenchymal stem cells are treated with a combination of guanidine and pungent glycoside, the expression levels of the above factors are further increased.

[0103] Therefore, it can be seen that the guanidine, punicin, or mixtures thereof according to the present invention have a pretreatment effect that enhances the immunosuppressive capacity or regenerative activity of stem cells.

[0104] Experimental Example 2: Evaluation of the Function Enhancement Effect of the Immune Regulation Activity of Stem Cells To confirm whether the treatment with guanidine and / or pungentin in Example 3 enhanced the function associated with the anti-inflammatory activity of mesenchymal stem cells, the supernatant obtained after secondary culture of stem cells was analyzed using the BD OptEIA™ Human Interleukin-6 ELISA Kit (Cat# 555220, BD Biosciences), R&D Human Basic Fibroblast Growth Factor (Cat# DY233, R&D), Epoch Microplate Spectrophotometer (BioTek), and Multiskan. TM Thermo Fisher Scientific used a GO microplate spectrophotometer (Thermo Fisher Scientific) to analyze the changes in IL-6 and FGF-2 cytokine levels in the cell culture media obtained in Examples 1, 2, and 3, following the manufacturer's operating procedures. The results are attached. Figure 7 and Figure 8 As shown in the figure. The statistical significance levels of each sample group relative to the control group (mean IL-6 content in the DW treatment group and DMSO treatment group) were marked in the figure by unpaired two-tailed t-test (*p≤0.05; **p≤0.01; ***p≤0.001).

[0105] like Figure 7 As shown, it can be confirmed that after treating mesenchymal stem cells with the combined use of guanidine and punicin according to the present invention, the expression level of the representative inflammatory cytokine IL-6 was significantly reduced compared with the control group.

[0106] In addition, such as Figure 8 As shown, after treating mesenchymal stem cells with guanidine and punicin according to the present invention, the expression level of the representative regeneration factor FGF-2 was significantly increased compared with the control group treated with solvent only.

[0107] Therefore, it can be seen that the guanidine, punicin, or mixtures thereof according to the present invention have a pretreatment effect that enhances the anti-inflammatory function of stem cells.

[0108] The above experiments demonstrate that pretreatment of stem cells with guanidine, pungent glycoside, or mixtures thereof according to the present invention activates the stem cells' immunomodulatory and regenerative activities, thereby enhancing their efficacy as a cell therapy agent. Furthermore, the pretreatment increases the expression levels of immunosuppressive and anti-inflammatory factors, as well as regenerative factors, in the stem cell culture medium. Therefore, the stem cell culture medium, or the conditioned medium and secretory medium from which cells are removed, can be used to treat various diseases.

[0109] The above description of the present invention is for illustrative purposes only. Those skilled in the art will understand that the present invention can be easily modified in other specific forms without altering its technical concept or essential features. Therefore, the above embodiments should be understood as exemplary in all respects and not as limiting in any way.

[0110] Industrial applicability This invention relates to a pretreatment composition capable of enhancing the immunomodulatory or regenerative activity of stem cells.

Claims

1. A composition for enhancing the immunomodulatory or regenerative activity of stem cells, characterized in that, Include: Guanidine or a pharmaceutically acceptable salt thereof; Punicin, its stereoisomers or pharmaceutically acceptable salts thereof; or A mixture of them.

2. The composition for enhancing the immunomodulatory or regenerative activity of stem cells according to claim 1, characterized in that, The stem cells mentioned are embryonic stem cells, adult stem cells, or induced pluripotent stem cells.

3. The composition for enhancing the immunomodulatory or regenerative activity of stem cells according to claim 1, characterized in that, The stem cells are mesenchymal stem cells derived from umbilical cord, umbilical cord blood, Wharton jelly, bone marrow, fat, muscle, nerve, skin, amnion, 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, characterized in that, The concentration of the guanidine or its pharmaceutically acceptable salt is from 10 μM to 1000 μM.

5. The composition for enhancing the immunomodulatory or regenerative activity of stem cells according to claim 1, characterized in that, The concentration of the punicalin, its stereoisomer, or a pharmaceutically acceptable salt thereof is from 0.1 nM to 100 nM.

6. The composition for enhancing the immunomodulatory or regenerative activity of stem cells according to claim 1, characterized in that, The composition induces an increase in the expression level of one or more proteins or their encoding mRNA selected from the group consisting of stem cell interleukin-1 receptor antagonists, interleukin-10, transforming growth factor-β, platelet-derived growth factor, vascular endothelial growth factor A, fibroblast growth factor 2, epidermal growth factor, and hepatocyte growth factor, or induces a decrease in the expression level of interleukin-6 or its encoding mRNA.

7. A method for enhancing the immunomodulatory or regenerative activity of stem cells, characterized in that, include: Stem cells were treated with guanidine or a pharmaceutically acceptable salt thereof; Processing steps of punicalin, its stereoisomers or pharmaceutically acceptable salts thereof; or mixtures thereof.

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