A composition containing endoplasmic reticulum derived from human tonsillar stem cells as an active ingredient

Abstract

The present invention relates to a composition containing endoplasmic reticulum derived from human tonsillar stem cells as an active ingredient, which promotes skin regeneration and expression of antioxidant-related genes in senescence-induced fibroblasts and exhibits the effect of suppressing fibroblast senescence.

Description

[Technical Field]

[0001] The present invention relates to a composition containing endoplasmic reticulum derived from human tonsillar stem cells as an active ingredient, in particular a composition for skin regeneration and anti-aging. [Background technology]

[0002] Aging is characterized by the time-dependent loss of the organism's functional and regenerative properties. Factors associated with skin aging damage cells, resulting in a delay in skin renewal and cell proliferation, known as cellular senescence. Cellular senescence is characterized by irreversible arrest of the cell cycle and alterations in the focal adhesive cytoskeleton.

[0003] Cellular aging in skin tissue is induced by a variety of factors, including oxidative stress, mitochondrial dysfunction, and ultraviolet radiation.

[0004] Over the past few decades, many researchers have been working to overcome this problem.

[0005] Recently, much research has focused on the tissue regenerative potential of exosomes to overcome cellular senescence.

[0006] Exosomes, nano-sized biomimetics produced by the endocytic pathway and secreted across the plasma membrane in cells, contain various components including miRNA, mRNA, and proteins, and have been investigated as a potential approach for skin rejuvenation and anti-aging.

[0007] However, despite the potential of exosomes for therapeutic purposes, there are several disadvantages, such as low efficiency, long procedure times and high technical expertise.

[0008] To overcome these drawbacks, many researchers have focused on producing exosome-mimetic nanovesicles directly from somatic cells. These biomimetic nanovesicles can be directly isolated from desired cells by sonication and / or extrusion and have been reported to share similar properties to exosomes. Given these similar properties, cell-derived biomimetic nanovesicles can be utilized for drug delivery, tissue regeneration, and cancer targeting. In particular, nanovesicles derived from human tonsil-derived mesenchymal stem cells (TMSCs) have been reported to attenuate liver fibrosis and inflammation. Furthermore, anticancer pharmaceutical compositions containing nanovesicles derived from human tonsil stem cells are known. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Republic of Korea Patent Publication No. 10-2020-0141868 Summary of the Invention [Problem to be solved by the invention]

[0010] The technical problem to be solved by the present invention is to provide a composition for skin regeneration and anti-aging, which contains endoplasmic reticulum derived from human tonsillar stem cells. [Means for solving the problem]

[0011] According to one aspect of the present invention, there is provided a composition for skin regeneration and anti-aging, which comprises endoplasmic reticulum derived from human tonsillar stem cells as an active ingredient. [Effects of the Invention]

[0012] The composition according to one embodiment of the present invention has skin regeneration and anti-aging effects and can be efficiently manufactured. [Brief explanation of the drawings]

[0013] [Figure 1A] FIG. 1A is a diagram showing the morphology of human tonsillar mesenchymal stem cells produced in Production Example 1 (scale bar=200 μm). [Figure 1B] FIG. 1B shows the expression of surface markers of human tonsillar intermediate layer stem cells produced in Production Example 1. [Figure 2A] Figure 2A shows protein expression and SEM images of nanovesicles prepared from human tonsil-derived mesenchymal stem cells (TMSC-NV). [Figure 2B] FIG. 2B shows the results of dynamic light scattering analysis of nanovesicles produced from human tonsillar mesenchymal stem cells produced in Preparation Example 1. [Figure 3A] Figure 3: Regulation of proliferation and senescence by TMSC-NV treatment in a passage-associated senescence model. Figure 3A shows morphological changes in human dermal fibroblasts (HDFs) (scale bar = 200 μm). [Figure 3B] FIG. 3B shows the proliferation of passage-associated senescent HDFs after TMSC-NV treatment. [Figure 3C] FIG. 3C shows the results of senescence-associated β-galactosidase analysis (scale bar=200 μm). [Figure 3D] FIG. 3D shows the quantitative analysis of the SA-β-galactosidase assay. [Figure 3E] FIG. 3E shows the expression of vinculin in focal attachments of HDFs. [Figure 3F] FIG. 3F shows quantitative data for vinculin expression in focal adhesions. [Figure 4A] Figure 4 shows the regulation of antioxidant genes in the extracellular matrix and HDFs treated with TMSC-NV in a passage-associated senescence model. Figure 4A shows the mRNA expression of COL1, ELASTIN, SOD2, and HMOX1 in passage-associated senescent HDFs. [Figure 4B] FIG. 4B shows the results of immunofluorescence analysis of collagen type 1 in passage-associated senescent HDFs. [Figure 4C] FIG. 4C shows quantitative data from immunofluorescence analysis. [Figure 5A] FIG. 5 shows the modulation of proliferation and senescence by TMSC-NV treatment in a UV-induced senescence model. FIG. 5A shows the morphological changes in HDFs due to treatment (scale bar = 200 μm). [Figure 5B] FIG. 5B shows a proliferation test of UV-induced HDFs after TMSC-NV treatment. [Figure 5C] FIG. 5C shows SA-β-galactosidase analysis of UV-induced senescent HDFs after TMSC-NV treatment (scale bar = 200 μm). [Figure 5D] FIG. 5D shows quantitative data from the SA-β-galactosidase assay. [Figure 5E] FIG. 5E shows vinculin expression in focal attachments of HDFs. [Figure 5F] FIG. 5F shows quantitative data for vinculin expression in focal adhesions. [Figure 6A] Figure 6 shows the regulation of extracellular matrix and antioxidant genes by TMSC-NV treatment in a UV-induced aging model. Figure 6A shows the mRNA expression of COL1, ELASTIN, SOD2, and HMOX1 in UV-induced aging HDFs. [Figure 6B] FIG. 6B shows the results of immunofluorescence analysis of collagen type 1 in UV-induced senescent HDFs. [Figure 6C] FIG. 6C shows quantitative data from immunofluorescence analysis. [Figure 7A] FIG. 7 shows the regulation of proliferation and senescence by CD146+ TMSC-NV treatment in a passage-associated senescence model. FIG. 7A shows the mRNA expression of COL1 and HMOX1 in senescent HDFs. [Figure 7B]FIG. 7B shows the results of senescence-associated β-galactosidase analysis (scale bar=200 μm). [Figure 7C] FIG. 7C shows the quantitative analysis of the SA-β-galactosidase assay. [Figure 8A] Figure 8 shows the results of immunohistochemistry after treatment with CD146+ TMSC-NV in a skin aging model in which human skin tissue was irradiated with ultraviolet B (UVB). Figure 8A shows the results of immunohistochemistry for collagen type 1, collagen type 3, involucrin, and filaggrin. [Figure 8B] FIG. 8B shows the quantitative results of the immunostaining. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention will now be described in more detail.

[0015] A composition for skin regeneration and anti-aging according to one embodiment of the present invention contains endoplasmic reticulum derived from human tonsillar stem cells as an active ingredient.

[0016] As used herein, the term "stem cell" refers to an undifferentiated cell that has the ability to self-renew and differentiate into two or more different types of cells.

[0017] As used herein, the term "active ingredient" refers to an ingredient that exhibits a desired activity alone or that can exhibit a desired activity together with a carrier or the like that is inactive by itself.

[0018] As used herein, the term "nanovesicle" refers to a nano-sized vesicle obtained from adult stem cells, and refers to a vesicle having a nano-size similar to exosomes, which are extracellular vesicles.

[0019] Furthermore, nanovesicles are formed as lipid membranes separated from the outside using phospholipids, the basic structure of biological membranes. Nanovesicles can not only carry water-soluble molecules (including DNA) or drugs, but also attach lipid-soluble drugs or bind positively and negatively charged substances. Phospholipids are amphipathic substances with a molecular structure consisting of an anionic or zwitterionic polar molecular group and two non-polar lipid-soluble chains with various degrees of unsaturation (approximately 16 hydrocarbons). Therefore, phospholipids disperse in water and spontaneously form vesicles.

[0020] In the field of applied science, nanovesicles are used in the cosmetics industry, drug delivery, and as a model for transferring genetic material to cells in vitro. Currently, nanovesicles can capture all water-soluble and lipid-soluble substances, are easy to target to specific tissues, are easy to size and deform, and use phospholipids poses almost no toxicity issues. They can also capture more drugs than other drug carriers.

[0021] As used herein, the term "endoplasmic reticulum" primarily refers to the extracellular endoplasmic reticulum, which may refer to the lipid bilayer-enclosed endoplasmic reticulum secreted by all cells into the external environment.

[0022] Extracellular vesicles are called by various names, such as exosomes, microvesicles, ectosomes, microparticles, membrane vesicles, nanovesicles, and outer membrane vesicles, depending on their origin, secretion mechanism, and size.

[0023] The endoplasmic reticulum may be one selected from extracellular endoplasmic reticulum, microvesicles and nanovesicles, and in particular may be nanovesicles.

[0024] According to one embodiment of the present invention, nanovesicles derived from human tonsillar stem cells express cell surface markers that are specifically expressed in exosomes.

[0025] According to one embodiment of the present invention, the human tonsillar stem cells may be, but are not limited to, human tonsillar mesoderm stem cells.

[0026] According to another embodiment of the present invention, the human tonsillar stem cells may be CD146 positive.

[0027] The nanovesicles may have a diameter of 50 nm to 250 nm, or 30 nm to 200 nm.

[0028] More specifically, the nanovesicles may have a diameter of 30 nm or more, 32 nm or more, 34 nm or more, 36 nm or more, 38 nm or more, 40 nm or more, 42 nm or more, 44 nm or more, 46 nm or more, 48 nm or more, or 50 nm or more, and 200 nm or less, 190 nm or less, 180 nm or less, 170 nm or less, 160 nm or less, 150 nm or less, 140 nm or less, 130 nm or less, 120 nm or less, 110 nm or less, 100 nm or less, 95 nm or less, 90 nm or less, 85 nm or less, 80 nm or less, 75 nm or less, or 70 nm or less. For example, the nanovesicles may have a diameter of 30 to 100 nm, 40 to 80 nm, 50 to 100 nm, or 50 to 80 nm.

[0029] According to one embodiment, the nanovesicles may have a diameter of 40 to 55 nm. More specifically, the nanovesicles may have an average diameter of 40 nm or more, 42 nm or more, 44 nm or more, 46 nm or more, 48 nm or more, or 50 nm or more, and 55 nm or less, 54 nm or less, 53 nm or less, 52 nm or less, 51 nm or less, or 50 nm or less. For example, the nanovesicles may have an average diameter of 42 to 53 nm, 46 to 52 nm, 48 to 52 nm, or 50 nm.

[0030] According to one embodiment of the present invention, the endoplasmic reticulum may additionally contain one or more immunogens selected from the group consisting of CD14, CD34, CD45, CD73, CD90, and CD146.

[0031] According to one embodiment of the present invention, the human tonsillar stem cells can be produced by a method comprising the steps of: digesting human tonsillar tissue with collagenase type 1 and DNase 1; filtering and centrifuging the digested product to remove the supernatant and obtain a cell pellet; and culturing the cells obtained from the cell pellet to obtain human tonsillar stem cells. In one embodiment, the step of digesting human tonsillar tissue with collagenase type 1 and DNase 1 is performed in low-glucose Dulbecco's modified Eagle's medium (DMEM). The step of culturing the cells obtained from the cell pellet can be performed in DMEM containing 10% fetal bovine serum, antibiotics, and antimycotics.

[0032] The nanovesicles can be produced by a method including the steps of suspending subcultured human tonsillar mesenchymal stem cells in a culture medium, centrifuging the suspension to remove the supernatant, resuspending the cell pellet from which the supernatant has been removed, and then passing the resuspended cell pellet through two or more filters with different pore sizes in an extruder.

[0033] The two or more filters having different pore sizes may be used in the order of larger pore size to smaller pore size. For example, the two or more filters having different pore sizes may be composed of a filter having a pore size of 8 to 12 μm, a filter having a pore size of 3 to 7 μm, and a filter having a pore size of 0.2 to 0.6 μm. For example, the two or more filters having different pore sizes may be used in the order of pore sizes of 10 μm, 5 μm, and 0.4 μm.

[0034] Meanwhile, a method for obtaining CD146-positive endoplasmic reticulum may further include the steps of treating the obtained human tonsillar stem cells with an FcR blocking reagent, treating them with CD146 microbeads, and then reacting them, treating them with a magnetic-activated cell sorting (MACS) buffer, centrifuging them to remove the supernatant, and separating CD146-positive and -negative cells using a MACS separator and column, for example, an LS column. According to one embodiment, the step of treating them with CD246 microbeads and then reacting them is performed under light-blocking conditions.

[0035] Alternatively, the method for obtaining CD146-positive endoplasmic reticulum may further include treating and reacting the obtained human tonsillar stem cells with an anti-CD146 antibody or a fluorescently conjugated anti-CD146 antibody, and separating CD146-positive and -negative cells using flow cytometry after the reaction. Alternatively, the method may further include capturing and separating the cells on a surface onto which an anti-CD146 antibody has been applied. The surface onto which the anti-CD146 antibody has been applied may be any surface to which an antibody can be attached, including, for example, a plastic plate, a metal plate, a metal alloy plate, polymer nanoparticles, or metal nanoparticles.

[0036] Furthermore, CD146-positive nanovesicles derived from human tonsillar stem cells can be produced by a method including a step of producing CD146-positive human tonsillar stem cell-derived nanovesicles from cells selected for the CD146 cell surface marker, and the step of producing nanovesicles can be produced by a method similar to the method for producing nanovesicles from human tonsillar stem cells.

[0037] According to one embodiment, the skin regenerating and anti-aging composition may be a pharmaceutical composition or a cosmetic composition.

[0038] According to one embodiment, the composition may be a pharmaceutical composition.

[0039] The pharmaceutical composition may further contain, in addition to the vesicles, pharmaceutical adjuvants such as preservatives, stabilizers, hydrating agents or emulsifying agents, salts for adjusting osmotic pressure and / or buffers, and other therapeutically useful substances, and may be formulated into various oral or parenteral dosage forms by conventional methods.

[0040] The oral dosage forms include, for example, tablets, pills, hard and soft capsules, liquids, suspensions, emulsions, syrups, powders, fine granules, granules, and pellets. These dosage forms may contain, in addition to the active ingredient, surfactants, diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine), and lubricants (e.g., silica, talc, stearic acid and its magnesium or calcium salts, and polyethylene glycol). Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidine, and may optionally contain pharmaceutical additives such as disintegrants (e.g., starch, agar, alginic acid, or its sodium salt), absorbents, colorants, flavorings, and sweeteners. The tablets can be manufactured by conventional mixing, granulating, or coating methods.

[0041] Furthermore, the parenteral administration form may be a transdermal administration form, or may be, for example, an injection, a drip infusion, an ointment, a lotion, a gel, a cream, a spray, a suspension, an emulsion, a suppository, a patch, or the like, but is not limited thereto.

[0042] The pharmaceutical compositions of the present invention can be prepared in unit dose form or in multi-dose containers by formulating them with pharmaceutically acceptable carriers and / or excipients in a manner easily understood by those skilled in the art to which this invention pertains. In this case, the dosage form may be a solution, suspension, or emulsion in an oily or aqueous medium, or may be in the form of an extract, powder, suppository, powder, granule, tablet, or capsule, and may additionally contain a dispersing agent or stabilizer.

[0043] Pharmaceutically acceptable carriers contained in the pharmaceutical compositions of the present invention are those commonly used in formulations, including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above ingredients, the pharmaceutical compositions of the present invention may additionally contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0044] The pharmaceutical compositions of the present invention may be administered orally and parenterally, for example, by intravenous, subcutaneous, intramuscular, intraperitoneal, topical, intranasal, pulmonary, rectal, intrathecal, ocular, dermal and transdermal administration.

[0045] The suitable 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, diet of the patient, administration time, administration route, excretion rate and reaction sensitivity, and an ordinary skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention.

[0046] Determining the dosage of the active ingredient is within the level of a person of ordinary skill in the art, and the daily dosage of the drug varies depending on various factors such as the stage of progression, time of onset, age, health condition, and complications of the subject to be administered. However, based on an adult, in one embodiment, the composition may be administered at 1 μg / kg to 200 mg / kg, and in another embodiment, at 50 μg / kg to 50 mg / kg, in 1 to 3 divided doses per day, and the dosage is not intended to limit the scope of the present invention in any way.

[0047] According to one embodiment of the present invention, the composition may be a cosmetic composition. For example, the cosmetic composition may be formulated into, but is not limited to, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleanser, oil, powder foundation, emulsion foundation, wax foundation, leave-on type, mist, spray, etc. More specifically, the cosmetic composition may be formulated into a cleanser such as shampoo, rinse, or body cleanser; a hair styling agent such as hair tonic, gel, or mousse; a hair cosmetic composition such as a hair nourishing lotion, hair essence, hair serum scalp treatment, hair treatment, hair conditioner, hair shampoo, hair lotion, hair tonic, or hair dye; or a basic cosmetic such as an oil-in-water (O / W) type or water-in-oil (O / W) type.

[0048] In addition to the essential ingredients, the composition may contain other ingredients that can be easily selected and blended by those skilled in the art depending on the type or intended use of the other topical preparation, etc. For example, the composition may further contain UV protection agents, hair conditioning agents, fragrances, etc.

[0049] The cosmetic composition may comprise a cosmetically acceptable vehicle or base, which may be in any suitable form for topical application, such as a solution, a gel, a solid or pasty anhydrous product, an emulsion obtained by dispersing an oily phase in an aqueous phase, a suspension, a microemulsion, a microcapsule, a microgranule or an ionic (liposome) and / or non-ionic vesicular dispersion, or in the form of a cream, skin, lotion, powder, ointment, spray or concealer stick. These compositions may be prepared by conventional methods known in the art.

[0050] When the dosage form of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, aliphatic esters of glycerol, polyethylene glycol or fatty acid esters of sorbitan.

[0051] When the dosage form of the present invention is a suspension, the carrier component may be a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester or polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant.

[0052] When the dosage form of the present invention is a paste, cream or gel, the carrier component may be animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide.

[0053] When the dosage form of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder is used as a carrier component, and particularly in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether can be additionally contained.

[0054] In one embodiment of the present invention, the cosmetic composition may further include a thickener. The thickener included in the cosmetic composition may be methylcellulose, carboxymethylcellulose, carboxymethylhydroxyguanine, hydroxymethylcellulose, hydroxyethylcellulose, carboxyvinyl polymer, polyquaternium, cetearyl alcohol, stearic acid, carrageenan, etc., and preferably, one or more of carboxymethylcellulose, carboxyvinyl polymer, and polyquaternium may be used, and most preferably, carboxyvinyl polymer.

[0055] In one embodiment of the present invention, the cosmetic composition may contain various suitable bases and additives as needed, and the types and amounts of these components can be easily selected by the inventor. If necessary, acceptable additives may be included, such as preservatives, colorants, additives, and other components commonly used in the art.

[0056] Specifically, the preservative may be phenoxyethanol or 1,2-hexanediol, and the fragrance may be an artificial fragrance.

[0057] In one embodiment of the present invention, the cosmetic composition may contain a composition selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymeric peptides, polymeric polysaccharides, sphingolipids, and seaweed extracts. Other ingredients that may be added include milk fat components, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, UV absorbers, preservatives, disinfectants, antioxidants, plant extracts, pH adjusters, alcohol, colorants, fragrances, blood circulation enhancers, cooling agents, antiperspirants, purified water, etc.

[0058] Furthermore, other ingredients that may be added are not limited to those mentioned above, and any of the ingredients mentioned above can be added within a range that does not impair the objects and effects of the present invention.

[0059] The present invention will be described in detail below with reference to examples for better understanding of the present invention. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

[0060] Manufacturing Example 1: Extraction and culture of human tonsillar mesenchymal stem cells (TMSC) Tonsillar mesenchymal stem cells (TMSCs) were isolated from human tonsil tissue obtained by tonsillectomy as follows.

[0061] Human tonsil tissue was washed with phosphate-buffered saline (PBS; Welgene, Seoul, South Korea) containing 2% antibiotic-antimycotic (Gibco, New York, NY, USA). The tissue was then minced and digested in low-glucose Dulbecco's modified Eagle's medium (DMEM; Gibco, New York, NY, USA) with 210 U / mL collagenase type 1 (Gibco, New York, NY, USA) and 4 KU / mL DNase 1 (Sigma, St. Louis, MO, USA) at 37°C for 1 hour and 30 minutes. The digested product was treated with stem cell culture medium in low-glucose Dulbecco's medium (DMEM / low glucose) supplemented with 10% fetal bovine serum and 1% antibiotics, then filtered through a 40 μm strainer and centrifuged at 1,300 rpm for 3 minutes.

[0062] The resulting pellet was washed twice with fresh DMEM. The cells obtained after washing were cultured in DMEM containing 10% fetal bovine serum (Gibco, New York, NY, USA) and 1% antibiotic and antimycotic (Gibco, New York, NY, USA) at 37°C and 5% CO2. The medium was changed every two days. All mesenchymal stem cells were subcultured using TrypLE express (Gibco, New York, NY, USA) at 5-6 day intervals.

[0063] FIG. 1A is a diagram showing the cell morphology of the human tonsillar stem cells prepared in Preparation Example 1, analyzed under an optical microscope (scale bar=200 μm, LSM700, ZEISS).

[0064] As shown in Figure 1A, TMSCs had a fibroblastic morphology similar to previously known mesenchymal stem cells.

[0065] Meanwhile, TMSCs were characterized using flow cytometry (FACSVerse II, BD Biosciences) with anti-CD90, anti-CD105, and anti-CD73 antibodies (Biolegend, San Diego, CA, USA).

[0066] Figure 1B shows the expression of surface markers for human tonsillar intermediate layer stem cells prepared in Preparation Example 1. As shown in Figure 1B, flow cytometry data showed that the TMSCs were positive (>90%) for common TMSC surface markers, including CD90, CD105, and CD73.

[0067] Manufacturing Example 2: Selection of CD146-expressing cells (CD146+ TMSCs) in human tonsillar mesenchymal stem cells CD146-positive tonsillar intermediate stem cells were selected from human tonsillar intermediate stem cells using a human CD146 MicroBead Kit (130-093-596, Miltenyi Biotec, Auburn, USA).

[0068] More specifically, human tonsillar mesenchymal stem cells cultured in Preparation Example 1 were washed once with phosphate buffered saline (PBS) and then treated with TrypLE express for 3 minutes to separate the cells. DMEM containing 10% fetal bovine serum (Gibco, New York, NY, USA) and 1% antibiotic and antimycotic (Gibco, New York, NY, USA) was added to the separated cells, and the cells were centrifuged at 1,300 rpm for 3 minutes. The supernatant was removed, the cells were resuspended in PBS, and centrifuged under the same conditions. After removing the supernatant, 60 μL / 10 ml of PBS supplemented with 0.5% fetal bovine serum and 2 mM EDTA was added. 7 The cell pellet was dissociated by treating the same number of cells as above. The cells obtained from the cell pellet were cultured and the resulting human tonsillar stem cells were added at 20 μL / 10 7 The cells were treated with FcR Blocking Reagent (BD Biosciences, Franklin Lakes, NJ, USA) at 20 μL / 10 7After treatment with CD146 microbeads, the cells were incubated for 15 minutes at 4°C in the dark. After incubation, the cells were treated with 1 mL of MACS buffer and centrifuged at 1,300 rpm for 3 minutes. The supernatant was removed. CD146-positive tonsillar mesenchymal stem cells (CD146+ TMSCs) and CD146-negative tonsillar mesenchymal stem cells (CD146- TMSCs) were separated using a MACS separator and an LS column (Miltenyi Biotec, Bergisch Gladbach, Germany).

[0069] Production Example 3: Production of TMSC-derived nanovesicles (TMSC-NV) To prepare TMSC-derived nanovesicles, the TMSCs obtained in Preparation Example 1 were isolated by treatment with TrypLE express solution (Gibco, New York, NY, USA) at 37°C for 3 minutes. The isolated cells were suspended in DMEM containing 10% fetal bovine serum (Gibco, New York, NY, USA) and 1% antibiotic and antimycotic agent (Gibco, New York, NY, USA) and centrifuged at 1300 rpm for 2 minutes. The supernatant was removed, and the resulting cell pellet was washed twice with PBS and diluted to 1 x 10 cells / ml in PBS at 10°C. 6 The cells were resuspended at a density of 1000 cells / mL.

[0070] The resuspended cells were passed through three successive porous polycarbonate filter papers with pore sizes of 10 μm, 5 μm, and 0.4 μm (sandwiched between the retainer and the extruder) using a mini-extruder (Avanti® Polar Lipids Mini Extruder, Alabaster, AL, USA) to produce nanovesicles.

[0071] Production Example 4: Production of nanovesicles derived from CD146+ TMSCs (CD146+ TMSC-NV) Nanovesicles were prepared in the same manner as in Preparation Example 3, except that the CD146+ TMSCs prepared in Preparation Example 2 were used instead of the TMSCs prepared in Preparation Example 1.

[0072] The size and shape of the nanovesicles obtained in Preparation Examples 3 and 4 were determined by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. 1) Transmission Electron Microscopy (TEM) Purified nanovesicles were applied to glow-discharged carbon-coated copper grids (Electron Microscopy Sciences, Fort Washington, PA). After allowing the nanovesicles to absorb onto the grids for 1 hour, the grids were fixed with 4% paraformaldehyde for 10 minutes, washed with drops of deionized water, and negatively stained with 2% uranyl acetate (Ted Pella, Redding, CA). Electron micrographs were recorded with a JEM 1011 microscope (JEOL, Tokyo, Japan) at an accelerating voltage of 100 kV. 2)Dynamic light scattering (DLS) The size distribution of nanovesicles was measured with a Zetasizer Nano ZS (Malvern Instrument Ltd., Malvern, UK).

[0073] Also, Micro BCA TM The concentration of nanovesicles was measured using a Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA).

[0074] Meanwhile, Western blotting method was used to measure the protein expression of nanovesicles. 3) Western blotting TMSCs and TMSC-NVs were harvested and lysed in Radioimmunoprecipitation assay (RIPA) buffer (Sigma, St. Louis, MO, USA). To remove cell debris, the lysates were centrifuged at 13,000 rpm for 20 minutes. The amount of protein in the supernatant was determined using a Micro BCA. TM Protein Assay Kit was used to measure total protein. 20 μg of total protein was loaded and separated on a 10% SDS-PAGE gel. After loading, the separated proteins were transferred to a membrane blocked with 5% BSA solution for 30 minutes. For immunoblotting, rabbit anti-CD9 (1:2000), anti-CD63 (1:2000), and anti-beta-actin (1:5000) primary antibodies (Abcam, Cambridge, UK) were applied overnight at 4°C.

[0075] A horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H+L) (1:5,000) secondary antibody (Invitrogen, Carlsbad, CA, USA) for chemiluminescent detection of proteins was applied for 2 h at room temperature, and Amersham™ ECL Select™ (Thermo Fisher Scientific, Waltham, MA, USA) was used for detection.

[0076] Figure 2A shows the protein expression of TMSC-NV and a TEM (JEM 1011 microscope (JEOL, Tokyo, Japan) image. Protein levels were normalized by β-actin.

[0077] FIG. 2B shows the results of dynamic light scattering analysis of TMSC-NV.

[0078] Figure 2A confirms that TMSC-NVs express exosome markers such as CD9 and CD63. Meanwhile, TMSC-NVs have a spherical shape, and Figure 2B shows that the diameter of TMSC-NVs is represented by two peaks (88.5 and 228.3 nm). These results indicate that TMSC-NVs have similar properties to exosomes.

[0079] Production Example 5: Production of nanovesicles derived from adipose stem cells Nanovesicles were prepared in the same manner as in Preparation Example 3, except that adipose stem cells were used instead of the TMSCs prepared in Preparation Example 1.

[0080] Production Example 6: Production of nanovesicles derived from bone marrow stem cells Nanovesicles were prepared in the same manner as in Preparation Example 3, except that bone marrow stem cells were used instead of the TMSCs prepared in Preparation Example 1.

[0081] Experimental Example Cell culture Human dermal fibroblasts (HDFs) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in DMEM supplemented with 10% fetal bovine serum (Gibco, New York, NY, USA) and 1% antibiotic-antimycotic (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C and 5% CO2. The medium was replaced every 2 days. Intrinsic replicative senescent cells were generated by repeated passaging. Cells at passage 3 and passage 15 were identified as the "juvenile" and "senescent" states, respectively. Extrinsic senescent cells were generated by irradiating them with 200 mJ / cm2 of irradiance. 2 The sample was treated with UV irradiation.

[0082] Experimental Example 1: Comparison of cell proliferation ability Cell proliferation was confirmed using the Cell Counting Kit-8 assay (CCK-8, Dojindo, Japan). HDFs purchased from ATCC were plated in 12-well plates at a density of 5,000 cells / cm. 2The HDFs were then cultured in stem cell culture medium (DMEM containing 10% fetal bovine serum (Gibco, New York, NY, USA) and 1% antibiotic and antimycotic (Gibco, New York, NY, USA)). After 24 hours of dispensing, the HDFs were treated once with nanovesicles from Preparation 3 and Preparation 4 at a protein concentration of 50 μg / mL, and then cultured for 6 days. To compare cell proliferation, the stem cell culture medium was mixed with CCK-8 at a 1:10 ratio and then added to each well. After incubation at 37°C for 1 hour and 30 minutes, the absorbance at 450 nm was measured to compare the degree of cell proliferation.

[0083] Experimental Example 2: Immunostaining analysis For immunostaining analysis, the cells from Preparation Examples 3 and 4 were fixed with 4% paraformaldehyde for 30 minutes, and then permeabilized with 0.05% Triton X-100 (Sigma, St. Louis, MO, USA) for 15 minutes.

[0084] After permeabilization, cells were blocked with 1% bovine serum albumin (BSA) (Sigma, St. Louis, MO, USA) for 30 minutes at room temperature and then incubated with anti-vinculin (1:200) for 1 hour at room temperature. After washing with PBS, goat anti-rabbit IgG H&L Alexa Fluor 488 secondary antibody (1:200) and tetramethylrhodamine-conjugated phalloidin (1:200) were applied for 1 hour in the dark. To confirm ECM production, cells were fixed, blocked, and incubated with anti-collagen 1 primary antibody (1:200) for 1 hour followed by goat anti-rabbit IgG H&L Alexa Fluor 488 (1:200). All antibodies used for immunocytochemical analysis were purchased from Abcam (Cambridge, MA, USA). Immunocytochemical analysis was counterstained with DAPI nuclear stain and examined under a ZEISS LSM700 confocal microscope (Zeiss, Oberkochen, Germany).

[0085] Experimental Example 3: Quantitative Real-Time Polymerase Chain Reaction (qPCR) For quantitative real-time polymerase chain reaction (qPCR), senescent fibroblasts were treated with the nanovesicles of Production Examples 3 and 4 for 6 days, and the cells were cultured in a 6-well plate.

[0086] Each well was washed with 1 mL of PBS, treated with 500 μL of Trizol reagent, and then transferred to a 1.75 mL tube.

[0087] The reagent was treated with 200 μL of chloroform and placed on ice for 10 minutes. The mixture was centrifuged at 13,000 rpm for 15 minutes, and the aqueous supernatant was carefully collected and mixed with an equal volume of isopropanol and incubated on ice for 10 minutes.

[0088] The RNA sample was centrifuged at 13,000 rpm for 15 minutes, the supernatant was removed, and the RNA pellet was obtained. The RNA pellet was washed with 75% EtOH and dried.

[0089] The clear RNA pellet was diluted with nuclease-free water, the RNA concentration was confirmed with Nanodrop2000, and cDNA was synthesized using 1 μg of RNA.

[0090] cDNA was synthesized using the PrimeScript RT Reagent kit (TAKARA, Japan), and the ΔΔCT values ​​were confirmed using a Step-One plus qPCR machine (ThermoFisher Scientific, USA).

[0091] Experimental Example 4: Senescence-associated beta-galactosidase assay (SA-β-galactosidase assay) Senescence-associated beta-galactosidase is an enzyme that catalyzes the hydrolysis of galactosides to monosaccharides. In senescent cells and tissues, it is detectable only at pH 6.0, but not at pH 4.0. As a biomarker of cellular senescence, its activity can be detected using a chromogenic assay that uses 5-bromo-4-chloro-3-indoyl-D-galactopyranoside (X-gal), which is converted to an insoluble blue compound.

[0092] Here, SA-β-galactosidase analysis was performed using a cell senescence staining kit (Cell Biolabs, San Diego, CA, USA).

[0093] To compare the degree of cellular senescence, the activity of SA-β-galactosidase in the cells was measured using a Cellular Senescence Staining Kit (CBA-230, Cell biolabs, USA).

[0094] More specifically, HDFs were treated with the nanovesicles of Preparation Examples 3 to 6, and after 6 days, the HDFs were washed once with PBS and then treated with 10% glycerol at room temperature for 5 minutes to fix the cells. The supernatant was removed, and the cells were washed three times with PBS. They were then stained for 14 hours at 37°C using a Cellular Senescence Staining Kit. After the reaction, the supernatant was removed, the cells were washed three times with PBS, and then photographed and quantitatively analyzed using a microscope. Quantitative data was measured by recording the coloration ratio of senescent cells.

[0095] Experimental Example 5: Confirmation of ex vivo tissue regeneration ability (immunostaining method) The donated human skin tissue was de-lipidated, washed three times with PBS, and then cut into 1cm x 1cm pieces. The skin tissue was cultured in a semi-agarose DMEM medium environment at 37°C and 5% CO2. 300mJ / cm2 was applied to the cultured human skin tissue. 2 After irradiation with ultraviolet B (UVB) equivalent to 100μg / ml, 20μL of CD146+ TMSC-NV was applied at 50μg / ml and 100μg / ml, respectively. 24 hours later, UVB irradiation and CD146+ TMSC-NV application were repeated under the same conditions. After repeated treatment, the human skin tissue was transferred to fresh semi-agarose DMEM medium, and after a third UVB irradiation and CD146+ TMSC-NV application, the tissue was cultured for 24 hours, after which it was fixed and immunostained.

[0096] Using immunostaining, collagen type 1, collagen type 3, involucrin, and filaggrin, which are proteins that make up human skin tissue, were stained.

[0097] Figure 3 shows the modulation of proliferation and senescence by TMSC-NV treatment in a passage-associated senescence model. Figure 3A shows morphological changes in HDFs (scale bar = 200 μm). Figure 3B shows the proliferation of passage-associated senescent HDFs after TMSC-NV treatment. Figure 3C shows the results of senescence-associated SA-β-galactosidase analysis (scale bar = 200 μm). Figure 3D shows quantitative analysis of SA-β-galactosidase analysis. Figure 3E shows vinculin expression in focal attachments of HDFs. Figure 3F shows quantitative data of vinculin expression in focal attachments. Significant differences between groups were determined by one-way ANOVA (ns > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001).

[0098] As shown in Figures 3A and 3B, TMSC-NV treatment increased the proliferation of senescent HDF cells. SA-β-galactosidase analysis was performed to confirm the anti-senescence role of TMSC-NV. As shown in Figure 3C, TMSC-NV treatment reduced the β-galactosidase activity of senescent HDFs. The quantitative data of SA-β-galactosidase analysis in Figure 3D showed that TMSC-NV treatment reduced the proportion of senescent cells, confirming that TMSC-NV treatment reduced the level of senescence in HDFs.

[0099] We also examined vinculin protein expression and morphological changes in the actin cytoskeleton at focal adhesions after treatment with TMSC-NV. Immunofluorescence analysis showed increased vinculin expression at focal adhesions in senescent HDFs, as shown in Figures 3E and 3F, which was reduced by TMSC-NV treatment.

[0100] These results indicate that TMSC-NV increases the proliferation of HDFs and reduces passaging-induced senescence.

[0101] To confirm the anti-aging properties of TMSC-NV in terms of molecular biology, we examined extracellular matrix (ECM) production and gene expression of aging-related antioxidant genes after TMSC-NV treatment.

[0102] Figure 4 shows the regulation of antioxidant genes in the extracellular matrix and HDFs treated with TMSC-NV in a passage-associated aging model. Figure 4A shows the mRNA expression of COL1, ELASTIN, SOD2, and HMOX1 in passage-associated aging HDFs. Figure 4B shows the results of immunofluorescence analysis of collagen type 1 in passage-associated aging HDFs. Figure 4C shows quantitative data from the immunofluorescence analysis. Significant differences between groups were determined by one-way ANOVA (*p<0.05, **p<0.01, ***p<0.001).

[0103] As shown in Figure 4A, the mRNA levels of collagen type 1 (COL1) and ELASTIN were decreased in senescent HDFs compared with young HDFs, but treatment with TMSC-NV revealed upregulation of ECM production.

[0104] Similarly, mRNA expression of antioxidant genes SOD2 and HMOX1 was increased in senescent HDFs by treatment with TMSC-NV. Furthermore, protein expression of COL1 was examined by immunofluorescence, as shown in Figures 4B and 4C. This indicates that treatment with TMSC-NV significantly increased ECM production in passage-associated senescent HDFs. These results suggest that treatment with TMSC-NV restores ECM production and antioxidant genes that are downregulated in senescent cells.

[0105] Figure 5 shows the modulation of proliferation and senescence by TMSC-NV treatment in a UV-induced senescence model. Figure 5A shows morphological changes in HDFs (scale bar = 200 μm). Figure 5B shows the proliferation of passage-associated senescent HDFs after TMSC-NV treatment. Figure 5C shows the results of senescence-associated SA-β-galactosidase analysis (scale bar = 200 μm). Figure 5D shows quantitative analysis of SA-β-galactosidase analysis. Figure 5E shows vinculin expression in focal attachments of HDFs. Figure 5F shows quantitative data of vinculin expression in focal attachments. Significant differences between groups were determined by one-way ANOVA (ns > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001).

[0106] As shown in Figures 5A and 5B, TMSC-NV treatment increased the proliferation of UV-induced senescent HDF cells. SA-β-galactosidase analysis was performed to confirm the anti-senescence role of TMSC-NV. As shown in Figure 5C, TMSC-NV treatment reduced β-galactosidase activity in UV-induced senescent HDFs. The quantitative data of SA-β-galactosidase analysis in Figure 5D showed that TMSC-NV treatment reduced the proportion of senescent cells, confirming that TMSC-NV treatment reduced the level of senescence in HDFs.

[0107] We also examined vinculin protein expression and morphological changes in the actin cytoskeleton at focal adhesions after treatment with TMSC-NV. Immunofluorescence analysis showed increased vinculin expression in focal adhesions of UV-induced senescent HDFs, as shown in Figures 5E and 5F, which was reduced by TMSC-NV treatment.

[0108] These results indicate that TMSC-NV increases the proliferation of HDFs and reduces passaging-induced senescence.

[0109] From a molecular biological perspective, to confirm the anti-aging properties of TMSC-NV in a UV-induced aging model, the production of senescence-related ECM and mRNA expression of antioxidant genes were examined by qPCR.

[0110] Figure 6 shows the regulation of extracellular matrix and antioxidant genes by TMSC-NV treatment in a UV-induced aging model. Figure 6A shows the mRNA expression of COL1, ELASTIN, SOD2, and HMOX1 in UV-induced aging HDFs. Figure 6B shows the results of immunofluorescence analysis of collagen type 1 in UV-induced aging HDFs. Figure 6C shows quantitative data from the immunofluorescence analysis.

[0111] As shown in Figure 6A, qPCR results indicated that COL1 and ELASTIN were decreased after UV irradiation, while COL1 significantly increased with TMSC-NV treatment. However, ELASTIN did not increase. The antioxidant genes SOD2 and HMOX1 were decreased by UV irradiation and increased with TMSC-NV treatment. Furthermore, as shown in Figures 6B and 6C, immunofluorescence analysis demonstrated decreased expression of collagen type 1 in UV-induced senescent HDFs, which increased with TMSC-NV treatment. These results indicate that TMSC-NV HDFs increase ECM production and antioxidant genes that reduce senescence.

[0112] To confirm the anti-aging properties of CD16+ TMSC-NVs in terms of molecular biology, we examined extracellular matrix (ECM) production and gene expression of aging-related antioxidant genes after CD146+ TMSC-NV treatment.

[0113] FIG. 7 shows the regulation of proliferation and senescence by CD146+ TMSC-NV treatment in a passage-associated senescence model. FIG. 7A shows the mRNA expression of COL1 and HMOX1 in senescent HDFs.

[0114] Referring to Figure 7A, when CD146+ TMSC-NV was treated for 6 days, the mRNA expression level of collagen type 1, a marker related to skin regeneration, increased, and the expression level of HMOX1, an antioxidant marker, increased to the highest level compared to treatment with adipose stem cell nanovesicles (ASC-NV) and bone marrow stem cell nanovesicles (BMMSC-NV).

[0115] FIG. 7B shows the results of the senescence-associated β-galactosidase assay (scale bar=200 μm), and FIG. 7C shows the quantitative analysis of the SA-β-galactosidase assay.

[0116] As shown in Figures 7B and 7C, CD146+ TMSC-NV-treated senescent fibroblasts and TMSC-NV-treated senescent fibroblasts were stained to a similar extent as fibroblasts with a relatively low passage number.

[0117] Based on the above results, it was confirmed that CD146+ TMSC-NV has high anti-aging and skin regeneration efficacy.

[0118] Figures 8A and 8B show the results of immunohistochemistry of collagen type 1, collagen type 3, involucrin, and filaggrin, which make up the human skin tissue, after treating the skin aging model with ultraviolet B (UVB) and treating it with CD146+ TMSC-NVs.

[0119] Based on the above results, it was confirmed that treatment with CD146+ TMSC-NVs can repair UV-induced damage to human skin tissue.

Claims

1. A composition for skin regeneration and anti-aging, comprising endoplasmic reticulum derived from CD146-positive human tonsillar mesoderm stem cells as an active ingredient, wherein the endoplasmic reticulum is a nanovesicle.

2. The composition of claim 1, wherein the nanovesicles have a diameter of 50 nm to 250 nm.

3. The composition of claim 1, wherein the endoplasmic reticulum additionally contains one or more immunogens selected from the group consisting of CD14, CD34, CD45, CD73, and CD90.

4. The composition of claim 1 , wherein the composition is a pharmaceutical composition or a cosmetic composition.

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