Compositions for promoting production of stem cell-derived exosomes and enhancing stemness of stem cells

Abstract

To provide compositions for promoting the production of exosomes derived from stem cells and enhancing stemness of stem cells so as to more efficiently produce high-quality stem cells and stem cell-derived exosomes.SOLUTION: Provided herein is a composition comprising interferon-γ for promoting the production of exosomes derived from a stem cell, where the stem cell is a mesenchymal stem cell derived from an induced pluripotent stem cell (iPSC).SELECTED DRAWING: Figure 47

Description

[Technical field]

[0001] The present invention relates to a composition for promoting the production of exosomes derived from stem cells and enhancing stem cell potential. [Background technology]

[0002] Extracellular vesicles (EXTRACs) are vesicles composed of spherical lipid bilayers with a size of 30 to 1000 nm, including microvesicles and exosomes.

[0003] The lipid bilayer of exosomes has a phospholipid bilayer structure like that of the source cell (donor cell), and is a component of substances secreted by cells outside the cell, and is known to play functional roles such as cell-cell communication and cellular immune regulation.

[0004] Exosomes contain cell-specific components that reflect the unique biological functions of the source cell (donor cell), including phospholipids, mRNA, miRNA, as well as various water-soluble proteins, exogenous proteins, and transmembrane protein components.

[0005] Such exosomes are excreted from all animal cells, including mast cells, lymphocytes, astrocytes, platelets, nerve cells, endothelial cells, and epithelial cells, and are found in various body fluids, including blood, urine, mucus, saliva, bile, ascites, and cerebrospinal fluid. Exosomes can pass through the blood-brain barrier (BBB) ​​and have high selective permeability to the extent that they can permeate the cell membranes of epidermal and endothelial cells, so they are also used in the development of DDS (drug delivery system), which is a nanocarrier for specific drugs.

[0006] Exosomes and microvesicles secreted from mesenchymal stem cells are known to be involved in cell-to-cell communication and to exert the therapeutic effects of stem cells in regenerative medicine.

[0007] Exosomes are known to have a trophic effect on the paracrine factors secreted by stem cells that do not survive for a long time and are transplanted into the body. Such secreted factors are small molecules such as growth factors, chemokines, and cytokines secreted by extracellular vesicles such as exosomes, and such exosomes are derived from stem cells. For this reason, exosomes are used to characterize stem cells and evaluate their therapeutic efficacy.

[0008] Recently, active research has been conducted into the therapeutic effects of various diseases using exosomes secreted by mesenchymal stem cells, rather than mesenchymal stem cells themselves, and it is anticipated in academia and industry that this could become a new alternative that can overcome the limitations of existing stem cell therapies.

[0009] To use such exosomes commercially, a large amount of high-quality exosomes is required, but currently, the amount of exosomes obtained from stem cells is extremely small, and there is insufficient development of substances that can increase the production of stem cell-derived exosomes.

[0010] On the other hand, it is known that mesenchymal stem cells lose their proliferation ability and differentiation ability into various lineages as the culture period is prolonged and they are subcultured several times. It is also known that the colony formation ability of late passage mesenchymal stem cells is significantly reduced compared to early passage mesenchymal stem cells. The aging of mesenchymal stem cells is not related to the age of the bone marrow donor, but is related to the weakened proliferation ability due to the long culture period, and is also thought to be related to the decrease in telomerase activity.

[0011] Thus, currently, the amount of mesenchymal stem cells obtained from humans is limited, and when inducing differentiation of the obtained cells into specific lineages, inconveniences such as a decrease in differentiation ability due to subculture are unavoidable. Such inconveniences are unrealistic for clinical applications that require precise induction of differentiation into target cells and the securing of a large amount of appropriate stem cells for this purpose. For this reason, there is an emerging need for a method that can maintain the proliferation rate over a long period of time and the differentiation ability into various lineages.

[0012] Therefore, a need has arisen for the development of new methods and materials that can promote the production of stem cell-derived exosomes and also maintain the proliferation rate and differentiation potential of stem cells for a long period of time. Summary of the Invention [Problem to be solved by the invention]

[0013] The present inventors have endeavored to develop a method and material that can promote the production of stem cell-derived exosomes as well as maintain the proliferation rate and differentiation ability of stem cells for a long period of time.As a result, the inventors have found that when stem cells are pretreated with one or more selected from the group consisting of Exendin-4, Phorbol 12-myristate 13-acetate (PMA), Interferon-γ, Tetrandrine, Hyaluronic acid, Substance P, Resveratrol, and Lanifibranor, the stemness and proliferation of stem cells are enhanced, and the number of stem cell-derived exosomes and the protein and RNA contents in the exosomes are significantly increased, thereby completing the present invention.

[0014] Therefore, an object of the present invention is to provide a composition for promoting the production of stem cell-derived exosomes, which contains exendin-4.

[0015] Another object of the present invention is to provide a composition for enhancing the stemness of stem cells, comprising Exendin-4.

[0016] It is still another object of the present invention to provide a stem cell therapeutic agent comprising the composition for promoting the production of stem cell-derived exosomes or enhancing the stem cell potential of stem cells.

[0017] Yet another object of the present invention is to provide stem cell-derived exosomes pretreated with Exendin-4.

[0018] Yet another object of the present invention is to provide a stem cell therapeutic agent comprising the stem cell-derived exosomes.

[0019] Yet another object of the present invention is to provide a method for producing stem cell-derived exosomes, which includes a pretreatment step of culturing stem cells in a cell culture medium containing Exendin-4.

[0020] Yet another object of the present invention is to provide a method for enhancing the stemness of stem cells, comprising a pretreatment step of culturing stem cells in a cell culture medium containing Exendin-4.

[0021] Yet another object of the present invention is to provide a use of a composition containing Exendin-4 for promoting the production of exosomes derived from stem cells.

[0022] It is still another object of the present invention to provide a use of a composition containing Exendin-4 for enhancing stemness of stem cells.

[0023] Yet another object of the present invention is to provide a composition for promoting the production of stem cell-derived exosomes, comprising phorbol 12-myristate 13-acetate (PMA).

[0024] It is still another object of the present invention to provide a composition for enhancing stemness of stem cells, comprising phorbol 12-myristate 13-acetate (PMA).

[0025] It is still another object of the present invention to provide a stem cell therapeutic agent comprising the composition for promoting the production of stem cell-derived exosomes or enhancing the stem cell potential of stem cells.

[0026] Yet another object of the present invention is to provide stem cell-derived exosomes pretreated with phorbol 12-myristate 13-acetate (PMA).

[0027] Yet another object of the present invention is to provide a stem cell therapeutic agent comprising the stem cell-derived exosomes.

[0028] Yet another object of the present invention is to provide a method for producing stem cell-derived exosomes, which comprises a pretreatment step of culturing stem cells in a cell culture medium containing phorbol 12-myristate 13-acetate (PMA).

[0029] Yet another object of the present invention is to provide a method for enhancing the stemness of stem cells, comprising a pretreatment step of culturing stem cells in a cell culture medium containing phorbol 12-myristate 13-acetate (PMA).

[0030] Yet another object of the present invention is to provide a use of a composition containing phorbol 12-myristate 13-acetate (PMA) for promoting the production of stem cell-derived exosomes.

[0031] Yet another object of the present invention is to provide a use of a composition containing phorbol 12-myristate 13-acetate (PMA) for enhancing stemness of stem cells.

[0032] Yet another object of the present invention is to provide a composition for promoting the production of stem cell-derived exosomes, comprising interferon-gamma.

[0033] It is still another object of the present invention to provide a composition for enhancing the stemness of stem cells, comprising interferon-gamma.

[0034] It is still another object of the present invention to provide a stem cell therapeutic agent comprising the composition for promoting the production of stem cell-derived exosomes or enhancing the stem cell potential of stem cells.

[0035] Yet another object of the present invention is to provide stem cell-derived exosomes pretreated with Interferon-γ.

[0036] Yet another object of the present invention is to provide a stem cell therapeutic agent comprising the stem cell-derived exosomes.

[0037] Yet another object of the present invention is to provide a method for producing stem cell-derived exosomes, which includes a pretreatment step of culturing stem cells in a cell culture medium containing interferon-gamma.

[0038] Yet another object of the present invention is to provide a method for enhancing the stemness of stem cells, comprising a pretreatment step of culturing stem cells in a cell culture medium containing interferon-gamma.

[0039] Yet another object of the present invention is to provide a use of a composition containing interferon-gamma for promoting the production of exosomes derived from stem cells.

[0040] Yet another object of the present invention is to provide a use of a composition containing interferon-gamma for enhancing stemness of stem cells.

[0041] Yet another object of the present invention is to provide a composition for promoting the production of stem cell-derived exosomes, comprising tetrandrine.

[0042] It is still another object of the present invention to provide a composition for enhancing stemness of stem cells, comprising tetrandrine.

[0043] It is still another object of the present invention to provide a stem cell therapeutic agent comprising the composition for promoting the production of stem cell-derived exosomes or enhancing the stem cell potential of stem cells.

[0044] Yet another object of the present invention is to provide stem cell-derived exosomes pretreated with tetrandrine.

[0045] Yet another object of the present invention is to provide a stem cell therapeutic agent comprising the stem cell-derived exosomes.

[0046] Yet another object of the present invention is to provide a method for producing stem cell-derived exosomes, which includes a pretreatment step of culturing stem cells in a cell culture medium containing tetrandrine.

[0047] Yet another object of the present invention is to provide a method for enhancing the stemness of stem cells, comprising a pretreatment step of culturing stem cells in a cell culture medium containing tetrandrine.

[0048] Yet another object of the present invention is to provide a use of a composition containing tetrandrine for promoting the production of stem cell-derived exosomes.

[0049] Yet another object of the present invention is to provide a use of a composition containing tetrandrine for enhancing stemness of stem cells.

[0050] Yet another object of the present invention is to provide a composition for promoting the production of stem cell-derived exosomes, which contains hyaluronic acid.

[0051] It is still another object of the present invention to provide a composition for enhancing stemness of stem cells, comprising hyaluronic acid.

[0052] It is still another object of the present invention to provide a stem cell therapeutic agent comprising the composition for promoting the production of stem cell-derived exosomes or enhancing the stem cell potential of stem cells.

[0053] Yet another object of the present invention is to provide stem cell-derived exosomes pretreated with hyaluronic acid.

[0054] Yet another object of the present invention is to provide a stem cell therapeutic agent comprising the stem cell-derived exosomes.

[0055] Yet another object of the present invention is to provide a method for producing stem cell-derived exosomes, comprising a pretreatment step of culturing stem cells in a cell culture medium containing hyaluronic acid.

[0056] Yet another object of the present invention is to provide a method for enhancing stemness of stem cells, comprising a pretreatment step of culturing stem cells in a cell culture medium containing hyaluronic acid.

[0057] Yet another object of the present invention is to provide a use of a composition containing hyaluronic acid for promoting the production of exosomes derived from stem cells.

[0058] Yet another object of the present invention is to provide a use of a composition containing hyaluronic acid for enhancing stemness of stem cells.

[0059] Yet another object of the present invention is to provide a composition for promoting the production of stem cell-derived exosomes, which contains Substance P.

[0060] It is still another object of the present invention to provide a composition for enhancing stemness of stem cells, comprising Substance P.

[0061] It is still another object of the present invention to provide a stem cell therapeutic agent comprising the composition for promoting the production of stem cell-derived exosomes or enhancing the stem cell potential of stem cells.

[0062] Yet another object of the present invention is to provide stem cell-derived exosomes pretreated with Substance P.

[0063] Yet another object of the present invention is to provide a stem cell therapeutic agent comprising the stem cell-derived exosomes.

[0064] Yet another object of the present invention is to provide a method for producing stem cell-derived exosomes, which includes a pretreatment step of culturing stem cells in a cell culture medium containing Substance P.

[0065] It is yet another object of the present invention to provide a method for enhancing stemness of stem cells, comprising a pretreatment step of culturing stem cells in a cell culture medium containing Substance P.

[0066] Yet another object of the present invention is to provide a use of a composition containing Substance P for promoting the production of stem cell-derived exosomes.

[0067] Yet another object of the present invention is to provide a use of a composition containing Substance P for enhancing stemness of stem cells.

[0068] Yet another object of the present invention is to provide a composition for promoting the production of stem cell-derived exosomes, comprising resveratrol.

[0069] It is still another object of the present invention to provide a composition for enhancing stemness of stem cells, comprising resveratrol.

[0070] It is still another object of the present invention to provide a stem cell therapeutic agent comprising the composition for promoting the production of stem cell-derived exosomes or enhancing the stem cell potential of stem cells.

[0071] Yet another object of the present invention is to provide stem cell-derived exosomes pretreated with resveratrol.

[0072] Yet another object of the present invention is to provide a stem cell therapeutic agent comprising the stem cell-derived exosomes.

[0073] Yet another object of the present invention is to provide a method for producing stem cell-derived exosomes, comprising a pretreatment step of culturing stem cells in a cell culture medium containing resveratrol.

[0074] Yet another object of the present invention is to provide a method for enhancing stemness of stem cells, comprising a pretreatment step of culturing stem cells in a cell culture medium containing resveratrol.

[0075] Yet another object of the present invention is to provide a use of a composition containing resveratrol for promoting the production of stem cell-derived exosomes.

[0076] Yet another object of the present invention is to provide a use of a composition containing resveratrol for enhancing stemness of stem cells.

[0077] Yet another object of the present invention is to provide a composition for promoting the production of stem cell-derived exosomes, comprising Lanifibranor.

[0078] Yet another object of the present invention is to provide a stem cell therapeutic agent for promoting the production of stem cell-derived exosomes, comprising lanifibranor.

[0079] It is yet another object of the present invention to provide a composition for enhancing stemness of stem cells, comprising lanifibranor.

[0080] It is yet another object of the present invention to provide a stem cell therapeutic agent comprising lanifibranor for enhancing stem cell potential.

[0081] Yet another object of the present invention is to provide stem cell-derived exosomes pretreated with lanifibranor.

[0082] Yet another object of the present invention is to provide a stem cell therapeutic agent comprising, as an active ingredient, exosomes derived from stem cells pretreated with lanifibranor.

[0083] Yet another object of the present invention is to provide a method for producing stem cell-derived exosomes, which includes a pretreatment step of culturing stem cells in a cell culture medium containing lanifibranor.

[0084] It is yet another object of the present invention to provide a method for enhancing stem cell potential, comprising a pretreatment step of culturing stem cells in a cell culture medium containing lanifibranor.

[0085] Yet another object of the present invention is to provide a use of a composition containing lanifibranor for promoting the production of exosomes derived from stem cells.

[0086] Yet another object of the present invention is to provide a use of a composition containing lanifibranor for enhancing stemness of stem cells. [Means for solving the problem]

[0087] The present inventors have endeavored to develop methods and materials that can promote the production of stem cell-derived exosomes as well as maintain the proliferation rate and differentiation ability of stem cells for a long period of time.As a result, it has been found that when stem cells are pretreated with one or more selected from the group consisting of Exendin-4, Phorbol 12-myristate 13-acetate (PMA), Interferon-γ, Tetrandrine, Hyaluronic acid, Substance P, Resveratrol, and Lanifibranor, the stemness and proliferation of stem cells are enhanced, and the number of stem cell-derived exosomes and the protein and RNA content in the exosomes are significantly increased.

[0088] The present invention relates to a composition for promoting the production of stem cell-derived exosomes or enhancing the stem cell potential of stem cells, the composition comprising one or more selected from the group consisting of Exendin-4, Phorbol 12-myristate 13-acetate (PMA), interferon-gamma (Interferon-γ), Tetrandrine, hyaluronic acid, Substance P, resveratrol, and Lanifibranor; a stem cell therapeutic agent comprising the composition; and a composition for enhancing the stem cell potential of stem cells, the composition comprising one or more selected from the group consisting of Exendin-4, Phorbol 12-myristate 13-acetate (PMA), interferon-gamma (Interferon-γ), Tetrandrine, hyaluronic acid, Substance P, resveratrol, and Lanifibranor. The present invention relates to stem cell-derived exosomes pretreated with one or more selected from the group consisting of phenylalanine, resveratrol, and lanifibranor, a stem cell therapeutic agent containing the stem cell-derived exosomes, a method for producing stem cell-derived exosomes, and a method for enhancing the stem cell potential of stem cells.

[0089] The present invention will now be described in more detail.

[0090] One aspect of the present invention relates to a composition for promoting the production of stem cell-derived exosomes, which comprises exendin-4.

[0091] The exendin-4 of the present invention is a peptide agonist of the glucagon-like peptide (GLP) receptor. Exendin-4 is known to promote insulin secretion and has been used clinically to treat type 2 diabetes and Parkinson's disease.

[0092] One aspect of the present invention relates to a composition for promoting the production of stem cell-derived exosomes, comprising phorbol 12-myristate 13-acetate (PMA).

[0093] The phorbol 12-myristate 13-acetate (PMA) of the present invention is also known as 12-O-tetradecanoylphorbol-13-acetate (TPA) and plays a role in activating protein kinase C (PKC), a signal transduction enzyme. PMA is also used as a tumor promoter, stimulating the division of B cells and other cells, and inducing activation in various cells to produce cytokines. For this reason, it has been used in research on inflammatory response inducers and the like.

[0094] One aspect of the present invention relates to a composition for promoting the production of stem cell-derived exosomes, comprising interferon-gamma.

[0095] Interferon-gamma of the present invention is a cytokine produced by activated T cells, NK cells, and CD4 and CD8 positive lymphocytes, and is involved in the activation, proliferation and differentiation of various cells including T lymphocytes as a mitogenic factor or growth factor, and in enhancing the expression of MHC on antigen-presenting cells, and plays an important role in immune and inflammatory responses. Interferon-gamma has also been used clinically for the treatment of infections and autoimmunity.

[0096] One aspect of the present invention relates to a composition for promoting the production of stem cell-derived exosomes, comprising tetrandrine.

[0097] Tetrandrine of the present invention is a calcium channel broker, which non-selectively inhibits calcium channel activity, induces G1 blockade of the G1 stage of cell cycle and cell death in various cell types, and has immunosuppressive, anti-inflammatory, anti-fibrotic, and anticoagulant effects, and can increase hepatic glycogen synthesis, increase glucose utilization, and lower plasma glucose concentration. Tetrandrine has also been used clinically as a combination treatment for diabetes, hypertension, arrhythmia, analgesic and antispasmodic, and treatment for liver sclerosis and rheumatoid arthritis.

[0098] One aspect of the present invention relates to a composition for promoting the production of stem cell-derived exosomes, which contains hyaluronic acid.

[0099] The hyaluronic acid of the present invention is a natural linear polysaccharide in which disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine are linked by β(1→4) interglycosidic linkage. Hyaluronic acid is known as one of the components constituting the dermis, and is known as an excellent biomaterial with excellent biocompatibility and biodegradability, and no immune rejection or toxicity in the body. In addition, hyaluronic acid is a natural glycosaminoglycan macromolecule with high viscosity and elasticity, and has various functions and physicochemical properties, so it has been used clinically for arthritis treatment, ophthalmic surgery, fillers, wound treatment, drug delivery vehicles, adhesion inhibitors, tissue engineering, and cosmetics.

[0100] One aspect of the present invention relates to a composition for promoting the production of stem cell-derived exosomes, which contains Substance P.

[0101] The substance P of the present invention is an undecapeptide of the tachykinin neuropeptide family, a neuropeptide that acts on the NK1 receptor and acts as a neurotransmitter and neuromodulator. In addition, substance P is known to be a strong vasodilator and promote cell growth, and is known to be effective in promoting the healing of difficult-to-treat ulcers, chronic urticaria, atopic dermatitis, chronic kidney disease, and in regulating inflammation and pain. Furthermore, substance P is useful for skin cell proliferation and regeneration, and is used as a raw material for cosmetics and has been used clinically for the treatment of hypertension, cirrhosis, and other diseases.

[0102] One aspect of the present invention relates to a composition for promoting the production of stem cell-derived exosomes, comprising resveratrol.

[0103] Resveratrol of the present invention is a polyphenolic substance that is a phytoalexin produced when plants are exposed to harmful environments such as mold and pests. It can activate Sirt1 (Sirtuin-1), a deacetylase, to promote mitochondrial function and promote the flow of sugar metabolism. Resveratrol also has anti-disease effects including anti-cancer, anti-virus, neuroprotection, anti-aging, anti-inflammatory, and life-prolonging effects. Resveratrol also exhibits antioxidant effects, and has the effect of protecting against reperfusion injury and suppressing arrhythmia by stimulating the production of nitric oxide (NO) and dilating blood vessels. As a result, resveratrol has been continuously studied as a therapeutic agent for cardiovascular and brain diseases, and has been used clinically in various fields such as anti-aging and anti-cancer related health functional foods and wrinkle-improving cosmetic raw materials.

[0104] One aspect of the present invention is a composition for promoting the production of stem cell-derived exosomes, comprising lanifibranor.

[0105] The lanifibranor of the present invention is a PPAR (peroxisome proliferator-activated receptors) agonist, a small molecule known to activate three PPAR isoforms, known as PPARα, PPARδ, and PPARγ, and induce anti-fibrotic, anti-inflammatory, and beneficial metabolic changes in the body. PPARs are ligand-activated transcription factors that belong to the nuclear hormone receptor family that regulate gene expression. PPARs play an essential role in regulating cell differentiation, development, and tumorigenesis. Lanifibranor is known to activate PPARs that regulate fibrosis and reduce abnormal growth of connective tissue. Lanifibranor has also been used clinically as a therapeutic agent for systemic sclerosis, idiopathic pulmonary fibrosis, and the like.

[0106] The composition according to the present invention may further contain a known exosome production promoter in addition to the above-mentioned components.

[0107] As used herein, "exosome" refers to a cell-derived endoplasmic reticulum that is present in the body fluids of most eukaryotic organisms. The diameter of an exosome is about 30 to 200 nm, which is larger than LDL protein but much smaller than red blood cells.

[0108] The exosomes may be released from the cell when multivesicular bodies fuse with the cell membrane, or may be immediately released from the cell membrane.

[0109] It is well known that such exosomes play important yet specialized functions, such as coagulation and intercellular signaling.

[0110] As used herein, the term "stem cell" refers to an undifferentiated cell that has the ability to self-replicate and to differentiate into two or more different types of cells.

[0111] The stem cells may be autologous or allogeneic and may be derived from any type of animal, including humans and non-human mammals, including but not limited to, whether the stem cells are derived from an adult or embryo.

[0112] Specifically, the stem cells may be, but are not limited to, embryonic stem cells, adult stem cells, or induced pluripotent stem cells.

[0113] As used herein, the term "embryonic stem cell" refers to a cell extracted during the development of an embryo, which is the inner cell mass extracted from an embryo at the blastula stage, just before the fertilized egg implants in the mother's uterus, and cultured in vitro.

[0114] The embryonic stem cells are pluripotent, meaning that they can differentiate into any tissue cell in an individual, or totipotent, meaning that they have the ability to self-renew, and in a broad sense also include embryoid bodies derived from embryonic stem cells.

[0115] The embryonic stem cells may be of any origin, including, but not limited to, human, monkey, pig, horse, cow, sheep, dog, cat, mouse, rabbit, and the like.

[0116] As used herein, the term "adult stem cell" refers to a cell extracted from umbilical cord blood, adult bone marrow, blood, etc., which is immediately prior to differentiating into a specific organ cell, and refers to an undifferentiated cell that retains the ability to develop into tissues within the body when needed.

[0117] The adult stem cells may include, but are not limited to, adult stem cells of various human or animal tissue origins, mesenchymal stromal cells derived from human or animal tissues, mesenchymal stem cells derived from induced pluripotent stem cells of various human or animal tissue origins, and multipotent stem cells.

[0118] The human or animal tissue may be, but is not limited to, umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane and / or placenta.

[0119] The stem cells of various human or animal tissue origins may be, for example, but are not limited to, hematopoietic stem cells, mammary stem cells, intestinal stem cells, vascular endothelial stem cells, neural stem cells, olfactory neural stem cells and / or testicular stem cells.

[0120] As used herein, the term "induced pluripotent stem cell (iPSC)" refers to a cell that has been induced to have pluripotent differentiation ability from a differentiated cell through an artificial reverse differentiation process, and may be used interchangeably with "reverse differentiated stem cell."

[0121] The artificial retrodifferentiation process may be carried out by using viral or non-viral vectors using retroviruses, lentiviruses, and Sendai viruses, or by introducing non-viral-mediated retrodifferentiation factors using proteins and cell extracts, etc., or may include a retrodifferentiation process using stem cell extracts, compounds, etc.

[0122] Said induced pluripotent stem cells exhibit almost the same properties as embryonic stem cells, specifically, they share aspects including cytomorphological similarity, similar gene and protein expression patterns, totipotency in vitro and in vivo, teratoma formation, generation of chimera mice upon insertion into mouse blastocysts, and germline transmission of genes.

[0123] The induced pluripotent stem cells may include, but are not limited to, induced pluripotent stem cells of any origin, such as human, monkey, pig, horse, cow, sheep, dog, cat, mouse, and rabbit.

[0124] As used herein, the term "promotion of production" refers to an increase in the production, synthesis, release, etc. of a particular substance within the same period of time compared to a control group.

[0125] Specifically, this means an increase in the amount of exosomes produced from stem cells and the content of proteins, RNA, etc. within the exosomes.

[0126] Another aspect of the present invention relates to a composition for enhancing the stemness of stem cells, comprising Exendin-4.

[0127] Another aspect of the present invention relates to a composition for enhancing stemness of stem cells, comprising phorbol 12-myristate 13-acetate (PMA).

[0128] Another aspect of the present invention relates to a composition for enhancing the stemness of stem cells, comprising interferon-gamma.

[0129] Another aspect of the present invention relates to a composition for enhancing stemness of stem cells, comprising tetrandrine.

[0130] Another aspect of the present invention relates to a composition for enhancing the stemness of stem cells, comprising hyaluronic acid.

[0131] Another aspect of the present invention relates to a composition containing Substance P for enhancing the stemness of stem cells.

[0132] Another aspect of the present invention relates to a composition for enhancing the stemness of stem cells, comprising resveratrol.

[0133] Another aspect of the present invention relates to a composition for enhancing stemness of stem cells, comprising Lanifibranor.

[0134] As used herein, the term "stem cell potential" is used in the art to collectively refer to the pluripotency to differentiate into all types of cells, and the self-renewal potential to divide indefinitely to produce more identical stem cells.

[0135] Specifically, it refers to enhancing the proliferation of stem cells while maintaining the undifferentiated state, improving telomerase activity, improving the expression of stemness acting signals, and improving cell motility, and can include the appearance of one or more of these characteristics.

[0136] Each of the compositions of the present invention may be used as a medium composition for stem cell culture, and may be administered in vivo together with stem cells as a pharmaceutical composition for promoting in vivo exosome production, or may be administered prior to or after the administration of the composition, thereby enhancing the in vivo efficacy of stem cells.

[0137] The enhancement of the in vivo efficacy of stem cells means that, under the assumption that the efficacy (in vivo efficacy) of stem cells for treating certain diseases such as arthritis and peripheral neuropathy is mediated by exosomes secreted by stem cells, co-administration of the composition of the present disclosure with stem cells promotes the production of exosomes from stem cells, thereby enhancing the therapeutic effect (in vivo efficacy) of stem cells against the disease.

[0138] When each of the compositions of the present invention is a medium composition, the "medium composition" refers to a composition containing essential components necessary for cell growth, survival, and proliferation in vitro, and includes any stem cell culture medium commonly used in the art, such as DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, DMEM / F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10), DMEM / F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), α-MEM (α-Minimal essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Isocove's Modified Dulbecco's Medium), KnockOut Commercially produced media such as DMEM and E8 (Essential 8 Medium) or artificially synthesized media can be used, but are not limited thereto.

[0139] The medium composition generally contains a carbon source, a nitrogen source, and trace element components, and may further contain amino acids, antibiotics, and the like.

[0140] In addition, the medium composition can be prepared by adding Exendin-4, a component of the present invention, to a conventional medium for culturing stem cells.

[0141] The medium composition can also be prepared by adding phorbol 12-myristate 13-acetate (PMA), a component of the present invention, to a conventional medium for culturing stem cells.

[0142] In addition, the medium composition can be prepared by adding interferon-gamma, a component of the present invention, to a conventional medium for culturing stem cells.

[0143] In addition, the medium composition can be prepared by adding tetrandrine, a component of the present invention, to a conventional medium for culturing stem cells.

[0144] In addition, the medium composition can be prepared by adding hyaluronic acid, a component of the present invention, to a conventional medium for culturing stem cells.

[0145] In addition, the medium composition can be prepared by adding the component P substance of the present invention to a conventional medium for culturing stem cells.

[0146] In addition, the medium composition can be prepared by adding resveratrol, a component of the present invention, to a conventional medium for culturing stem cells.

[0147] In addition, the medium composition can be prepared by adding Lanifibranor, a component of the present invention, to a conventional medium for culturing stem cells.

[0148] The component exendin-4 of the present invention may be added to the culture medium at a concentration of 10 to 1000 nM, 10 to 100 nM, 10 to 90 nM, 10 to 80 nM, 10 to 70 nM, 10 to 60 nM, 10 to 50 nM, 10 to 40 nM, 10 to 30 nM, or 20 nM, but is not limited thereto.

[0149] The component phorbol 12-myristate 13-acetate (PMA) of the present invention may be added to the culture medium at a concentration of 1 to 1000 nM, 1 to 500 nM, 1 to 100 nM, 1 to 90 nM, 1 to 80 nM, 1 to 70 nM, 1 to 60 nM, 1 to 50 nM, 1 to 40 nM, 1 to 30 nM, 1 to 20 nM, or 10 nM, but is not limited thereto.

[0150] The component interferon-gamma of the present invention may be added to the culture medium at a concentration of 1 to 1000 ng / mL, 1 to 500 ng / mL, 1 to 100 ng / mL, 1 to 90 ng / mL, 1 to 80 ng / mL, 1 to 70 ng / mL, 1 to 60 ng / mL, 1 to 50 ng / mL, 1 to 40 ng / mL, 1 to 30 ng / mL, 1 to 20 ng / mL, or 10 ng / mL, but is not limited thereto.

[0151] Tetrandrine, a component of the present invention, may be added to the culture medium at a concentration of 1 to 1000 μM, 1 to 500 μM, 1 to 100 μM, 1 to 90 μM, 1 to 80 μM, 1 to 70 μM, 1 to 60 μM, 1 to 50 μM, 1 to 40 μM, 1 to 30 μM, 1 to 20 μM, or 10 μM, but is not limited thereto.

[0152] The hyaluronic acid, a component of the present invention, may be added to the culture medium at a concentration of 1 to 1000 μg / mL, 1 to 500 μg / mL, 1 to 100 μg / mL, 1 to 90 μg / mL, 1 to 80 μg / mL, 1 to 70 μg / mL, 1 to 60 μg / mL, 1 to 50 μg / mL, 1 to 40 μg / mL, 1 to 30 μg / mL, 1 to 20 μg / mL, or 10 μg / mL, but is not limited thereto.

[0153] The component P substance of the present invention may be added to the culture medium at a concentration of 10 to 1000 nM, 10 to 100 nM, 10 to 90 nM, 10 to 80 nM, 10 to 70 nM, 10 to 60 nM, 10 to 50 nM, 10 to 40 nM, 20 to 40 nM, or 30 nM, but is not limited thereto.

[0154] The component resveratrol of the present invention may be added to the medium at a concentration of 1 to 1000 nM, 1 to 500 nM, 1 to 100 nM, 1 to 90 nM, 1 to 80 nM, 1 to 70 nM, 1 to 60 nM, 1 to 50 nM, 1 to 40 nM, 1 to 30 nM, 1 to 20 nM, or 10 nM, but is not limited thereto.

[0155] The component Lanifibranor of the present invention may be added to the culture medium at a concentration of 1 to 1000 μM, 1 to 500 μM, 1 to 100 μM, 1 to 90 μM, 1 to 80 μM, 1 to 70 μM, 1 to 60 μM, 1 to 50 μM, 1 to 40 μM, 1 to 30 μM, 1 to 20 μM, or 10 μM, but is not limited thereto.

[0156] When each of the compositions of the present invention is a pharmaceutical composition, the pharma- ceutically acceptable carriers that may be included in the pharmaceutical composition of the present invention are those that are commonly used in formulation, including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharma-ceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0157] The pharmaceutical compositions of the present invention can be administered orally and parenterally, for example, by intravenous, subcutaneous, intramuscular, intraperitoneal, topical, intranasal, pulmonary, rectal, intrathecal, ocular, dermal, and transdermal administration.

[0158] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity of the patient, and a skilled ordinary physician can easily determine and prescribe an effective dosage for the desired treatment or prevention. According to one embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.0001 to 1000 mg / kg.

[0159] The pharmaceutical composition of the present invention can be formulated using pharma- ceutical acceptable carriers and / or excipients by a method that can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. The composition of the present disclosure can be prepared in a unit dose form or can be contained in a multi-dose container. In this case, the dosage form can be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and can further include a dispersing agent or stabilizer.

[0160] Yet another aspect of the present invention relates to stem cell-derived exosomes pretreated with Exendin-4.

[0161] Yet another aspect of the present invention relates to stem cell-derived exosomes pretreated with phorbol 12-myristate 13-acetate (PMA).

[0162] Yet another aspect of the present invention relates to exosomes derived from stem cells pretreated with Interferon-γ.

[0163] Yet another aspect of the present invention relates to stem cell-derived exosomes pretreated with tetrandrine.

[0164] Yet another aspect of the present invention relates to stem cell-derived exosomes pretreated with hyaluronic acid.

[0165] Yet another aspect of the present invention relates to stem cell-derived exosomes pretreated with Substance P.

[0166] Yet another aspect of the present invention relates to stem cell-derived exosomes pretreated with resveratrol.

[0167] Yet another aspect of the present invention relates to stem cell-derived exosomes pretreated with Lanifibranor.

[0168] Description of the contents common to each composition of the present invention and the stem cell-derived exosomes will be omitted to avoid excessive complexity of this specification.

[0169] Yet another aspect of the present invention relates to a stem cell therapeutic agent comprising a composition containing the above-mentioned Exendin-4, stem cells pretreated with Exendin-4, or exosomes derived from stem cells pretreated with Exendin-4.

[0170] Yet another aspect of the present invention relates to a stem cell therapeutic agent comprising a composition comprising the above-mentioned phorbol 12-myristate 13-acetate (PMA), stem cells pretreated with phorbol 12-myristate 13-acetate (PMA), or exosomes derived from stem cells pretreated with phorbol 12-myristate 13-acetate (PMA).

[0171] Yet another aspect of the present invention relates to a stem cell therapeutic agent comprising a composition containing the above-mentioned interferon-gamma, stem cells pretreated with interferon-gamma, or exosomes derived from stem cells pretreated with interferon-gamma.

[0172] Yet another aspect of the present invention relates to a stem cell therapeutic agent comprising a composition containing the above-mentioned tetrandrine, stem cells pretreated with tetrandrine, or exosomes derived from stem cells pretreated with tetrandrine.

[0173] Yet another aspect of the present invention relates to a stem cell therapeutic agent comprising a composition containing the above-mentioned hyaluronic acid, stem cells pretreated with hyaluronic acid, or exosomes derived from stem cells pretreated with hyaluronic acid.

[0174] Yet another aspect of the present invention relates to a stem cell therapeutic agent comprising a composition containing the above-mentioned substance P, stem cells pretreated with substance P, or exosomes derived from stem cells pretreated with substance P.

[0175] Yet another aspect of the present invention relates to a stem cell therapeutic agent comprising a composition containing the above-mentioned resveratrol, stem cells pretreated with resveratrol, or exosomes derived from stem cells pretreated with resveratrol.

[0176] Yet another aspect of the present invention relates to a stem cell therapeutic agent comprising a composition comprising the above-mentioned Lanifibranor, stem cells pretreated with Lanifibranor, or exosomes derived from stem cells pretreated with Lanifibranor.

[0177] In the present invention, the term "cell therapy agent" refers to a pharmaceutical product used for therapeutic, diagnostic, and preventive purposes by a series of actions such as expanding and selecting living autologous, allogenic, or xenogenic cells in vitro, or by changing the biological properties of cells in other ways, in order to restore the functions of cells and tissues.

[0178] Such cell therapeutic agents can be broadly divided into two types: the first is "stem cell therapeutic agents" for tissue regeneration, organ function recovery, or regulating the function of immune cells, and the second is "immune cell therapeutic agents" for regulating immune responses, such as suppressing immune responses in the body or enhancing immune responses.

[0179] The compositions of the present invention and their components, Exendin-4, Phorbol 12-myristate 13-acetate (PMA), Interferon-γ, Tetrandrine, Hyaluronic acid, Substance P, Resveratrol, and Lanifibranor, have the effect of enhancing the stem cell activity of stem cells and promoting the production of stem cell-derived exosomes, thereby enhancing the therapeutic efficacy of stem cells. Therefore, the "cell therapeutic agent" as used herein means a "stem cell therapeutic agent."

[0180] The stem cell therapeutic agent may be used for treating diseases in a subject in need thereof, including, but not limited to, cardiovascular diseases such as myocardial infarction and heart failure, cancer diseases such as liver cancer, gastric cancer, colon cancer, prostate cancer, bladder cancer, and lung cancer, inflammatory diseases such as atopic dermatitis, arthritis, autoimmune encephalomyelitis, systemic lupus erythematosus, colitis, and multiple sclerosis, and autoimmune diseases.

[0181] The stem cell therapeutic agent shares components with the above-mentioned compositions of the present invention, and therefore, the contents common to both compositions will be omitted in order to avoid overly complicating this specification.

[0182] Yet another aspect of the present invention relates to a method for producing stem cell-derived exosomes, comprising the steps of:

[0183] a) culturing stem cells in a cell culture medium containing Exendin-4.

[0184] Yet another aspect of the present invention relates to a method for producing stem cell-derived exosomes, comprising the steps of:

[0185] a) Culturing stem cells in a cell culture medium containing phorbol 12-myristate 13-acetate (PMA).

[0186] Yet another aspect of the present invention relates to a method for producing stem cell-derived exosomes, comprising the steps of:

[0187] a) culturing stem cells in a cell culture medium containing interferon-gamma.

[0188] Yet another aspect of the present invention relates to a method for producing stem cell-derived exosomes, comprising the steps of:

[0189] a) Culturing stem cells in a cell culture medium containing tetrandrine.

[0190] Yet another aspect of the present invention relates to a method for producing stem cell-derived exosomes, comprising the steps of:

[0191] a) culturing stem cells in a cell culture medium containing hyaluronic acid;

[0192] Yet another aspect of the present invention relates to a method for producing stem cell-derived exosomes, comprising the steps of:

[0193] a) culturing stem cells in a cell culture medium containing Substance P;

[0194] Yet another aspect of the present invention relates to a method for producing stem cell-derived exosomes, comprising the steps of:

[0195] a) culturing stem cells in a cell culture medium containing resveratrol.

[0196] Yet another aspect of the present invention relates to a method for producing stem cell-derived exosomes, comprising the steps of:

[0197] a) culturing stem cells in a cell culture medium containing Lanifibranor.

[0198] The method may further comprise the following steps:

[0199] b) washing the cultured stem cells and then further culturing them in a cell culture medium; and

[0200] c) Isolating exosomes.

[0201] The method for producing stem cell-derived exosomes of the present invention will be described in detail below.

[0202] a) Stages

[0203] This step is a process of stimulating stem cells by pretreating them with Exendin-4, Phorbol 12-myristate 13-acetate (PMA), Interferon-gamma, Tetrandrine, Hyaluronic acid, Substance P, Resveratrol, or Lanifibranor. This process causes the stem cells to produce a greater number of exosomes and increase the content of proteins and RNA in the exosomes compared to stem cells not pretreated with the substances.

[0204] b) Stage

[0205] This step is a process of inducing exosome secretion or production from stem cells by washing the stem cells pretreated with the substance according to the present invention to remove the pretreatment substance, and culturing them in new cell culture medium. The stem cell pretreatment substance (exendin-4) in step a) is removed by the washing process in step b) and does not remain in the stem cells, exosomes produced by the stem cells, or culture medium after washing. Therefore, the stem cells treated with the stem cell pretreatment substance of the present invention in step a), the exosomes produced by the stem cells, and the culture medium can be usefully used for subsequent research or disease treatment purposes without being affected by the residue of the above-mentioned pretreatment substance.

[0206] The cell culture medium at this stage further contains exosome-removed fetal bovine serum. The reason for using exosome-removed FBS in the cell culture medium is to prevent the exosomes secreted by stem cells from being contaminated by FBS-derived exosomes, since the FBS commonly used contains a large amount of exosomes derived from bovine serum.

[0207] The additional culture in this stage may be carried out for 12 to 120 hours, 24 to 96 hours, 48 ​​to 96 hours, or 60 to 84 hours, most specifically, for 72 hours, but is not limited thereto.

[0208] c) Stage

[0209] In this step, the culture medium of the stem cells additionally cultured in step b) is centrifuged at 200 to 400×g for 5 to 20 minutes to remove remaining cells and cell debris, the supernatant is then taken and centrifuged at high speed at 9,000 to 12,000×g for 60 to 80 minutes, the supernatant is then taken again and centrifuged at 90,000 to 120,000×g for 80 to 100 minutes, and the supernatant is removed to obtain the exosomes remaining in the lower layer.

[0210] According to a specific example of the present invention, the mesenchymal stem cell culture medium is collected and centrifuged at 300×g for 10 minutes to remove remaining cells and cell debris, the supernatant is taken and filtered using a 0.22 μm filter, and then centrifuged using a high speed centrifuge at 10,000×g for 70 minutes at 4° C. The centrifuged supernatant is again taken and centrifuged using an ultracentrifuge at 100,000×g for 90 minutes at 4° C. to remove the supernatant, thereby isolating the exosomes remaining in the lower layer.

[0211] Yet another aspect of the present invention relates to a method for enhancing stemness of stem cells, comprising the steps of:

[0212] a) culturing stem cells in a cell culture medium containing Exendin-4.

[0213] Yet another aspect of the present invention relates to a method for enhancing stemness of stem cells, comprising the steps of:

[0214] a) Culturing stem cells in a cell culture medium containing phorbol 12-myristate 13-acetate (PMA).

[0215] Yet another aspect of the present invention relates to a method for enhancing stemness of stem cells, comprising the steps of:

[0216] a) culturing stem cells in a cell culture medium containing interferon-gamma.

[0217] Yet another aspect of the present invention relates to a method for enhancing stemness of stem cells, comprising the steps of:

[0218] a) Culturing stem cells in a cell culture medium containing tetrandrine.

[0219] Yet another aspect of the present invention relates to a method for enhancing stemness of stem cells, comprising the steps of:

[0220] a) culturing stem cells in a cell culture medium containing hyaluronic acid;

[0221] Yet another aspect of the present invention relates to a method for enhancing stemness of stem cells, comprising the steps of:

[0222] a) culturing stem cells in a cell culture medium containing Substance P;

[0223] Yet another aspect of the present invention relates to a method for enhancing stemness of stem cells, comprising the steps of:

[0224] a) culturing stem cells in a cell culture medium containing resveratrol.

[0225] Yet another aspect of the present invention relates to a method for enhancing stemness of stem cells, comprising the steps of:

[0226] a) culturing stem cells in a cell culture medium containing Lanifibranor.

[0227] The method for enhancing the stem cell potential of stem cells has steps in common with the above-mentioned method for producing stem cell-derived exosomes of the present invention, and description of the contents common to both methods will be omitted to avoid excessive complexity of this specification.

[0228] Yet another aspect of the present invention is use of a composition containing Exendin-4 to promote the production of exosomes derived from stem cells.

[0229] Yet another embodiment of the present invention is the use of a composition containing Exendin-4 for enhancing stemness of stem cells.

[0230] Yet another aspect of the present invention is the use of a composition containing phorbol 12-myristate 13-acetate (PMA) to promote the production of stem cell-derived exosomes.

[0231] Yet another embodiment of the present invention is the use of a composition containing phorbol 12-myristate 13-acetate (PMA) for enhancing stemness of stem cells.

[0232] Yet another aspect of the present invention is use of a composition containing interferon-gamma to promote the production of exosomes derived from stem cells.

[0233] Yet another embodiment of the present invention is the use of a composition containing interferon-gamma (Interferon-γ) for enhancing the stemness of stem cells.

[0234] Yet another aspect of the present invention is the use of a composition containing tetrandrine for promoting the production of stem cell-derived exosomes.

[0235] Yet another aspect of the present invention is the use of a composition containing tetrandrine for enhancing stemness of stem cells.

[0236] Yet another aspect of the present invention is a use of a composition containing hyaluronic acid for promoting the production of exosomes derived from stem cells.

[0237] Yet another aspect of the present invention is the use of a composition containing hyaluronic acid for enhancing the stemness of stem cells.

[0238] Yet another aspect of the present invention is use of a composition containing Substance P for promoting the production of stem cell-derived exosomes.

[0239] Yet another embodiment of the present invention is the use of a composition containing Substance P for enhancing stemness of stem cells.

[0240] Yet another aspect of the present invention is a use of a composition containing resveratrol for promoting the production of stem cell-derived exosomes.

[0241] Yet another aspect of the present invention is the use of a composition containing resveratrol for enhancing stemness of stem cells.

[0242] Yet another aspect of the present invention is a use of a composition containing Lanifibranor for promoting the production of stem cell-derived exosomes.

[0243] Yet another aspect of the present invention is the use of a composition containing Lanifibranor for enhancing stemness of stem cells. Effect of the Invention

[0244] The present invention relates to a composition for promoting the production of stem cell-derived exosomes and enhancing the function of stem cells, comprising one selected from Exendin-4, Phorbol 12-myristate 13-acetate (PMA), Interferon-γ, Tetrandrine, Hyaluronic acid, Substance P, Resveratrol, and Lanifibranor. When the composition of the present invention is used in stem cell culture, the stem cell activity of stem cells and the production amount of stem cell-derived exosomes are increased, so that high-quality stem cells and stem cell-derived exosomes can be produced more efficiently, and the composition can be usefully used in related research and development and commercialization. [Brief description of the drawings]

[0245] [Figure 1] FIG. 1 is a diagram confirming an increase in stem cell proliferation rate by treatment with a stem cell pretreatment substance (Exendin-4) according to one embodiment of the present invention.

[0246] [Figure 2A] FIG. 1 is a diagram confirming that the stemness of stem cells is enhanced by treatment with a stem cell pretreatment substance (Exendin-4) according to one embodiment of the present invention.

[0247] [Figure 2B] FIG. 1 is a diagram confirming that the stemness of stem cells is enhanced by treatment with a stem cell pretreatment substance (Exendin-4) according to one embodiment of the present invention.

[0248] [Diagram 3] FIG. 1 is a diagram confirming the size distribution of exosomes after treatment with a stem cell pretreatment substance (Exendin-4) according to one embodiment of the present invention.

[0249] [Figure 4] FIG. 1 is a diagram confirming an increase in exosome number due to treatment with a stem cell pretreatment substance (Exendin-4) in accordance with one embodiment of the present invention.

[0250] [Diagram 5] FIG. 1 is a diagram confirming an increase in the amount of exosome-derived proteins by treatment with a stem cell pretreatment substance (Exendin-4) according to one embodiment of the present invention.

[0251] [Figure 6] FIG. 1 is a diagram confirming an increase in the amount of exosome-derived RNA by treatment with a stem cell pretreatment substance (Exendin-4) in accordance with one embodiment of the present invention.

[0252] [Figure 7]FIG. 1 is a diagram confirming an increase in stem cell proliferation rate by treatment with a stem cell pretreatment substance (phorbol 12-myristate 13-acetate (PMA)) according to one embodiment of the present invention.

[0253] [Figure 8A] FIG. 1 is a diagram confirming the enhancement of stemness of stem cells by treatment with a stem cell pretreatment substance (phorbol 12-myristate 13-acetate (PMA)) according to one embodiment of the present invention.

[0254] [Figure 8B] FIG. 1 is a diagram confirming the enhancement of stemness of stem cells by treatment with a stem cell pretreatment substance (phorbol 12-myristate 13-acetate (PMA)) according to one embodiment of the present invention.

[0255] [Figure 9] FIG. 1 is a diagram confirming the size distribution of exosomes following treatment with a stem cell pretreatment substance (phorbol 12-myristate 13-acetate (PMA)) in accordance with one embodiment of the present invention.

[0256] [Figure 10] FIG. 1 is a diagram confirming an increase in exosome number due to treatment with a stem cell pretreatment substance (phorbol 12-myristate 13-acetate (PMA)) in accordance with one embodiment of the present invention.

[0257] [Figure 11] FIG. 1 is a graph confirming an increase in the amount of exosome-derived proteins by treatment with a stem cell pretreatment substance (phorbol 12-myristate 13-acetate (PMA)) in accordance with one embodiment of the present invention.

[0258] [Figure 12] FIG. 1 is a diagram confirming an increase in the amount of exosome-derived RNA by treatment with a stem cell pretreatment substance (phorbol 12-myristate 13-acetate (PMA)) in accordance with one embodiment of the present invention.

[0259] [Figure 13] FIG. 1 is a diagram confirming an increase in stem cell proliferation rate by treatment with a stem cell pretreatment substance (Interferon-γ) according to one embodiment of the present invention.

[0260] [Figure 14A] FIG. 1 is a diagram confirming that the stemness of stem cells is enhanced by treatment with a stem cell pretreatment substance (Interferon-γ) according to one embodiment of the present invention.

[0261] [Figure 14B] FIG. 1 is a diagram confirming that the stemness of stem cells is enhanced by treatment with a stem cell pretreatment substance (Interferon-γ) according to one embodiment of the present invention.

[0262] [Figure 15] FIG. 1 is a diagram confirming the size distribution of exosomes after treatment with a stem cell pretreatment substance (Interferon-γ) according to one embodiment of the present invention.

[0263] [Figure 16] FIG. 1 is a diagram confirming an increase in exosome number due to treatment with a stem cell pretreatment substance (Interferon-γ) according to one embodiment of the present invention.

[0264] [Figure 17] FIG. 1 is a diagram confirming an increase in the amount of exosome-derived proteins by treatment with a stem cell pretreatment substance (Interferon-γ) according to one embodiment of the present invention.

[0265] [Figure 18] FIG. 1 is a diagram confirming an increase in the amount of exosome-derived RNA by treatment with a stem cell pretreatment substance (Interferon-γ) according to one embodiment of the present invention.

[0266] [Figure 19] FIG. 1 is a diagram confirming an increase in stem cell proliferation rate by treatment with a stem cell pretreatment substance (Tetrandrine) according to one embodiment of the present invention.

[0267] [Figure 20A] FIG. 1 is a diagram confirming the enhancement of stemness of stem cells by treatment with a stem cell pretreatment substance (Tetrandrine) according to one embodiment of the present invention.

[0268] [Figure 20B] FIG. 1 is a diagram confirming the enhancement of stemness of stem cells by treatment with a stem cell pretreatment substance (Tetrandrine) according to one embodiment of the present invention.

[0269] [Figure 21] FIG. 1 is a diagram confirming the size distribution of exosomes after treatment with a stem cell pretreatment substance (tetrandrine) according to one embodiment of the present invention.

[0270] [Figure 22] FIG. 1 is a diagram confirming an increase in the number of exosomes by treatment with a stem cell pretreatment substance (tetrandrine) in accordance with one embodiment of the present invention.

[0271] [Diagram 23] FIG. 1 is a diagram confirming an increase in the amount of exosome-derived proteins by treatment with a stem cell pretreatment substance (tetrandrine) in accordance with one embodiment of the present invention.

[0272] [Figure 24] FIG. 1 is a diagram confirming an increase in the amount of exosome-derived RNA by treatment with a stem cell pretreatment substance (tetrandrine) in accordance with one embodiment of the present invention.

[0273] [Diagram 25] FIG. 1 is a diagram confirming an increase in stem cell proliferation rate by treatment with a stem cell pretreatment material (hyaluronic acid) according to one embodiment of the present invention.

[0274] [Figure 26A] FIG. 1 is a diagram confirming that the stemness of stem cells is enhanced by treatment with a stem cell pretreatment material (hyaluronic acid) according to one embodiment of the present invention.

[0275] [Figure 26B] FIG. 1 is a diagram confirming that the stemness of stem cells is enhanced by treatment with a stem cell pretreatment material (hyaluronic acid) according to one embodiment of the present invention.

[0276] [Figure 27] FIG. 1 is a diagram confirming the size distribution of exosomes after treatment with a stem cell pretreatment material (hyaluronic acid) according to one embodiment of the present invention.

[0277] [Figure 28] FIG. 1 is a diagram confirming an increase in the number of exosomes due to treatment with a stem cell pretreatment substance (hyaluronic acid) in accordance with one embodiment of the present invention.

[0278] [Figure 29] FIG. 1 is a diagram confirming an increase in the amount of exosome-derived proteins by treatment with a stem cell pretreatment substance (hyaluronic acid) according to one embodiment of the present invention.

[0279] [Diagram 30] FIG. 1 is a diagram confirming an increase in the amount of exosome-derived RNA by treatment with a stem cell pretreatment substance (hyaluronic acid) according to one embodiment of the present invention.

[0280] [Diagram 31] FIG. 1 is a diagram confirming an increase in stem cell proliferation rate by treatment with a stem cell pretreatment substance (Substance P) according to one embodiment of the present invention.

[0281] [Figure 32A] FIG. 1 is a diagram confirming the enhancement of stemness of stem cells by treatment with a stem cell pretreatment substance (Substance P) according to one embodiment of the present invention.

[0282] [Figure 32B] FIG. 1 is a diagram confirming the enhancement of stemness of stem cells by treatment with a stem cell pretreatment substance (Substance P) according to one embodiment of the present invention.

[0283] [Diagram 33] FIG. 1 is a diagram confirming the size distribution of exosomes after treatment with a stem cell pretreatment substance (Substance P) according to one embodiment of the present invention.

[0284] [Diagram 34] FIG. 1 is a diagram confirming an increase in the number of exosomes due to treatment with a stem cell pretreatment substance (Substance P) in accordance with one embodiment of the present invention.

[0285] [Diagram 35] FIG. 1 is a diagram confirming an increase in the amount of exosome-derived proteins by treatment with a stem cell pretreatment substance (Substance P) according to one embodiment of the present invention.

[0286] [Diagram 36] FIG. 1 is a diagram confirming an increase in the amount of exosome-derived RNA by treatment with a stem cell pretreatment substance (Substance P) according to one embodiment of the present invention.

[0287] [Figure 37]FIG. 2 is a diagram confirming an increase in stem cell proliferation rate by treatment with a stem cell pretreatment substance (resveratrol) according to one embodiment of the present invention.

[0288] [Figure 38A] FIG. 2 is a diagram confirming that the stemness of stem cells is enhanced by treatment with a stem cell pretreatment substance (resveratrol) according to one embodiment of the present invention.

[0289] [Figure 38B] FIG. 2 is a diagram confirming that the stemness of stem cells is enhanced by treatment with a stem cell pretreatment substance (resveratrol) according to one embodiment of the present invention.

[0290] [Figure 39] FIG. 1 is a diagram confirming the size distribution of exosomes after treatment with a stem cell pretreatment substance (resveratrol) according to one embodiment of the present invention.

[0291] [Diagram 40] FIG. 1 is a diagram confirming an increase in exosome number due to treatment with a stem cell pretreatment substance (resveratrol) in accordance with one embodiment of the present invention.

[0292] [Diagram 41] FIG. 1 is a diagram confirming an increase in the amount of exosome-derived protein by treatment with a stem cell pretreatment substance (resveratrol) according to one embodiment of the present invention.

[0293] [Diagram 42] FIG. 1 is a diagram confirming an increase in the amount of exosome-derived RNA by treatment with a stem cell pretreatment substance (resveratrol) in accordance with one embodiment of the present invention.

[0294] [Diagram 43]1 is a graph confirming an increase in stem cell proliferation rate by treatment with a stem cell pretreatment material (Lanifibranor) according to one embodiment of the present invention.

[0295] [Figure 44A] FIG. 1 is a diagram confirming that the stemness of stem cells is enhanced by treatment with a stem cell pretreatment substance (Lanifibranor) according to one embodiment of the present invention.

[0296] [Figure 44B] FIG. 1 is a diagram confirming that the stemness of stem cells is enhanced by treatment with a stem cell pretreatment substance (Lanifibranor) according to one embodiment of the present invention.

[0297] [Diagram 45] FIG. 1 is a diagram confirming the size distribution of exosomes after treatment with a stem cell pretreatment substance (Lanifibranor) according to one embodiment of the present invention.

[0298] [Figure 46] FIG. 1 is a diagram confirming an increase in exosome number due to treatment with a stem cell pretreatment substance (Lanifibranor) according to one embodiment of the present invention.

[0299] [Figure 47] FIG. 1 is a diagram confirming an increase in the amount of exosome-derived protein by treatment with a stem cell pretreatment substance (Lanifibranor) according to one embodiment of the present invention.

[0300] [Figure 48] FIG. 1 is a diagram confirming an increase in the amount of exosome-derived RNA by treatment with a stem cell pretreatment substance (Lanifibranor) according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0301] A composition for promoting the production of stem cell-derived exosomes, comprising one or more selected from the group consisting of Exendin-4, Phorbol 12-myristate 13-acetate (PMA), Interferon-gamma, Tetrandrine, Hyaluronic acid, Substance P, Resveratrol, and Lanifibranor. EXAMPLES

[0302] The present invention will now be described in more detail with reference to the following examples. However, these examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0303] Example 1. Isolation of exosomes derived from stem cells by treatment with Exendin-4

[0304] Exendin-4 pretreatment

[0305] Umbilical cord tissue-derived mesenchymal stem cells (Primary Umbilical Cord-Derived Mesenchymal Stem Cells, ATCC No. PCS-500-010; Lot No. 63822428) were cultured for one week in culture medium containing 20 nM exendin-4 [high glucose DMEM (Gibco, Cat no. 11995-065); 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100x) (Gibco, Cat no. 11140-050)].

[0306] Additional culture

[0307] After completing the culture, the exendin-4 pretreated mesenchymal stem cells were washed and further cultured for 72 hours in culture medium supplemented with 10% exosome-removed fetal bovine serum (FBS). The reason for using exosome-removed FBS is to prevent the contamination of exosomes secreted by cells with exosomes derived from FBS, since the FBS normally used contains a large amount of exosomes derived from bovine serum.

[0308] Exosome isolation

[0309] After 72 hours of culture, the mesenchymal stem cell culture medium treated with the pretreatment substances was collected and centrifuged at 300 x g for 10 minutes to remove remaining cells and cell debris. The supernatant was taken and filtered using a 0.22 μm filter, then centrifuged at 10,000 x g for 70 minutes at 4°C using a high speed centrifuge. The centrifuged supernatant was again taken and centrifuged at 100,000 x g for 90 minutes at 4°C using an ultracentrifuge, and the supernatant was removed. The exosomes remaining in the lower layer were diluted in PBS (phosphate buffered saline) and used in subsequent experiments.

[0310] Example 2. Isolation of stem cell-derived exosomes by PMA treatment

[0311] PMA pretreatment

[0312] Umbilical cord tissue-derived mesenchymal stem cells (Primary Umbilical Cord-Derived Mesenchymal Stem Cells, ATCC No. PCS-500-010; Lot No. 63822428) were cultured for one week in culture medium containing 10 nM PMA [high glucose DMEM (Gibco, Cat no. 11995-065); 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100x) (Gibco, Cat no. 11140-050)].

[0313] Additional culture

[0314] After completing the culture, the PMA-pretreated mesenchymal stem cells were washed and further cultured for 72 hours in culture medium supplemented with 10% exosome-removed fetal bovine serum (FBS). The reason for using exosome-removed FBS is to prevent the contamination of exosomes secreted by cells with exosomes derived from FBS, since the FBS normally used contains a large amount of exosomes derived from bovine serum.

[0315] Exosome isolation

[0316] After 72 hours of culture, the mesenchymal stem cell culture medium treated with the pretreatment substances was collected and centrifuged at 300 x g for 10 minutes to remove remaining cells and cell debris. The supernatant was taken and filtered using a 0.22 μm filter, then centrifuged at 10,000 x g for 70 minutes at 4°C using a high speed centrifuge. The centrifuged supernatant was again taken and centrifuged at 100,000 x g for 90 minutes at 4°C using an ultracentrifuge, and the supernatant was removed. The exosomes remaining in the lower layer were diluted in PBS (phosphate buffered saline) and used in subsequent experiments.

[0317] Example 3. Isolation of exosomes derived from stem cells by treatment with interferon-γ

[0318] Interferon-γ pretreatment

[0319] Umbilical cord tissue-derived mesenchymal stem cells (Primary Umbilical Cord-Derived Mesenchymal Stem Cells, ATCC No. PCS-500-010; Lot No. 63822428) were cultured for one week in culture medium [high glucose DMEM (Gibco, Cat no. 11995-065); 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100x) (Gibco, Cat no. 11140-050)] containing 10 ng / mL interferon-γ.

[0320] Additional culture

[0321] After completing the culture, the interferon-γ pretreated mesenchymal stem cells were washed and further cultured for 72 hours in culture medium supplemented with 10% exosome-removed fetal bovine serum (FBS). The reason for using exosome-removed FBS is to prevent the contamination of exosomes secreted by cells with exosomes derived from FBS, since the FBS normally used contains a large amount of exosomes derived from bovine serum.

[0322] Exosome isolation

[0323] After 72 hours of culture, the mesenchymal stem cell culture medium treated with the pretreatment substances was collected and centrifuged at 300 x g for 10 minutes to remove remaining cells and cell debris. The supernatant was taken and filtered using a 0.22 μm filter, then centrifuged at 10,000 x g for 70 minutes at 4°C using a high speed centrifuge. The centrifuged supernatant was again taken and centrifuged at 100,000 x g for 90 minutes at 4°C using an ultracentrifuge, and the supernatant was removed. The exosomes remaining in the lower layer were diluted in PBS (phosphate buffered saline) and used in subsequent experiments.

[0324] Example 4. Isolation of stem cell-derived exosomes by tetrandrine treatment

[0325] Tetrandrine pretreatment

[0326] Umbilical cord tissue-derived mesenchymal stem cells (Primary Umbilical Cord-Derived Mesenchymal Stem Cells, ATCC No. PCS-500-010; Lot No. 63822428) were cultured for one week in culture medium containing 10 μM tetrandrine [high glucose DMEM (Gibco, Cat no. 11995-065); 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100x) (Gibco, Cat no. 11140-050)].

[0327] Additional culture

[0328] After completing the culture, the tetrandrine-pretreated mesenchymal stem cells were washed and further cultured for 72 hours in culture medium supplemented with 10% exosome-removed fetal bovine serum (FBS). The reason for using exosome-removed FBS is to prevent the contamination of exosomes secreted by cells with exosomes derived from FBS, since the FBS normally used contains a large amount of exosomes derived from bovine serum.

[0329] Exosome isolation

[0330] After 72 hours of culture, the mesenchymal stem cell culture medium treated with the pretreatment substances was collected and centrifuged at 300 x g for 10 minutes to remove remaining cells and cell debris. The supernatant was taken and filtered using a 0.22 μm filter, then centrifuged at 10,000 x g for 70 minutes at 4°C using a high speed centrifuge. The centrifuged supernatant was again taken and centrifuged at 100,000 x g for 90 minutes at 4°C using an ultracentrifuge, and the supernatant was removed. The exosomes remaining in the lower layer were diluted in PBS (phosphate buffered saline) and used in subsequent experiments.

[0331] Example 5. Isolation of stem cell-derived exosomes by hyaluronic acid treatment

[0332] Hyaluronic Acid Pretreatment

[0333] Umbilical cord tissue-derived mesenchymal stem cells (Primary Umbilical Cord-Derived Mesenchymal Stem Cells, ATCC No. PCS-500-010; Lot No. 63822428) were cultured for one week in culture medium containing 10 μg / mL hyaluronic acid [high glucose DMEM (Gibco, Cat no. 11995-065); 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100x) (Gibco, Cat no. 11140-050)].

[0334] Additional culture

[0335] After completing the culture, the hyaluronic acid-pretreated mesenchymal stem cells were washed and further cultured for 72 hours in culture medium supplemented with 10% exosome-removed fetal bovine serum (FBS). The reason for using exosome-removed FBS is to prevent the contamination of exosomes secreted by cells with exosomes derived from FBS, since the FBS normally used contains a large amount of exosomes derived from bovine serum.

[0336] Exosome isolation

[0337] After 72 hours of culture, the mesenchymal stem cell culture medium treated with the pretreatment substances was collected and centrifuged at 300 x g for 10 minutes to remove remaining cells and cell debris. The supernatant was taken and filtered using a 0.22 μm filter, then centrifuged at 10,000 x g for 70 minutes at 4°C using a high speed centrifuge. The centrifuged supernatant was again taken and centrifuged at 100,000 x g for 90 minutes at 4°C using an ultracentrifuge, and the supernatant was removed. The exosomes remaining in the lower layer were diluted in PBS (phosphate buffered saline) and used in subsequent experiments.

[0338] Example 6. Isolation of exosomes derived from stem cells by treatment with Substance P

[0339] P substance pretreatment

[0340] Umbilical cord tissue-derived mesenchymal stem cells (Primary Umbilical Cord-Derived Mesenchymal Stem Cells, ATCC No. PCS-500-010; Lot No. 63822428) were cultured for one week in culture medium containing 30 nM of P substance [high glucose DMEM (Gibco, Cat no. 11995-065); 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100x) (Gibco, Cat no. 11140-050)].

[0341] Additional culture

[0342] After completing the culture, the mesenchymal stem cells pretreated with substance P were washed and further cultured for 72 hours in culture medium supplemented with 10% exosome-removed fetal bovine serum (FBS). The reason for using exosome-removed FBS is to prevent the contamination of exosomes secreted by cells with exosomes derived from FBS, since the FBS normally used contains a large amount of exosomes derived from bovine serum.

[0343] Exosome isolation

[0344] After 72 hours of culture, the mesenchymal stem cell culture medium treated with the pretreatment substances was collected and centrifuged at 300 x g for 10 minutes to remove remaining cells and cell debris. The supernatant was taken and filtered using a 0.22 μm filter, then centrifuged at 10,000 x g for 70 minutes at 4°C using a high speed centrifuge. The centrifuged supernatant was again taken and centrifuged at 100,000 x g for 90 minutes at 4°C using an ultracentrifuge, and the supernatant was removed. The exosomes remaining in the lower layer were diluted in PBS (phosphate buffered saline) and used in subsequent experiments.

[0345] Example 7. Isolation of exosomes derived from stem cells by treatment with resveratrol

[0346] Resveratrol Pretreatment

[0347] Umbilical cord tissue-derived mesenchymal stem cells (Primary Umbilical Cord-Derived Mesenchymal Stem Cells, ATCC No. PCS-500-010; Lot No. 63822428) were cultured for one week in culture medium containing 10 nM resveratrol [high glucose DMEM (Gibco, Cat no. 11995-065); 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100x) (Gibco, Cat no. 11140-050)].

[0348] Additional culture

[0349] After completing the culture, the resveratrol-pretreated mesenchymal stem cells were washed and further cultured for 72 hours in culture medium supplemented with 10% exosome-removed fetal bovine serum (FBS). The reason for using exosome-removed FBS is to prevent the contamination of exosomes secreted by cells with exosomes derived from FBS, since the FBS normally used contains a large amount of exosomes derived from bovine serum.

[0350] Exosome isolation

[0351] After 72 hours of culture, the mesenchymal stem cell culture medium treated with the pretreatment substances was collected and centrifuged at 300 x g for 10 minutes to remove remaining cells and cell debris. The supernatant was taken and filtered using a 0.22 μm filter, then centrifuged at 10,000 x g for 70 minutes at 4°C using a high speed centrifuge. The centrifuged supernatant was again taken and centrifuged at 100,000 x g for 90 minutes at 4°C using an ultracentrifuge, and the supernatant was removed. The exosomes remaining in the lower layer were diluted in PBS (phosphate buffered saline) and used in subsequent experiments.

[0352] Example 8. Isolation of exosomes derived from stem cells by Lanifibranor treatment

[0353] Lanifibranol Pretreatment

[0354] Umbilical cord tissue-derived mesenchymal stem cells (Primary Umbilical Cord-Derived Mesenchymal Stem Cells, ATCC No. PCS-500-010; Lot No. 63822428) were cultured for one week in culture medium containing 10 μM lanifibranol [high glucose DMEM (Gibco, Cat no. 11995-065); 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100x) (Gibco, Cat no. 11140-050)].

[0355] Additional culture

[0356] After completing the culture, the Lanifibranol-pretreated mesenchymal stem cells were washed and further cultured for 72 hours in culture medium supplemented with 10% exosome-removed fetal bovine serum (FBS). The reason for using exosome-removed FBS is to prevent the contamination of exosomes secreted by cells with exosomes derived from FBS, since the FBS normally used contains a large amount of exosomes derived from bovine serum.

[0357] Exosome isolation

[0358] After 72 hours of culture, the mesenchymal stem cell culture medium treated with the pretreatment substances was collected and centrifuged at 300 x g for 10 minutes to remove remaining cells and cell debris. The supernatant was taken and filtered using a 0.22 μm filter, then centrifuged at 10,000 x g for 70 minutes at 4°C using a high speed centrifuge. The centrifuged supernatant was again taken and centrifuged at 100,000 x g for 90 minutes at 4°C using an ultracentrifuge, and the supernatant was removed. The exosomes remaining in the lower layer were diluted in PBS (phosphate buffered saline) and used in subsequent experiments.

[0359] Experimental example 1. Enhancement of stem cell function by exendin-4 treatment

[0360] 1-1. Increase in stem cell proliferation by exendin-4 treatment

[0361] In a 96-well plate containing 100uL of culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100x) (Gibco, Cat no. 11140-050)], the mesenchymal stem cells and the mesenchymal stem cells pretreated with exendin-4 of the above example were seeded at 3000 cells per well and then cultured in a CO2 incubator for 24 hours. Then, CCK solution (10uL / well) was added and the cells were cultured in a CO2 incubator for 4 hours, and the absorbance was measured at a wavelength of 450nm (420-480nm) to confirm the proliferation rate.

[0362] As can be seen from Figure 1, the cell proliferation rate of mesenchymal stem cells pretreated with the stem cell pretreatment substance [exendin-4] increased by approximately 340%, from 1 to 3.4, compared to mesenchymal stem cells that were not treated with any substance.

[0363] 1-2. Enhancement of stemness of stem cells by exendin-4 treatment

[0364] Mesenchymal stem cells and mesenchymal stem cells pretreated with exendin-4 of the above example were seeded at 1000 cells per dish in a 100 mm cell culture dish containing 10 mL of culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100x) (Gibco, Cat no. 11140-050)] and cultured for 21 days. The dish with the cultured cells attached was washed twice with PBS and fixed with 95% methanol at room temperature for about 2 minutes. The fixed cells were washed three times with PBS, stained for 5 minutes with 0.5% crystal violet solution [crystal violet (Sigma, C-3886, USA) 5g, methanol 100ml], washed with water, and dried at room temperature. The numbers of colonies consisting of 50 or more cells in each dish were counted and compared.

[0365] As can be seen from Figures 2A and 2B, the number of CFU-F colonies in mesenchymal stem cells pretreated with the stem cell pretreatment substance [exendin-4] (185 cells) increased by approximately 647% compared to mesenchymal stem cells not treated with any substance (28.6 cells).

[0366] Experimental example 2. Increase in exosome production efficiency by exendin-4 treatment

[0367] 2-1. Increase in exosome number by exendin-4 treatment

[0368] The number of exosomes isolated in the above example was confirmed by a nanoparticle tracking assay (NanoSight NS300, Malvern). For comparison, the number of exosomes isolated in the same manner as in the example was confirmed, except that no pretreatment substance was used. The results are shown in Table 1, Figure 3, and Figure 4 below.

[0369] [Table 1]

[0370] (MSC: untreated mesenchymal stem cells, Exe4-MSC: exendin-4 treated mesenchymal stem cells)

[0371] As can be seen from Table 1 and FIG. 4, the number of exosomes produced was approximately 5.85 times higher in mesenchymal stem cells pretreated with the stem cell pretreatment substance [exendin-4] than in mesenchymal stem cells not treated with any substance.

[0372] These results demonstrate that treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present invention increases the number of exosomes produced by the mesenchymal stem cells.

[0373] 2-2. Increase in exosome-derived protein amount by exendin-4 treatment

[0374] Proteins were isolated from the exosomes isolated in the above examples using an exosome protein isolation kit (Total exosome RNA and protein isolation kit, Invitrogen), and the absorbance was measured at 595 nm using Bradford analysis to quantify the proteins. For comparison, proteins were isolated and quantified from exosomes isolated in the same manner as in the examples, except that no pretreatment substances were used. The results are shown in Table 2 and FIG. 5 below.

[0375] [Table 2]

[0376] (MSC: untreated mesenchymal stem cells, Exe4-MSC: exendin-4 treated mesenchymal stem cells)

[0377] As can be seen from Table 2 and FIG. 5, exosome-derived proteins were produced in an amount approximately 5.4 times greater in mesenchymal stem cells pretreated with the stem cell pretreatment substance [exendin-4] than in mesenchymal stem cells not treated with any substance.

[0378] From the above results, it can be seen that when mesenchymal stem cells are treated with the stem cell pretreatment material according to the present invention, not only the number of exosomes but also the content of exosomal proteins increases.

[0379] 2-3. Increase in exosome-derived RNA amount by exendin-4 treatment

[0380] Using an RNA isolation kit (Total exosome RNA and protein isolation kit, Invitrogen), total exosome RNA was isolated from the exosomes isolated in the above example, and the RNA concentration was measured using Nanodrop. For comparison, total exosome RNA was isolated from exosomes isolated in the same manner as in the example, except that no pretreatment substance was used, and the concentration was measured. The results are shown in Table 3 and Figure 6 below.

[0381] [Table 3]

[0382] (MSC: untreated mesenchymal stem cells, Exe4-MSC: exendin-4 treated mesenchymal stem cells)

[0383] As can be seen from Table 3 and FIG. 6, exosome-derived RNA was produced in an amount approximately 5.6 times greater in mesenchymal stem cells pretreated with the stem cell pretreatment substance [exendin-4] than in mesenchymal stem cells not treated with any substance.

[0384] From the above results, it can be seen that when mesenchymal stem cells are treated with the stem cell pretreatment substance according to the present invention, not only the number of exosomes but also the content of exosome-derived RNA (exosomal RNA) is increased.

[0385] Experimental example 3. Enhancement of stem cell function by PMA treatment

[0386] 3-1. Increase in stem cell proliferation rate by PMA treatment

[0387] In a 96-well plate containing 100uL of culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100x) (Gibco, Cat no. 11140-050)], the mesenchymal stem cells and the mesenchymal stem cells pretreated with PMA of the above example were seeded at 3000 cells per well and then cultured in a CO2 incubator for 24 hours. Then, CCK solution (10uL / well) was added and the cells were cultured in a CO2 incubator for 4 hours, and the absorbance was measured at a wavelength of 450nm (420~480nm) to confirm the proliferation rate.

[0388] As can be seen from Figure 7, the cell proliferation rate of mesenchymal stem cells pretreated with the stem cell pretreatment substance [PMA] increased by approximately 220%, from 1 to 2.2, compared to mesenchymal stem cells that were not treated with any substance.

[0389] 3-2. Enhancement of stemness of stem cells by PMA treatment

[0390] Mesenchymal stem cells and mesenchymal stem cells pretreated with PMA from the above example were seeded at 1000 cells per dish in 100 mm cell culture dishes containing 10 mL of culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100x) (Gibco, Cat no. 11140-050)] and cultured for 21 days. The dishes with the cultured cells were washed twice with PBS and fixed with 95% methanol at room temperature for about 2 minutes. The fixed cells were washed three times with PBS, stained for 5 minutes with 0.5% crystal violet solution [crystal violet (Sigma, C-3886, USA) 5 g, methanol 100 ml], washed with water, and dried at room temperature. The number of colonies consisting of 50 or more cells in each dish was counted and compared.

[0391] As can be seen from Figures 8A and 8B, the number of CFU-F colonies in mesenchymal stem cells pretreated with the stem cell pretreatment substance [PMA] (285.7 cells) increased by approximately 996.5% compared to mesenchymal stem cells not treated with any substance (28.6 cells).

[0392] Experimental Example 4. Increase in exosome production efficiency by PMA treatment

[0393] 4-1. Increase in exosome numbers by PMA treatment

[0394] The number of exosomes isolated in the above example was confirmed by a nanoparticle tracking assay (NanoSight NS300, Malvern). For comparison, the number of exosomes isolated in the same manner as in the example was confirmed, except that no pretreatment substance was used. The results are shown in Table 4, Figures 9 and 10 below.

[0395] [Table 4]

[0396] (MSC: untreated mesenchymal stem cells, PMA MSC: PMA-treated mesenchymal stem cells)

[0397] As can be seen from Table 4 and FIG. 10, the number of exosomes produced was about 5.2 times higher in mesenchymal stem cells pretreated with the stem cell pretreatment substance [PMA] than in mesenchymal stem cells not treated with any substance.

[0398] These results demonstrate that treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present invention increases the number of exosomes produced by the mesenchymal stem cells.

[0399] 4-2. Increase in exosome-derived protein amount by PMA treatment

[0400] Proteins were isolated from the exosomes isolated in the above examples using an exosome protein isolation kit (Total exosome RNA and protein isolation kit, Invitrogen), and the absorbance was measured at 595 nm using Bradford analysis to quantify the proteins. For comparison, proteins were isolated and quantified from exosomes isolated in the same manner as in the examples, except that no pretreatment substances were used. The results are shown in Table 5 and FIG. 11 below.

[0401] [Table 5]

[0402] (MSC: untreated mesenchymal stem cells, PMA MSC: PMA-treated mesenchymal stem cells)

[0403] As can be seen from Table 5 and FIG. 11, exosome-derived proteins were produced in approximately 5-fold greater amounts in mesenchymal stem cells pretreated with the stem cell pretreatment substance [PMA] than in mesenchymal stem cells not treated with any substance.

[0404] From the above results, it can be seen that when mesenchymal stem cells are treated with the stem cell pretreatment material according to the present invention, not only the number of exosomes but also the content of exosome-derived proteins increases.

[0405] 4-3. Increase in exosome-derived RNA amount by PMA treatment

[0406] Using an RNA isolation kit (Total exosome RNA and protein isolation kit, Invitrogen), total exosome RNA was isolated from the exosomes isolated in the above example, and the RNA concentration was measured using Nanodrop. For comparison, total exosome RNA was isolated from exosomes isolated in the same manner as in the example, except that no pretreatment substance was used, and the concentration was measured. The results are shown in Table 6 and Figure 12 below.

[0407] [Table 6]

[0408] (MSC: untreated mesenchymal stem cells, PMA MSC: PMA-treated mesenchymal stem cells)

[0409] As can be seen from Table 6 and FIG. 12, exosome-derived RNA was produced in an amount about four times greater in mesenchymal stem cells pretreated with the stem cell pretreatment substance [PMA] than in mesenchymal stem cells not treated with any substance.

[0410] From the above results, it can be seen that when mesenchymal stem cells are treated with the stem cell pretreatment substance according to the present invention, not only the number of exosomes but also the content of exosome-derived RNA (exosomal RNA) is increased.

[0411] Experimental Example 5. Enhancement of stem cell function by interferon-γ treatment

[0412] 5-1. Increase in stem cell proliferation rate by interferon-γ treatment

[0413] Mesenchymal stem cells and mesenchymal stem cells pretreated with interferon-γ of the above example were seeded at 3000 cells per well in a 96-well plate containing 100uL culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100x) (Gibco, Cat no. 11140-050)] and cultured in a CO2 incubator for 24 hours. CCK solution (10uL / well) was then added and cultured in a CO2 incubator for 4 hours, after which the absorbance was measured at 450nm (420~480nm) to confirm the proliferation rate.

[0414] As can be seen from Figure 13, the cell proliferation rate of mesenchymal stem cells pretreated with the stem cell pretreatment substance [interferon-γ] increased by approximately 242%, from 1 to 2.42, compared to mesenchymal stem cells that were not treated with any substance.

[0415] 5-2. Enhancement of stemness of stem cells by interferon-γ treatment

[0416] Mesenchymal stem cells and mesenchymal stem cells pretreated with interferon-γ of the above example were seeded at 1000 cells per dish in a 100 mm cell culture dish containing 10 mL of culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100x) (Gibco, Cat no. 11140-050)] and cultured for 21 days. The dish with the cultured cells was washed twice with PBS and fixed with 95% methanol at room temperature for about 2 minutes. The fixed cells were washed three times with PBS, stained for 5 minutes with 0.5% crystal violet solution [crystal violet (Sigma, C-3886, USA) 5 g, methanol 100 ml], washed with water, and dried at room temperature. The numbers of colonies consisting of 50 or more cells in each dish were counted and compared.

[0417] As can be seen from Figures 14A and 14B, the number of CFU-F colonies in mesenchymal stem cells pretreated with the stem cell pretreatment substance [interferon-γ] (136 cells) increased by approximately 476% compared to mesenchymal stem cells not treated with any substance (28.6 cells).

[0418] Experimental Example 6. Increase in exosome production efficiency by interferon-γ treatment

[0419] 6-1. Increase in exosome number by interferon-γ treatment

[0420] The number of exosomes isolated in the above example was confirmed by a nanoparticle tracking assay (NanoSight NS300, Malvern). For comparison, the number of exosomes isolated in the same manner as in the example was confirmed, except that no pretreatment substance was used. The results are shown in Table 7, Figure 15, and Figure 16 below.

[0421] [Table 7]

[0422] (MSC: untreated mesenchymal stem cells, IFNγ MSC: interferon-γ treated mesenchymal stem cells)

[0423] As can be seen from Table 7 and FIG. 16, the number of exosomes produced was approximately 6.7 times higher in mesenchymal stem cells pretreated with the stem cell pretreatment substance [interferon-γ] than in mesenchymal stem cells not treated with any substance.

[0424] These results demonstrate that treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present invention increases the number of exosomes produced by the mesenchymal stem cells.

[0425] 6-2. Increase in exosome-derived protein amount by interferon-γ treatment

[0426] Proteins were isolated from the exosomes isolated in the above examples using an exosome protein isolation kit (Total exosome RNA and protein isolation kit, Invitrogen), and the absorbance was measured at 595 nm using Bradford analysis to quantify the proteins. For comparison, proteins were isolated and quantified from exosomes isolated in the same manner as in the examples, except that no pretreatment substances were used. The results are shown in Table 8 and FIG. 17 below.

[0427] [Table 8]

[0428] (MSC: untreated mesenchymal stem cells, IFNγ MSC: interferon-γ treated mesenchymal stem cells)

[0429] As can be seen from Table 8 and FIG. 17, exosome-derived proteins were produced in an amount approximately 6.2 times greater in mesenchymal stem cells pretreated with the stem cell pretreatment substance [interferon-γ] than in mesenchymal stem cells not treated with any substance.

[0430] From the above results, it can be seen that when mesenchymal stem cells are treated with the stem cell pretreatment material according to the present invention, not only the number of exosomes but also the content of exosome-derived proteins increases.

[0431] 6-3. Increase in exosome-derived RNA amount by interferon-γ treatment

[0432] Using an RNA isolation kit (Total exosome RNA and protein isolation kit, Invitrogen), total exosome RNA was isolated from the exosomes isolated in the above example, and the RNA concentration was measured using Nanodrop. At this time, for comparison, total exosome RNA was isolated from exosomes isolated in the same manner as in the example, except that no pretreatment substance was used, and the concentration was measured. The results are shown in Table 9 and Figure 18 below.

[0433] [Table 9]

[0434] (MSC: untreated mesenchymal stem cells, IFNγ MSC: interferon-γ treated mesenchymal stem cells)

[0435] As can be seen from Table 9 and FIG. 18, exosome-derived RNA was produced in an amount approximately 5.0 times greater in mesenchymal stem cells pretreated with the stem cell pretreatment substance [interferon-γ] than in mesenchymal stem cells not treated with any substance.

[0436] From the above results, it can be seen that when mesenchymal stem cells are treated with the stem cell pretreatment substance according to the present invention, not only the number of exosomes but also the content of exosome-derived RNA (exosomal RNA) is increased.

[0437] Experimental Example 7. Enhancement of stem cell function by tetrandrine treatment

[0438] 7-1. Increase in stem cell proliferation rate by tetrandrine treatment

[0439] Mesenchymal stem cells and mesenchymal stem cells pretreated with tetrandrine in the above example were seeded at 3000 cells per well in a 96-well plate containing 100uL of culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100x) (Gibco, Cat no. 11140-050)] and then cultured in a CO2 incubator for 24 hours. CCK solution (10uL / well) was then added and cultured in a CO2 incubator for 4 hours, after which the absorbance was measured at 450nm (420~480nm) to confirm the proliferation rate.

[0440] As can be seen from Figure 19, the cell proliferation rate of mesenchymal stem cells pretreated with the stem cell pretreatment substance [tetrandrine] increased by approximately 260%, from 1 to 2.6, compared to mesenchymal stem cells that were not treated with any substance.

[0441] 7-2. Enhancement of stemness of stem cells by tetrandrine treatment

[0442] Mesenchymal stem cells and mesenchymal stem cells pretreated with tetrandrine from the above example were seeded at 1000 cells per dish in 100 mm cell culture dishes containing 10 mL of culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100x) (Gibco, Cat no. 11140-050)] and cultured for 21 days. The dishes with the cultured cells were washed twice with PBS and fixed with 95% methanol at room temperature for about 2 minutes. The fixed cells were washed three times with PBS, stained for 5 minutes with 0.5% crystal violet solution [crystal violet (Sigma, C-3886, USA) 5 g, methanol 100 ml], washed with water, and dried at room temperature. The number of colonies consisting of 50 or more cells in each dish was counted and compared.

[0443] As can be seen from Figures 20A and 20B, the number of CFU-F colonies in mesenchymal stem cells pretreated with the stem cell pretreatment substance [tetrandrine] (121.3 cells) increased by approximately 423% compared to mesenchymal stem cells not treated with any substance (28.7 cells).

[0444] Experimental Example 8. Increase in exosome production efficiency by tetrandrine treatment

[0445] 8-1. Increase in exosome number by tetrandrine treatment

[0446] The number of exosomes isolated in the above example was confirmed by a nanoparticle tracking assay (NanoSight NS300, Malvern). For comparison, the number of exosomes isolated in the same manner as in the example was confirmed, except that no pretreatment substance was used. The results are shown in Table 10, Figure 21 and Figure 22 below.

[0447] [Table 10]

[0448] (MSC: untreated mesenchymal stem cells, Tet MSC: tetrandrine-treated mesenchymal stem cells)

[0449] As can be seen from Table 10 and FIG. 22, the number of exosomes produced was approximately 4.3 times higher in mesenchymal stem cells pretreated with the stem cell pretreatment substance [tetrandrine] than in mesenchymal stem cells not treated with any substance.

[0450] These results demonstrate that treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present invention increases the number of exosomes produced by the mesenchymal stem cells.

[0451] 8-2. Increase in exosome-derived protein amount by tetrandrine treatment

[0452] Proteins were isolated from the exosomes isolated in the above examples using an exosome protein isolation kit (Total exosome RNA and protein isolation kit, Invitrogen), and the absorbance was measured at 595 nm using Bradford analysis to quantify the proteins. For comparison, proteins were isolated and quantified from exosomes isolated in the same manner as in the examples, except that no pretreatment substances were used. The results are shown in Table 11 and FIG. 23 below.

[0453] [Table 11]

[0454] (MSC: untreated mesenchymal stem cells, Tet MSC: tetrandrine-treated mesenchymal stem cells)

[0455] As can be seen from Table 11 and FIG. 23, exosome-derived proteins were produced in approximately 4.5 times greater amounts in mesenchymal stem cells pretreated with the stem cell pretreatment substance [tetrandrine] than in mesenchymal stem cells not treated with any substance.

[0456] From the above results, it can be seen that when mesenchymal stem cells are treated with the stem cell pretreatment material according to the present invention, not only the number of exosomes but also the content of exosome-derived proteins increases.

[0457] 8-3. Increase in exosome-derived RNA by tetrandrine treatment

[0458] Using an RNA isolation kit (Total exosome RNA and protein isolation kit, Invitrogen), total exosome RNA was isolated from the exosomes isolated in the above examples, and the RNA concentration was measured using Nanodrop. For comparison, total exosome RNA was isolated from exosomes isolated in the same manner as in the examples, except that no pretreatment substances were used, and the concentration was measured. The results are shown in Table 12 and Figure 24 below.

[0459] [Table 12]

[0460] (MSC: untreated mesenchymal stem cells, Tet MSC: tetrandrine-treated mesenchymal stem cells)

[0461] As can be seen from Table 12 and FIG. 24, exosome-derived RNA was produced in an amount approximately 4.4 times greater in mesenchymal stem cells pretreated with the stem cell pretreatment substance [tetrandrine] than in mesenchymal stem cells not treated with any substance.

[0462] From the above results, it can be seen that when mesenchymal stem cells are treated with the stem cell pretreatment substance according to the present invention, not only the number of exosomes but also the content of exosome-derived RNA (exosomal RNA) is increased.

[0463] Experimental Example 9. Enhancement of stem cell function by hyaluronic acid treatment

[0464] 9-1. Increase in stem cell proliferation rate by hyaluronic acid treatment

[0465] In a 96-well plate containing 100uL of culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100x) (Gibco, Cat no. 11140-050)], mesenchymal stem cells and mesenchymal stem cells pretreated with hyaluronic acid of the above example were seeded at 3000 cells per well, and then cultured in a CO2 incubator for 24 hours. Then, CCK solution (10uL / well) was added, and the cells were cultured in a CO2 incubator for 4 hours, and the absorbance was measured at 450nm (420~480nm) wavelength to confirm the proliferation rate.

[0466] As can be seen from Figure 25, compared to mesenchymal stem cells that were not treated with any substance, the cell proliferation rate of mesenchymal stem cells pretreated with the stem cell pretreatment substance [hyaluronic acid] increased by approximately 364%, from 1 to 3.6.

[0467] 9-2. Enhancement of stemness of stem cells by hyaluronic acid treatment

[0468] Mesenchymal stem cells and mesenchymal stem cells pretreated with hyaluronic acid of the above example were seeded at 1000 cells per dish in a 100 mm cell culture dish containing 10 mL of culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100x) (Gibco, Cat no. 11140-050)] and cultured for 21 days. The dish with the cultured cells was washed twice with PBS and fixed with 95% methanol at room temperature for about 2 minutes. The fixed cells were washed three times with PBS, stained for 5 minutes with 0.5% crystal violet solution [crystal violet (Sigma, C-3886, USA) 5g, methanol 100ml], washed with water, and dried at room temperature. The numbers of colonies consisting of 50 or more cells in each dish were counted and compared.

[0469] As can be seen from Figures 26A and 26B, the number of CFU-F colonies in mesenchymal stem cells pretreated with the stem cell pretreatment substance [hyaluronic acid] (167.6 cells) increased by approximately 586% compared to mesenchymal stem cells not treated with any substance (28.6 cells).

[0470] Experimental Example 10. Increase in exosome production efficiency by hyaluronic acid treatment

[0471] 10-1. Increase in exosome number by hyaluronic acid treatment

[0472] The number of exosomes isolated in the above example was confirmed by a nanoparticle tracking assay (NanoSight NS300, Malvern). For comparison, the number of exosomes isolated in the same manner as in the example was confirmed, except that no pretreatment substance was used. The results are shown in Table 13, Figure 27, and Figure 28 below.

[0473] [Table 13]

[0474] (MSC: untreated mesenchymal stem cells, HA MSC: hyaluronic acid-treated mesenchymal stem cells)

[0475] As can be seen from Table 13 and FIG. 28, the number of exosomes produced was approximately 5.2 times greater in mesenchymal stem cells pretreated with the stem cell pretreatment substance [hyaluronic acid] than in mesenchymal stem cells not treated with any substance.

[0476] These results demonstrate that the number of exosomes produced by mesenchymal stem cells increases when the stem cell pretreatment substance according to the present invention is treated with the mesenchymal stem cells.

[0477] 10-2. Increase in exosome-derived protein amount by hyaluronic acid treatment

[0478] Proteins were isolated from the exosomes isolated in the above examples using an exosome protein isolation kit (Total exosome RNA and protein isolation kit, Invitrogen), and the absorbance was measured at 595 nm using Bradford analysis to quantify the proteins. For comparison, proteins were isolated and quantified from exosomes isolated in the same manner as in the examples, except that no pretreatment substances were used. The results are shown in Table 14 and FIG. 29 below.

[0479] [Table 14]

[0480] (MSC: untreated mesenchymal stem cells, HA MSC: hyaluronic acid-treated mesenchymal stem cells)

[0481] As can be seen from Table 14 and FIG. 29, exosome-derived proteins were produced in an amount approximately 5.35 times greater in mesenchymal stem cells pretreated with the stem cell pretreatment substance [hyaluronic acid] than in mesenchymal stem cells not treated with any substance.

[0482] From the above results, it can be seen that when mesenchymal stem cells are treated with the stem cell pretreatment material according to the present invention, not only the number of exosomes but also the content of exosome-derived proteins increases.

[0483] 10-3. Increase in exosome-derived RNA amount by hyaluronic acid treatment

[0484] Using an RNA isolation kit (Total exosome RNA and protein isolation kit, Invitrogen), total exosome RNA was isolated from the exosomes isolated in the above examples, and the RNA concentration was measured using Nanodrop. At this time, for comparison, total exosome RNA was isolated from exosomes isolated in the same manner as in the examples, except that no pretreatment substance was used, and the concentration was measured. The results are shown in Table 15 and Figure 30 below.

[0485] [Table 15]

[0486] (MSC: untreated mesenchymal stem cells, HA MSC: hyaluronic acid-treated mesenchymal stem cells)

[0487] As can be seen from Table 15 and FIG. 30, exosome-derived RNA was produced in an amount approximately 5.3 times greater in mesenchymal stem cells pretreated with the stem cell pretreatment substance [hyaluronic acid] than in mesenchymal stem cells not treated with any substance.

[0488] From the above results, it can be seen that when mesenchymal stem cells are treated with the stem cell pretreatment substance according to the present invention, not only the number of exosomes but also the content of exosome-derived RNA (exosomal RNA) is increased.

[0489] Experimental Example 11. Enhancement of stem cell function by P substance treatment

[0490] 11-1. Increase in stem cell proliferation rate by P treatment

[0491] Mesenchymal stem cells and mesenchymal stem cells pretreated with the above-mentioned P substance were seeded at 3000 cells per well in a 96-well plate containing 100uL of culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100x) (Gibco, Cat no. 11140-050)] and cultured in a CO2 incubator for 24 hours. CCK solution (10uL / well) was then added and cultured in a CO2 incubator for 4 hours, after which the absorbance was measured at 450nm (420~480nm) to confirm the proliferation rate.

[0492] As can be seen from Figure 31, the cell proliferation rate of mesenchymal stem cells pretreated with stem cell pretreatment substance [substance P] increased by approximately 290%, from 1 to 2.9, compared to mesenchymal stem cells that were not treated with any substance.

[0493] 11-2. Enhancement of stemness of stem cells by treatment with P substances

[0494] Mesenchymal stem cells and mesenchymal stem cells pretreated with the above-mentioned P substance were seeded at 1000 cells per dish in 100 mm cell culture dishes containing 10 mL of culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100x) (Gibco, Cat no. 11140-050)] and cultured for 21 days. The dishes with the cultured cells were washed twice with PBS and fixed with 95% methanol at room temperature for about 2 minutes. The fixed cells were washed three times with PBS, stained with 0.5% crystal violet solution [crystal violet (Sigma, C-3886, USA) 5 g, methanol 100 ml] for 5 minutes, washed with water, and dried at room temperature. The number of colonies consisting of 50 or more cells in each dish was counted and compared.

[0495] As can be seen from Figures 32A and 32B, the number of CFU-F colonies in mesenchymal stem cells (116 cells) pretreated with stem cell pretreatment substance [substance P] increased by approximately 404% compared to mesenchymal stem cells (28.7 cells) that were not treated with any substance.

[0496] Experimental Example 12. Increase in exosome production efficiency by P substance treatment

[0497] 12-1. Increase in exosome number by P substance treatment

[0498] The number of exosomes isolated in the above example was confirmed by a nanoparticle tracking assay (NanoSight NS300, Malvern). For comparison, the number of exosomes isolated in the same manner as in the example was confirmed, except that no pretreatment substance was used. The results are shown in Table 16, Figure 33, and Figure 34 below.

[0499] [Table 16]

[0500] (MSC: untreated mesenchymal stem cells, SubsP MSC: P substance-treated mesenchymal stem cells)

[0501] As can be seen from Table 16 and FIG. 34, the number of exosomes produced was approximately 4.3 times higher in mesenchymal stem cells pretreated with the stem cell pretreatment substance [substance P] than in mesenchymal stem cells not treated with any substance.

[0502] These results demonstrate that treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present invention increases the number of exosomes produced by the mesenchymal stem cells.

[0503] 12-2. Increase in exosome-derived protein amount by P substance treatment

[0504] Proteins were isolated from the exosomes isolated in the above examples using an exosome protein isolation kit (Total exosome RNA and protein isolation kit, Invitrogen), and the absorbance was measured at 595 nm using Bradford analysis to quantify the proteins. For comparison, proteins were isolated and quantified from exosomes isolated in the same manner as in the examples, except that no pretreatment substances were used. The results are shown in Table 17 and FIG. 35 below.

[0505] [Table 17]

[0506] (MSC: untreated mesenchymal stem cells, SubsP MSC: P substance-treated mesenchymal stem cells)

[0507] As can be seen from Table 17 and FIG. 35, exosome-derived proteins were produced in approximately 5.2-fold greater amounts in mesenchymal stem cells pretreated with the stem cell pretreatment substance [substance P] than in mesenchymal stem cells not treated with any substance.

[0508] From the above results, it can be seen that when mesenchymal stem cells are treated with the stem cell pretreatment material according to the present invention, not only the number of exosomes but also the content of exosomal proteins increases.

[0509] 12-3. Increase in exosome-derived RNA by P treatment

[0510] Using an RNA isolation kit (Total exosome RNA and protein isolation kit, Invitrogen), total exosome RNA was isolated from the exosomes isolated in the above examples, and the RNA concentration was measured using Nanodrop. For comparison, total exosome RNA was isolated from exosomes isolated in the same manner as in the examples, except that no pretreatment substance was used, and the concentration was measured. The results are shown in Table 18 and Figure 36 below.

[0511] [Table 18]

[0512] (MSC: untreated mesenchymal stem cells, SubsP MSC: P substance-treated mesenchymal stem cells)

[0513] As can be seen from Table 18 and FIG. 36, exosome-derived RNA was produced in an amount approximately 4.9 times greater in mesenchymal stem cells pretreated with the stem cell pretreatment substance [substance P] than in mesenchymal stem cells not treated with any substance.

[0514] From the above results, it can be seen that when mesenchymal stem cells are treated with the stem cell pretreatment substance according to the present invention, not only the number of exosomes but also the content of exosome-derived RNA (exosomal RNA) is increased.

[0515] Experimental Example 13. Enhancement of stem cell function by resveratrol treatment

[0516] 13-1. Increase in stem cell proliferation rate by resveratrol treatment

[0517] In a 96-well plate containing 100uL of culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100x) (Gibco, Cat no. 11140-050)], mesenchymal stem cells and mesenchymal stem cells pretreated with resveratrol of the above example were seeded at 3000 cells per well and then cultured in a CO2 incubator for 24 hours. Then, CCK solution (10uL / well) was added and the cells were cultured in a CO2 incubator for 4 hours, and the absorbance was measured at 450nm (420~480nm) wavelength to confirm the proliferation rate.

[0518] As can be seen from Figure 37, the cell proliferation rate of mesenchymal stem cells pretreated with the stem cell pretreatment substance [resveratrol] increased by approximately 322%, from 1 to 3.22, compared to mesenchymal stem cells that were not treated with any substance.

[0519] 13-2. Enhancement of stemness of stem cells by resveratrol treatment

[0520] Mesenchymal stem cells and mesenchymal stem cells pretreated with resveratrol of the above example were seeded at 1000 cells per dish in a 100 mm cell culture dish containing 10 mL of culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100x) (Gibco, Cat no. 11140-050)] and cultured for 21 days. The dish with the cultured cells was washed twice with PBS and fixed with 95% methanol at room temperature for about 2 minutes. The fixed cells were washed three times with PBS, stained for 5 minutes with 0.5% crystal violet solution [crystal violet (Sigma, C-3886, USA) 5 g, methanol 100 ml], washed with water, and dried at room temperature. The numbers of colonies consisting of 50 or more cells in each dish were counted and compared.

[0521] As can be seen from Figures 38A and 38B, the number of CFU-F colonies in mesenchymal stem cells (311 cells) pretreated with the stem cell pretreatment substance [resveratrol] increased by approximately 1087% compared to mesenchymal stem cells (28.6 cells) that were not treated with any substance.

[0522] Experimental Example 14. Increase in exosome production efficiency by resveratrol treatment

[0523] 14-1. Increase in exosome number by resveratrol treatment

[0524] The number of exosomes isolated in the above example was confirmed by a nanoparticle tracking assay (NanoSight NS300, Malvern). For comparison, the number of exosomes isolated in the same manner as in the example was confirmed, except that no pretreatment substance was used. The results are shown in Table 19, Figure 39, and Figure 40 below.

[0525] [Table 19]

[0526] (MSC: untreated mesenchymal stem cells, Resv MSC: resveratrol-treated mesenchymal stem cells)

[0527] As can be seen from Table 19 and FIG. 40, the number of exosomes produced was approximately 5.2 times greater in mesenchymal stem cells pretreated with the stem cell pretreatment substance [resveratrol] than in mesenchymal stem cells not treated with any substance.

[0528] These results demonstrate that treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present invention increases the number of exosomes produced by the mesenchymal stem cells.

[0529] 14-2. Increase in exosome-derived protein amount by resveratrol treatment

[0530] Proteins were isolated from the exosomes isolated in the above examples using an exosome protein isolation kit (Total exosome RNA and protein isolation kit, Invitrogen), and the absorbance was measured at 595 nm using Bradford analysis to quantify the proteins. For comparison, proteins were isolated and quantified from exosomes isolated in the same manner as in the examples, except that no pretreatment substances were used. The results are shown in Table 20 and Figure 41 below.

[0531] [Table 20]

[0532] (MSC: untreated mesenchymal stem cells, Resv MSC: resveratrol-treated mesenchymal stem cells)

[0533] As can be seen from Table 20 and FIG. 41, exosome-derived proteins were produced in an amount approximately 5.4 times greater in mesenchymal stem cells pretreated with the stem cell pretreatment substance [resveratrol] than in mesenchymal stem cells not treated with any substance.

[0534] From the above results, it can be seen that when mesenchymal stem cells are treated with the stem cell pretreatment material according to the present invention, not only the number of exosomes but also the content of exosomal proteins increases.

[0535] 14-3. Increase in exosome-derived RNA by resveratrol treatment

[0536] Using an RNA isolation kit (Total exosome RNA and protein isolation kit, Invitrogen), total exosome RNA was isolated from the exosomes isolated in the above Example, and the RNA concentration was measured using Nanodrop. At this time, for comparison, total exosome RNA was isolated from the exosomes isolated in the same manner as in the Example, except that no pretreatment substance was used, and the concentration was measured. The results are shown in Table 21 and Figure 42 below.

[0537] [Table 21]

[0538] (MSC: untreated mesenchymal stem cells, Resv MSC: resveratrol-treated mesenchymal stem cells)

[0539] As can be seen from Table 21 and FIG. 42, exosome-derived RNA was produced in an amount approximately 4.7 times greater in mesenchymal stem cells pretreated with the stem cell pretreatment substance [resveratrol] than in mesenchymal stem cells not treated with any substance.

[0540] From the above results, it can be seen that when mesenchymal stem cells are treated with the stem cell pretreatment substance according to the present invention, not only the number of exosomes but also the content of exosome-derived RNA (exosomal RNA) is increased.

[0541] Experimental Example 15. Enhancement of stem cell function by Lanifibranol treatment

[0542] 15-1. Increase in stem cell proliferation rate by Lanifibranor treatment

[0543] Mesenchymal stem cells and mesenchymal stem cells pretreated with Lanifibranol in the above Example were seeded at 3000 cells per well in a 96-well plate containing 100uL culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100x) (Gibco, Cat no. 11140-050)] and cultured in a CO2 incubator for 24 hours. CCK solution (10uL / well) was then added and cultured in a CO2 incubator for 4 hours, after which the absorbance was measured at 450nm (420~480nm) wavelength to confirm the proliferation rate.

[0544] As can be seen from Figure 43, the cell proliferation rate of mesenchymal stem cells pretreated with the stem cell pretreatment substance [Lanifibranor] increased by approximately 181%, from 1 to 1.81, compared to mesenchymal stem cells that were not treated with any substance.

[0545] 15-2. Enhancement of stemness of stem cells by Lanifibranol treatment

[0546] Mesenchymal stem cells and mesenchymal stem cells pretreated with lanifibranol in the above Example were seeded at 1000 cells per dish in a 100 mm cell culture dish containing 10 mL of culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100x) (Gibco, Cat no. 11140-050)] and cultured for 21 days. The dish with the cultured cells was washed twice with PBS and fixed with 95% methanol at room temperature for about 2 minutes. The fixed cells were washed three times with PBS, stained for 5 minutes with 0.5% crystal violet solution [crystal violet (Sigma, C-3886, USA) 5 g, methanol 100 ml], washed with water, and dried at room temperature. The numbers of colonies consisting of 50 or more cells in each dish were counted and compared.

[0547] As can be seen from Figures 44A and 44B, the number of CFU-F colonies in mesenchymal stem cells (56.7 cells) pretreated with the stem cell pretreatment substance [Lanifibranor] increased by approximately 142% compared to mesenchymal stem cells (40 cells) that were not treated with any substance.

[0548] Experimental Example 16. Increase in exosome production efficiency by Lanifibranol treatment

[0549] 16-1. Increase in exosome number by Lanifibranol treatment

[0550] The number of exosomes isolated in the above example was confirmed by a nanoparticle tracking assay (NanoSight NS300, Malvern). For comparison, the number of exosomes isolated in the same manner as in the example was confirmed, except that no pretreatment substance was used. The results are shown in Table 22, Figure 45, and Figure 46 below.

[0551] [Table 22]

[0552] (MSC: untreated mesenchymal stem cells, Lani-MSC-Exo: lanifibranor-treated mesenchymal stem cells) As can be seen from Table 22 and FIG. 46, the number of exosomes produced was approximately 4.95 times higher in mesenchymal stem cells pretreated with the stem cell pretreatment substance [lanifibranol] than in mesenchymal stem cells not treated with any substance.

[0553] These results demonstrate that treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present invention increases the number of exosomes produced by the mesenchymal stem cells.

[0554] 16-2. Increase in exosome-derived protein amount by Lanifibranol treatment

[0555] Proteins were isolated from the exosomes isolated in the above examples using an exosome protein isolation kit (Total exosome RNA and protein isolation kit, Invitrogen), and the absorbance was measured at 595 nm using Bradford analysis to quantify the proteins. For comparison, proteins were isolated and quantified from exosomes isolated in the same manner as in the examples, except that no pretreatment substances were used. The results are shown in Table 23 and Figure 47 below.

[0556] [Table 23]

[0557] (MSC: untreated mesenchymal stem cells, Lani-MSC-Exo: lanifibranor-treated mesenchymal stem cells)

[0558] As can be seen from Table 23 and FIG. 47, exosome-derived proteins were produced in an amount approximately 4.6 times greater in mesenchymal stem cells pretreated with the stem cell pretreatment substance [lanifibranol] than in mesenchymal stem cells not treated with any substance.

[0559] From the above results, it can be seen that when mesenchymal stem cells are treated with the stem cell pretreatment material according to the present invention, not only the number of exosomes but also the content of exosome-derived proteins increases.

[0560] 16-3. Increase in exosome-derived RNA by Lanifibranol treatment

[0561] Using an RNA isolation kit (Total exosome RNA and protein isolation kit, Invitrogen), total exosome RNA was isolated from the exosomes isolated in the above Example, and the RNA concentration was measured using Nanodrop. At this time, for comparison, total exosome RNA was isolated from the exosomes isolated in the same manner as in the Example, except that no pretreatment substance was used, and the concentration was measured. The results are shown in Table 24 and Figure 48 below.

[0562] [Table 24]

[0563] (MSC: untreated mesenchymal stem cells, Lani-MSC-Exo: lanifibranor-treated mesenchymal stem cells)

[0564] As can be seen from Table 24 and FIG. 48, exosome-derived RNA was produced in an amount approximately 4.1 times greater in mesenchymal stem cells pretreated with a stem cell pretreatment substance (lanifibranol) than in mesenchymal stem cells not treated with any substance.

[0565] From the above results, it can be seen that when mesenchymal stem cells are treated with the stem cell pretreatment substance according to the present invention, not only the number of exosomes but also the content of exosome-derived RNA (exosomal RNA) is increased.

[0566] Conclusion

[0567] It has been found that when mesenchymal stem cells are treated with the stem cell pretreatment substance of the present invention (Exendin-4, Phorbol 12-myristate 13-acetate (PMA), Interferon-gamma, Tetrandrine, Hyaluronic acid, Substance P, Resveratrol, or Lanifibranor), the stemness of the stem cells is increased. It has also been found that when mesenchymal stem cells are treated with the stem cell pretreatment substance of the present invention, in addition to the number of exosomes, the production amount of exosomal protein and the content of exosomal RNA are also increased.

[0568] Therefore, the stem cell pretreatment substance according to the present invention (Exendin-4, Phorbol 12-myristate 13-acetate (PMA), Interferon-γ, Tetrandrine, Hyaluronic acid, Substance P, Resveratrol, or Lanifibranor) not only enhances the stem cell potential of stem cells but also promotes the production of stem cell-derived exosomes, and is therefore expected to be used in the production of stem cells with excellent functionality and in the mass production of exosomes. [Industrial Applicability]

[0569] The present invention relates to a composition for promoting the production of exosomes derived from stem cells and enhancing stem cell potential.

Claims

1. A composition for promoting the production of stem cell-derived exosomes, comprising interferon-gamma, The composition for promoting the production of stem cell-derived exosomes, wherein the stem cells are mesenchymal stem cells derived from induced pluripotent stem cells (iPSCs).

2. The composition for promoting the production of stem cell-derived exosomes according to claim 1 , wherein the induced pluripotent stem cells are induced pluripotent stem cells of human or animal tissue origin.

3. The composition for promoting the production of stem cell-derived exosomes according to claim 2, wherein the human or animal tissue is selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta.

4. A composition for enhancing stemness of stem cells, comprising interferon-gamma, The composition for enhancing stem cell potency of stem cells, wherein the stem cells are mesenchymal stem cells derived from induced pluripotent stem cells (iPSCs).

5. A culture step of culturing induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells in a cell culture medium containing interferon-gamma; a further culturing step of washing the cultured cells obtained by the culturing step and then culturing them further in a cell culture medium; A separation step of isolating exosomes; A method for producing stem cell-derived exosomes, comprising:

6. The method for producing stem cell-derived exosomes according to claim 5 , wherein the cell culture medium comprises exosome-depleted fetal bovine serum.

7. A method for enhancing stemness of stem cells, comprising the step of culturing mesenchymal stem cells derived from induced pluripotent stem cells (iPSCs) in a cell culture medium containing interferon-gamma.

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