Skin aging inhibiting composition comprising stem cell-derived exosome and method for preparing same

Abstract

The present invention relates to a composition for inhibiting skin aging comprising exosomes derived from stem cells, and a method for preparing the same, the composition comprising exosomes of the present invention having excellent effects of preventing skin wrinkles, increasing skin elasticity, preventing skin aging, or promoting skin regeneration, and thus being useful as a variety of cosmetic materials and pharmaceutical compositions.

Description

Technical Field

[0001] The present invention relates to a skin aging inhibitory composition containing exosomes derived from stem cells and a method for preparing the same. More specifically, the present invention relates to a composition containing exosomes isolated from mesenchymal stem cells or their cultures, which are excellent in preventing skin striae atrophicae, increasing skin elasticity, preventing skin aging, or promoting skin regeneration.

Background Art

[0002] Extracellular Vesicles are vesicles composed of a spherical lipid bilayer with a size of 30 nm to 1000 nm, including Micro vesicles and Exosomes.

[0003] It is known that the lipid bilayer of exosomes has a phospholipid bilayer membrane structure like the source cell (donor cell), and the lipid bilayer of exosomes functions as a constitutive body for cells to secrete substances extracellularly, playing functional roles such as cell-to-cell communication and cell immune intervention.

[0004] Exosomes contain cell-specific components that reflect the unique biological functions of the source cells, and in addition to phospholipids, mRNA, and miRNA, they include various water-soluble proteins, extrinsic proteins, and transmembrane protein components.

[0005] Such exosomes are released from all animal cells such as mast cells, lymphocytes, stellate cells, platelets, nerve cells, endothelial cells, and epithelial cells, and are found in various body fluids such as blood, urine, mucus, saliva, bile, ascites, and cerebrospinal fluid. Exosomes can also cross the Blood-Brain Barrier (BBB) and have a high selective permeability capable of penetrating the cell membranes of epidermal cells and endothelial cells. Therefore, they can also be used for the development of a drug delivery system (DDS) as a nano carrier for specific drugs.

[0006] It is known that exosomes and micro vesicles secreted from mesenchymal stem cells are involved in cell-to-cell communication and exhibit the therapeutic efficacy of stem cells in regenerative medicine.

[0007] When transplanted into the body, known stem cells bring a trophic effect to paracrine factors secreted by cells that cannot survive long term. Among these factors, low-molecular-weight substances (such as growth factors, chemokines, and cytokines) are secreted via extracellular vesicles (such as exosomes), and these exosomes are derived from stem cells. Therefore, exosomes are used to identify the characteristics of stem cells and evaluate the therapeutic efficacy of stem cells. Furthermore, in recent years, there has been active research on the effect of using exosomes secreted by mesenchymal stem cells instead of mesenchymal stem cells themselves to treat various diseases, and the academic and industrial communities expect that this approach could become a new alternative that can overcome the limitations of existing stem cell therapies.

Summary of the Invention

[0008]

Problems to be Solved by the Invention

[0009] Accordingly, the present inventors have developed a composition isolated from stem cells or their culture media, and have confirmed that the composition of the present invention has significantly excellent effects in preventing striae atrophicae, improving the skin, and in particular, increasing skin elasticity, preventing skin aging, and promoting skin regeneration.

[0010] Accordingly, an object of the present invention is to provide a composition comprising exosomes derived from stem cells.

[0011] Another object of the present invention is to provide a method for preparing a composition comprising exosomes derived from stem cells.

[0012] Yet another object of the present invention is to provide a composition comprising exosomes derived from stem cells for preventing striae atrophicae, increasing skin elasticity, preventing skin aging, or promoting skin regeneration.

[0013] Yet another object of the present invention is to provide a method for preventing striae atrophicae, increasing skin elasticity, preventing skin aging, or promoting skin regeneration using exosomes derived from stem cells.

[0014] Yet another object of the present invention is to provide the use of a composition comprising exosomes derived from stem cells in preventing striae atrophicae, increasing skin elasticity, preventing skin aging, or promoting skin regeneration.

[0015]

Means for Solving the Problems

[0016] Accordingly, the present inventors have confirmed that the composition of the present invention has excellent effects in preventing striae atrophicae, increasing skin elasticity, preventing skin aging, or promoting skin regeneration.

[0017] Hereinafter, the present invention will be described in more detail.

[0018] One aspect of the present invention is a composition comprising exosomes derived from stem cells.

[0019] The term "exosome" in this specification is a cell-derived vesicle present in the body fluids of almost all eukaryotes, and refers to a vesicle with a diameter of about 30 nm to 100 nm, that is, larger than LDL protein but significantly smaller than red blood cells. As is well known, when multivesicular bodies fuse with the cell membrane, exosomes can be released from cells, or can be directly released from the cell membrane, and perform important and special functions such as coagulation and intercellular signal transduction.

[0020] The term "stem cell" in this specification is an undifferentiated cell, which refers to a cell that has the ability of self-renewal and the ability to differentiate into two or more different types of cells.

[0021] In one embodiment of the present invention, the stem cells can be autologous or allogeneic stem cells, can be of any type of animal origin including humans and non-human mammals, and can be adult-derived stem cells or embryonic-derived stem cells. For example, the stem cells can be selected from the group consisting of embryonic stem cells, adult stem cells, induced pluripotent stem cells (iPSCs), mesenchymal stem cells derived from induced pluripotent stem cells, BxC stem cells, mesenchymal stem cells derived from induced pluripotent stem cells pretreated with hyaluronic acid (HA), and BxC-R11 stem cells, but are not limited thereto.

[0022] The term "adult stem cell" in this specification is a cell extracted from umbilical cord blood, adult bone marrow, blood, etc., which refers to a cell before differentiating into specific organ cells, and refers to an undifferentiated cell with the ability to develop into tissues in the body when necessary.

[0023] In one embodiment of the present invention, the adult stem cells can be selected from the group consisting of adult stem cells derived from humans, animals or animal tissues, mesenchymal stromal cells derived from humans, animals or animal tissues, and mesenchymal stem cells derived from induced pluripotent stem cells derived from humans, animals or animal tissues, but are not limited thereto.

[0024] In the present invention, humans, animals or animal tissues can be selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amnion and placenta, but are not limited thereto.

[0025] In the present invention, stem cells derived from various tissues of humans or animals can be selected from the group consisting of, but not limited to, hematopoietic progenitor cells, mammary stem cells, intestinal stem cells, vascular endothelial stem cells, neural stem cells, olfactory neural stem cells, and testicular stem cells.

[0026] The term "embryonic stem cell" in this specification refers to a cell extracted during the development of an embryo, which means a cell obtained by extracting the inner cell mass from the blastocyst stage germ before the fertilized egg implants in the mother's uterus and culturing it in vitro.

[0027] Embryonic stem cells refer to cells with the ability of self-renewal, which have pluripotency or totipotency to differentiate into all tissue cells of an individual, and in a broad sense, also include embryoid bodies derived from embryonic stem cells.

[0028] In this specification, stem cells can include embryonic stem cells from all sources, such as humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice, and rabbits, etc., but not limited to these.

[0029] The term "induced pluripotent stem cell (iPSC)" in this specification refers to a cell with pluripotent differentiation ability induced from differentiated cells through an artificial dedifferentiation process, and can be used with the same meaning as "dedifferentiated stem cells".

[0030] The artificial dedifferentiation process can be carried out by virus-mediated using retroviruses, lentiviruses, and Sendai viruses, or by introducing dedifferentiation factors mediated by non-viral vectors, using proteins and cell extracts, etc., or includes a dedifferentiation process based on stem cell extracts and compounds, etc.

[0031] Induced pluripotent stem cells have almost the same characteristics as embryonic stem cells. Specifically, they have similar cell shapes, similar gene and protein expressions, are pluripotent in vitro and in vivo, form teratomas, form chimeric mice when inserted into the blastocysts of mice, and are capable of achieving germline transmission of genes.

[0032] In one implementation example of the present invention, the stem cells can be mesenchymal stem cells derived from induced pluripotent stem cells.

[0033] In one embodiment of the present invention, the composition may comprise exosomes derived from mesenchymal stem cells derived from induced pluripotent stem cells.

[0034] The term "mesenchymal stem cell (MSC)" in this specification refers to stem cells derived from mesenchyme. Mesenchymal stem cells can differentiate into one or more cells selected from the group consisting of osteoblasts, chondrocytes, adipocytes, or myocytes. Mesenchymal stem cells can be isolated from all types of adult tissues. For example, they can be isolated from bone marrow, adipose tissue, umbilical cord, or peripheral blood. A mesenchymal stem cell population (MSC population) can be defined as exhibiting a specific phenotype. The mesenchymal stem cell population differentiated from induced pluripotent stem cells can exhibit the same phenotypic characteristics as the conventional mesenchymal stem cell population. The mesenchymal stem cell population can be understood as a stem cell population that expresses more than 95% of the CD105, CD73, and CD90 markers and expresses less than 2% of the CD45, CD34, and SSEA-4 markers.

[0035] In one embodiment of the present invention, the stem cells can be BxC stem cells.

[0036] In one embodiment of the present invention, the composition may comprise exosomes derived from BxC stem cells.

[0037] The term "BxC stem cell" in this specification refers to the stem cells prepared by further culturing after separating the population of induced pluripotent stem cells (iPSCs) that do not express stage-specific embryonic antigen 4 (SSEA-4) protein after culturing the induced pluripotent stem cells. BxC stem cells are cells at the stage prior to the complete differentiation into mesenchymal stem cells among induced pluripotent stem cells, and can obtain the properties of complete mesenchymal stem cells through further culturing. Therefore, the phenotype of the BxC stem cell population does not show exactly the same phenotype as that of the mesenchymal stem cell population, and can show a phenotypic similarity range of 96% to 99.9% with that of the mesenchymal stem cell population. For example, 0.3% of the induced pluripotent stem cell population can express CD90 protein, while 99.7% of the mesenchymal stem cell population can express CD90 protein, and the BxC stem cell population can express it at a ratio of 96.9%, which is about 98% of that of the mesenchymal stem cells. Thus, BxC stem cells can be defined as follows: after culturing induced pluripotent stem cells, further culturing the induced pluripotent stem cells that do not express SSEA-4 protein, without completely differentiating them into mesenchymal stem cells, but differentiating 96% to 99.9% of the stem cells. Similarly, compared with the mesenchymal stem cells differentiated from induced pluripotent stem cells, BxC stem cells can have excellent cell stemness and secrete a large amount of proteins related to functionality. Specifically, when the BxC stem cells of the present invention are passaged more than 9 times, the difference in proliferation ability shows more than 10 times compared with the mesenchymal stem cells (MSCs) of the same tissue origin, and even when passaged more than 12 times, no decrease in proliferation ability is observed. In addition, compared with conventional mesenchymal stem cells, the expression level of the marker Ki67 related to cell proliferation ability of BxC stem cells also shows a high level more than 2 times. Moreover, similarly, compared with the mesenchymal stem cells differentiated from induced pluripotent stem cells, BxC stem cells can express at a higher level one or more genes selected from the group consisting of ANKRD1, CPE, NKAIN4, LCP1, CCDC3, MAMDC2, CLSTN2, SFTA1P, EPB41L3, PDE1C, EMILIN2, SULT1C4, TRIM58, DENND2A, CADM4, AIF1L, NTM, SHISA2, RASSF4, and ACKR3, and can express at a lower level one or more genes selected from the group consisting of DHRS3, BMPER, IFI6, PRSS12, RDH10, and KCNE4.

[0038] The term "stem cell ability" in this specification refers to pluripotency, which has the ability to generate all cells, and self-renewal ability, which can infinitely create cells similar to itself. For example, it can refer to maintaining the undifferentiated state of undifferentiated cells and increasing the proliferation ability of stem cells, or increasing telomerase activity, or increasing the expression of stemness acting signals, or increasing cell migration activity, and may include exhibiting one or more of these characteristics.

[0039] In one implementation example of the present invention, the stem cells can be mesenchymal stem cells derived from induced pluripotent stem cells pretreated with hyaluronic acid (HA).

[0040] In one implementation example of the present invention, the composition may contain exosomes derived from mesenchymal stem cells derived from induced pluripotent stem cells pretreated with hyaluronic acid (HA).

[0041] The term "pretreatment" in this specification refers to the process of culturing by adding a specific substance to the culture medium of mesenchymal stem cells. For example, pretreatment refers to the process of further culturing mesenchymal stem cells that have completed the differentiation of induced pluripotent stem cells in a culture medium supplemented with hyaluronic acid (HA).

[0042] In the present invention, hyaluronic acid can increase the stemness and proliferation ability of stem cells, and can increase the number of exosomes derived from stem cells, as well as the content of proteins and RNAs in the exosomes.

[0043] In one implementation example of the present invention, the stem cells can be BxC-R11 stem cells.

[0044] In one implementation example of the present invention, the composition may contain exosomes derived from BxC-R11 stem cells (BxC-R11e).

[0045] The term "BxC-R11 stem cell" in this specification refers to mesenchymal stem cells that are cultured from the BxC stem cells of the present invention and fully differentiated into mesenchymal stem cells, and then cultured (pretreated) in a medium containing hyaluronic acid. For example, BxC-R11 stem cells can be prepared by further culturing BxC stem cells and fully differentiating them into mesenchymal stem cells, and then culturing them in a medium containing 0.1 μg / ml to 1000 μg / ml of hyaluronic acid, for example, in a medium containing 40 μg / ml of hyaluronic acid for 12 hours to 48 hours. The cell proliferation rate of BxC-R11 stem cells increases by about 360%, the exosome production efficiency increases by about 5 times, and the amount of exosome-derived proteins increases by more than about 5 times.

[0046] In one embodiment of the present invention, hyaluronic acid can be pretreated at a concentration of 0.1 μg / ml to 1000 μg / ml, 0.5 μg / ml to 1000 μg / ml, 1 μg / ml to 500 μg / ml, 1 μg / ml to 200 μg / ml, 1 μg / ml to 100 μg / ml, 1 μg / ml to 80 μg / ml, 1 μg / ml to 60 μg / ml, 10 μg / ml to 60 μg / ml. For example, it can be pretreated at a concentration of 40 μg / ml, but is not limited thereto.

[0047] Another aspect of the present invention is a cosmetic material composition for preventing striae atrophicae, increasing skin elasticity, preventing skin aging, or promoting skin regeneration, the cosmetic material composition comprising exosomes isolated from mesenchymal stem cells (MSC) derived from induced pluripotent stem cells (iPSC) as an active ingredient.

[0048] Another aspect of the present invention is a cosmetic material composition for increasing skin elasticity, the cosmetic material composition comprising exosomes isolated from mesenchymal stem cells (MSC) derived from induced pluripotent stem cells (iPSC) as an active ingredient.

[0049] Another aspect of the present invention is a cosmetic material composition for preventing skin aging, the cosmetic material composition comprising exosomes isolated from mesenchymal stem cells (MSC) derived from induced pluripotent stem cells (iPSC) as an active ingredient.

[0050] Another aspect of the present invention is a cosmetic material composition for promoting skin regeneration, the cosmetic material composition comprising exosomes isolated from mesenchymal stem cells (MSCs) derived from induced pluripotent stem cells (iPSCs) as an active ingredient.

[0051] Another aspect of the present invention is a cosmetic material composition for preventing striae atrophicae, the cosmetic material composition comprising exosomes isolated from mesenchymal stem cells (MSCs) derived from induced pluripotent stem cells (iPSCs) as an active ingredient.

[0052] The term "regeneration" in the present specification refers to the process of repairing skin tissue damaged by external and internal causes of the skin (cells). Damage based on the external cause can be caused by ultraviolet rays, external pollutants, trauma, injuries, etc., and damage based on the internal cause can be caused by stress, etc. When the composition of the present invention comes into contact with normal or young skin fibroblasts and aged skin fibroblasts (human dermal fibroblasts (HDFs)), a significant improvement in the cell proliferation rate can be confirmed (refer to Example 3 and Figure 1 ), and the expression of the NANOG gene closely related to cell regeneration is also promoted (refer to Example 4 and Figure 2 ), and the expression of epidermal growth factor (EFG) and elastin genes in skin fibroblasts is promoted (refer to Example 6 and Figure 5 ). Thus, it is confirmed that the composition has a very excellent skin regeneration promoting effect, and thus can be used for the purpose of promoting skin regeneration.

[0053] The term "skin elasticity" in the present specification is presented by elastin or collagen present in the dermis layer. When skin fibroblasts are activated, the state rich in elastin or collagen can be maintained, thereby improving skin elasticity. Therefore, when the composition containing exosomes of the present invention comes into contact with aged fibroblasts (human dermal fibroblasts (HDFs)) of animals including humans, a significant improvement in the cell proliferation rate is confirmed (refer to Example 3 and Figure 1 ), and the expression of epidermal growth factor (EFG) and elastin genes in fibroblasts is promoted (refer to Example 6 and Figure 5), and not only promotes elastin expression but also promotes the assembly of elastin fibers, thus confirming that the composition has a very excellent effect of increasing skin elasticity.

[0054] The term "skin aging" in this specification refers to symptoms such as reduced elasticity, loss of luster, appearance of wrinkles, weakened regeneration ability, or severe dryness on the skin, which can be caused by the passage of time or external environment, etc. Therefore, the composition containing exosomes of the present invention has excellent fibroblast proliferation activity (refer to Example 3, Example 4, and Figure 1 ), SA-β-Gal activity reduction ability (refer to Example 5 and Figure 4 ), and promotes the expression of epidermal growth factor and elastin genes (refer to Example 6 and Figure 5 ), and thus can be used for the purpose of improving or preventing skin aging.

[0055] The term "preventing striae atrophicae" in this specification is called striae distensae in medical terms, which refers to the loosening or breakage of the binding of collagen fibers due to skin expansion, and at the same time forms an atrophic linear band generated by skin splitting. It is known that collagen and elastin are reduced in the skin lesions of patients with striae distensae. In particular, collagen-elastin hydrolysate shows an effect in preventing striae distensae, etc., and collagen and elastin play an important role in preventing striae distensae. On the other hand, it was confirmed that the composition containing exosomes of the present invention promotes the expression of elastin gene (refer to Example 6 and Figure 5 ), and not only promotes elastin expression but also promotes the assembly of elastin fibers. Therefore, it was confirmed that it also has an excellent effect of preventing striae atrophicae. In particular, although the composition containing exosomes of the present invention was treated with dexamethasone that inhibits the expression of collagen and fibroblasts in skin cells, compared with hyaluronic acid, it was confirmed that the composition can significantly promote the expression of collagen and elastin genes (Example 8 and Figures 9 to 10 ).

[0056] In this specification, the term "comprising as an active ingredient" means containing an amount sufficient to achieve the activity of a specific effect (such as preventing striae atrophicae, increasing skin elasticity, preventing skin aging, or promoting skin regeneration effect) of exosomes isolated from stem cells or their cultures.

[0057] In one implementation example of the present invention, the mesenchymal stem cells derived from induced pluripotent stem cells can be differentiated from the precursor cells of the mesenchymal stem cells derived from induced pluripotent stem cells that do not express stage-specific embryonic antigen 4 (SSEA-4) protein.

[0058] In one implementation example of the present invention, the cosmetic material composition may have a dosage form selected from the group consisting of solutions, topical ointments, creams, foams, nutritious lotions, softening lotions, facial masks, softening skin lotions, emulsions, makeup primers, serums, soaps, liquid detergents, bath agents, sunscreen creams, sunscreen oils, suspensions, emulsions, pastes, gels, emollient lotions, powders, soaps, surfactant-containing facial cleansers, oils, powder foundations, emulsion foundations, wax foundations, patches, and sprays, but is not limited thereto.

[0059] When the dosage form of the present invention is an ointment, paste, cream, or gel, carrier components such as animal oils, vegetable oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silica, talc, and zinc oxide can be used, but are not limited thereto. They can be used alone or in combination of two or more.

[0060] When the dosage form of the present invention is a powder or a spray, carrier components such as lactose, talc, silica, aluminum hydroxide, calcium silicate, and polyamide powder can be used. In particular, when the dosage form is a spray, a propellant such as chlorofluorocarbon, propane / butane, or dimethyl ether can also be included, but is not limited thereto. They can be used alone or in combination of two or more.

[0061] When the dosage form of the present invention is a solution or an emulsion, carrier components such as solvents, solubilizers, or emulsifiers can be used. For example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol oil, etc. can be used. In particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, sesame oil, glycerol fatty acid esters, polyethylene glycol, or sorbitan can be used, but is not limited thereto. They can be used alone or in combination of two or more.

[0062] When the dosage form of the present invention is a suspension, carrier components such as liquid diluents (such as water, ethanol, or propylene glycol), suspending agents (such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitan ester, and polyoxyethylene dehydrated sorbitol ester), microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tragacanth can be used, but is not limited thereto. They can be used alone or in combination of two or more.

[0063] Another aspect of the present invention is a pharmaceutical composition for preventing striae atrophicae, increasing skin elasticity, preventing skin aging, or promoting skin regeneration, which contains exosomes isolated from mesenchymal stem cells (MSCs) derived from induced pluripotent stem cells (iPSCs) as an active ingredient.

[0064] The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable carrier.

[0065] The term "pharmaceutically acceptable" in this specification refers to a compound commonly used in the pharmaceutical field that does not stimulate the organism and does not inhibit the biological activity and characteristics of the administered compound when administered.

[0066] In the present invention, any carrier commonly used in the technical field can be used. Non-limiting examples of the carrier may be saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, maltodextrin, glycerol, ethanol, or a combination thereof, etc.

[0067] In the present invention, other pharmaceutically acceptable additives (such as excipients, diluents, antioxidants, buffers, or bacteriostatic agents, etc.) can be added to the pharmaceutical composition as needed, and fillers, extenders, wetting agents, disintegrants, dispersants, surfactants, binders, or lubricants, etc. can also be added for use.

[0068] Another aspect of the present invention is a quasi-drug for preventing striae atrophicae, increasing skin elasticity, preventing skin aging, or promoting skin regeneration, which contains exosomes isolated from mesenchymal stem cells (MSCs) derived from induced pluripotent stem cells (iPSCs) as an active ingredient.

[0069] The dosage form of the quasi-drug composition of the present invention can be a body shower gel, a disinfectant cleaner, a detergent, a kitchen detergent, a wet wipe, a soap, a hand sanitizer, or an ointment, but is not limited thereto.

[0070] Another aspect of the present invention is a skin external preparation for preventing striae atrophicae, increasing skin elasticity, preventing skin aging, or promoting skin regeneration, which contains exosomes isolated from mesenchymal stem cells (MSCs) derived from induced pluripotent stem cells (iPSCs) as an active ingredient.

[0071] Another aspect of the present invention is a method for preparing a cosmetic material composition for preventing striae cutis, increasing skin elasticity, preventing skin aging or promoting skin regeneration, the preparation method comprising the following steps:

[0072] A separation step of separating exosomes from stem cells or their cultures.

[0073] In one implementation example of the present invention, the separation step may be a step of separating exosomes from mesenchymal stem cells derived from induced pluripotent stem cells or their cultures.

[0074] In one implementation example of the present invention, the stem cells may be autologous or allogeneic stem cells, may be of any type of animal origin including humans and non-human mammals, and may be adult-derived stem cells or embryonic-derived stem cells. For example, the stem cells may be embryonic stem cells, adult stem cells, induced pluripotent stem cells (iPSCs), mesenchymal stem cells derived from induced pluripotent stem cells, BxC stem cells, and BxC-R11 stem cells, but are not limited thereto.

[0075] In the separation step, the culture medium of the stem cells is centrifuged at 200xg to 400xg for 5 minutes to 20 minutes, and after removing the remaining cells and cell residues, the supernatant is taken, and then centrifuged at 9000xg to 12000xg at high speed for 60 minutes to 80 minutes, and the supernatant is taken again, and centrifuged at 90000xg to 120000xg for 80 minutes to 100 minutes, and the supernatant is removed, thereby obtaining the exosomes remaining in the lower layer.

[0076] In one implementation example of the present invention, the method may further include a pretreatment step of pretreating mesenchymal stem cells derived from induced pluripotent stem cells with hyaluronic acid.

[0077] In one implementation example of the present invention, the method may further include a screening and culturing step of separating and culturing SSEA-4(-) cells in cultured induced pluripotent stem cells to differentiate them into BxC stem cells.

[0078] In one implementation example of the present invention, the method may further include a production step of culturing extracellular exosomes of stem cells through a cell culture medium.

[0079] The exosome production step of the present invention is a process of inducing the secretion or production of exosomes from stem cells. In the present invention, the cell culture medium may include all the culture media for stem cell culture commonly used in the art. For example, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10 (DMEM / F-10), Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 (DMEM / F-12), α-Minimal essential Medium (α-MEM), Glasgow's Minimal Essential Medium (G-MEM), Isocove's Modified Dulbecco's Medium (IMDM), KnockOut DMEM, and E8 (Essential8Medium) and other commercially prepared media or artificially synthesized media can be used, but it is not limited thereto.

[0080] In one implementation example of the present invention, the cell culture medium may further contain components such as a carbon source, a nitrogen source, trace element components, amino acids, and antibiotics.

[0081] In one implementation example of the present invention, the exosome production step may include a further culturing step of culturing stem cells with fetal bovine serum (FBS) from which exosomes have been removed.

[0082] The FBS from which exosomes have been removed in the cell culture medium, different from ordinary FBS that contains a large amount of exosomes derived from bovine serum, can prevent the contamination of exosomes derived from FBS other than the exosomes secreted by stem cells due to the removal of exosomes.

[0083] Another aspect of the present invention is a method for preparing a composition containing exosomes for preventing striae atrophicae, increasing skin elasticity, preventing skin aging, or promoting skin regeneration, the preparation method including the following steps:

[0084] The first culture step of culturing induced pluripotent stem cells in a culture medium;

[0085] The screening and culture step of isolating and culturing SSEA-4(-) cells from the cultured induced pluripotent stem cells to differentiate into BxC stem cells;

[0086] The second culture step of culturing BxC stem cells and differentiating them into mesenchymal stem cells;

[0087] The pretreatment step of pretreating hyaluronic acid (HA) in mesenchymal stem cells;

[0088] The production step of producing exosomes by culturing the pretreated mesenchymal stem cells; and

[0089] The isolation step of isolating exosomes from mesenchymal stem cells or their culture.

[0090] In one implementation example of the present invention, the first culture step may be a step of culturing induced pluripotent stem cells in a culture medium containing FBS and bFGF for 1 day to 10 days.

[0091] In one implementation example of the present invention, the screening and culture step may be a step of isolating SSEA-4(-) cells from induced pluripotent stem cells and culturing them in a culture medium containing FBS and bFGF for 1 day to 10 days to differentiate them into BxC stem cells.

[0092] In one implementation example of the present invention, the pretreatment step may be a step of culturing mesenchymal stem cells in a culture medium containing hyaluronic acid at a concentration of 0.1 μg / ml to 1000 μg / ml, 0.5 μg / ml to 1000 μg / ml, 1 μg / ml to 500 μg / ml, 1 μg / ml to 200 μg / ml, 1 μg / ml to 100 μg / ml, 1 μg / ml to 80 μg / ml, 1 μg / ml to 60 μg / ml, 10 μg / ml to 60 μg / ml, for example, at a concentration of 40 μg / ml.

[0093] In one implementation example of the present invention, the production step may include a further culture step of culturing mesenchymal stem cells with fetal bovine serum (FBS) from which exosomes have been removed.

[0094] Another aspect of the present invention is a method for preventing striae atrophicae, increasing skin elasticity, preventing skin aging or promoting skin regeneration, the method comprising the following steps:

[0095] A step of contacting exosomes isolated from mesenchymal stem cells (MSCs) derived from induced pluripotent stem cells (iPSCs) with the skin of a subject.

[0096] In one embodiment of the present invention, the exosomes may comprise exosomes derived from BxC-R11 stem cells (BxC-R11e).

[0097] The term "administration" in this specification refers to providing a prescribed substance to a patient by any suitable method, and the administration route of the cosmetic material composition or pharmaceutical composition of the present invention may be oral or parenteral administration through all conventional routes as long as it can reach the target tissue. In addition, the composition of the present invention may also be administered using any device capable of delivering the active ingredient to the target cell.

[0098] The term "subject" in this specification is not particularly limited but includes, for example, humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs. For example, it may be a human, but is not limited thereto.

[0099] In one embodiment of the present invention, the pharmaceutical composition of the present invention may be administered alone, but usually, in consideration of the administration method and standard pharmaceutical practice, it may be administered in admixture with a selected pharmaceutical carrier.

[0100] Another aspect of the present invention is the use of exosomes isolated from mesenchymal stem cells (MSCs) derived from induced pluripotent stem cells (iPSCs) in preventing skin striae distensae, increasing skin elasticity, preventing skin aging, or promoting skin regeneration.

[0101]

Effects of the Invention

[0102] The present invention relates to a skin aging-inhibiting composition containing exosomes derived from stem cells and a method for preparing the same. The composition containing exosomes of the present invention has excellent effects of preventing skin striae distensae, increasing skin elasticity, preventing skin aging, or promoting skin regeneration, and thus can be used as various cosmetic materials and pharmaceutical compositions.

Description of the Drawings

[0103] Figure 1It is a graph for confirming the degree of improvement in the cell proliferation rate in aged human dermal fibroblasts (aged HDFs) of the control group (vehicle and hyaluronic acid) and the experimental group [R11e (HA-iMSC-EVs) exosomes] according to an embodiment of the present invention.

[0104] Figure 2 It is a graph for confirming the degree of NANOG gene expression in aged human dermal fibroblasts (aged HDFs) of the control group [vehicle, hyaluronic acid, and young human dermal fibroblasts] and the experimental group [R11e (HA-iMSC-EVs) exosomes] according to an embodiment of the present invention.

[0105] Figure 3 It is a graph for confirming the degree of senescence associated beta galactosidase activity (SA-β-Gal) in aged human dermal fibroblasts (aged HDFs) of the control group [vehicle, hyaluronic acid, and young human dermal fibroblasts] and the experimental group [R11e (HA-iMSC-EVs) exosomes] according to an embodiment of the present invention.

[0106] Figure 4 It is a graph showing the results of confirming the SA-β-Gal activity (senescence associated beta galactosidase activity) in aged human dermal fibroblasts (aged HDFs) of the control group [vehicle, hyaluronic acid, and young human dermal fibroblasts] and the experimental group [R11e (HA-iMSC-EVs) exosomes] according to an embodiment of the present invention.

[0107] Figure 5It is a graph showing the degree of epidermal growth factor (EGF) gene expression in skin fibroblasts of the control group [vehicle, hyaluronic acid, and young human dermal fibroblasts] and the experimental group [R11e (HA-iMSC-EVs) exosomes] according to an embodiment of the present invention.

[0108] Figure 6 It is a graph showing the degree of elastin gene expression in skin fibroblasts of the control group [vehicle, hyaluronic acid, and young human dermal fibroblasts] and the experimental group [R11e (HA-iMSC-EVs) exosomes] according to an embodiment of the present invention.

[0109] Figure 7 It is a graph showing the results of observing elastin protein expression in skin fibroblasts of the control group [vehicle, hyaluronic acid, and young human dermal fibroblasts] and the experimental group [R11e (HA-iMSC-EVs) exosomes] according to an embodiment of the present invention.

[0110] Figure 8 It is a graph showing the results of observing elastin protein expression in skin fibroblasts of the control group [vehicle, hyaluronic acid, and young human dermal fibroblasts] and the experimental group [R11e (HA-iMSC-EVs) exosomes] according to an embodiment of the present invention.

[0111] Figure 9 It is a graph showing the results of measuring the collagen expression level after treating fibroblasts with dexamethasone according to an embodiment of the present invention.

[0112] Figure 10 It is a graph showing the results of measuring the elastin expression level after treating fibroblasts with dexamethasone according to an embodiment of the present invention.

[0113] Figure 11 It is a graph showing the results of measuring the cell viability after treating fibroblasts with dexamethasone according to an embodiment of the present invention.

[0114] Figure 12 It is a graph showing the results of measuring the expression of collagen (COL1a) gene in skin fibroblasts cultured in a control group [skin fibroblasts cultured in serum-free DMEM] according to an embodiment of the present invention, a negative control group treated with a vehicle (PBS), a positive control group treated with 100 μg / ml of hyaluronic acid, and skin fibroblasts treated with a test substance [R11e (HA-iMSC-EVs) exosomes].

[0115] Figure 13 It is a graph showing the results of measuring the expression of elastin (ELN) gene in skin fibroblasts cultured in a control group [skin fibroblasts cultured in serum-free DMEM] according to an embodiment of the present invention, a negative control group treated with a vehicle (PBS), a positive control group treated with 100 μg / ml of hyaluronic acid, and skin fibroblasts treated with a test substance [R11e (HA-iMSC-EVs) exosomes].

Detailed Description of the Invention

[0116] A cosmetic material composition for preventing striae cutis, increasing skin elasticity, preventing skin aging or promoting skin regeneration, comprising exosomes isolated from induced pluripotent stem cell (iPSC)-derived mesenchymal stem cell (MSC) as an active ingredient.

Specific Example

[0118] Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited to these examples.

[0119]

Example 1: Culture of Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells

[0120] Induced pluripotent stem cells (iPSCs) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 10 ng / ml bFGF for 7 days. Then, by FACS analysis, SSEA-4(-) cells that did not express stage-specific embryonic antigen 4 (SSEA-4) protein on the cell surface were isolated from the cultured induced pluripotent stem cells, and precursor cells of mesenchymal stem cells derived from induced pluripotent stem cells were obtained. Next, the isolated SSEA-4(-) cells were passaged and further cultured in DMEM medium supplemented with 10% FBS and 10 ng / ml bFGF for 7 days to prepare BxC stem cells.

[0121] Then, the BxC stem cells were further cultured in a medium containing high-glucose DMEM (Gibco, USA), 10% FBS (HyClone, USA), and 1% MEM non-essential amino acid solution (100X, Gibco, USA) to fully differentiate into mesenchymal stem cells derived from induced pluripotent stem cells.

[0122]

Example 2: Isolation of exosomes pretreated with hyaluronic acid (BxC-R11e)

[0123] The mesenchymal stem cells derived from induced pluripotent stem cells prepared in Example 1 were cultured in high-glucose DMEM medium containing 10% fetal bovine serum, 1% MEM non-essential amino acid solution, and 40 μg / ml hyaluronic acid for 24 hours to prepare mesenchymal stem cells derived from induced pluripotent stem cells pretreated with hyaluronic acid (BxC-R11 stem cells).

[0124] After the culture was completed, the BxC-R11 stem cells were washed and further cultured in a medium supplemented with 10% exosome-depleted FBS for 72 hours.

[0125] After 72 hours of culture, the medium treated with the pre-treated substance was recovered and centrifuged at 300 x g for 10 minutes to remove the remaining cells and cell residues. Next, the supernatant was taken and filtered through a 0.22 μm filter, and then centrifuged at 10,000 x g and 4 °C for 70 minutes using a high speed centrifuge. Then, the supernatant obtained by centrifugation was taken again and centrifuged at 100,000 x g and 4 °C for 90 minutes using an ultracentrifuge to remove the supernatant, and the exosomes remaining in the lower layer were diluted in PBS to isolate hyaluronic acid-pretreated exosomes (hereinafter, BxC-R11e exosomes), which were used in the following experiments.

[0126]

Example 3: Confirmation of improvement in the reduced cell proliferation rate in senescent fibroblasts

[0127] To confirm whether the reduced cell proliferation rate can be improved in senescent cells, after treating fibroblasts (human dermal fibroblasts (HDFs)) with the exosomes (BxC-R11e) pretreated with hyaluronic acid produced in Example 2, the cell proliferation rate within 24 hours was compared.

[0128] 5000 senescent fibroblasts (Aged HDF) were seeded in each well of a 96-well plate, and in order for the cells to attach, they were cultured for more than 16 hours at 37 °C and 5% CO2 using a basal medium (High glucose DMEM, 10% FBS, 100 U / mL penicillin-streptomycin). In serum-free DMEM, the experimental group was treated with exosomes (BxC-R11e) pretreated with 1 - 100 μg / ml of the test drug (hyaluronic acid), the negative control group was treated with PBS, and the positive control group was treated with 100 μg / ml of hyaluronic acid (HA) and then cultured for 24 hours.

[0129] Then, the cell proliferation rate was determined using the Cell Count Kit-8 (CCK-8) assay (Enzo Life Sciences, Inc., New York, NY, USA). After culturing for 24 hours, the CCK-8 solution was added, and after culturing for 2 hours at 37 °C, the optical density (OD) was measured at a wavelength of 450 nm using a microplate reader. After averaging, the measured values for each well (n = 3 / group) were expressed as relative values based on the average value of the vehicle group, which served as the negative control group, and are shown in Figure 1 and Table 1.

[0130]

Table 1

[0131]

[0132] As Figure 1 shown in Table 1, with the number of cells in the negative control group (PBS-treated group) after 24 hours of drug treatment set as 1, the results of measuring the relative proliferation rate showed that the positive control group (HA-treated group) increased by approximately 18% to 1.18. On the other hand, it was confirmed that compared with the negative control group, the proliferation rate of the group treated with 1 μg / ml of R11e increased by 18%, the proliferation rate of the group treated with 10 μg / ml of R11e increased by 26% compared with the negative control group, and the group treated with 100 μg / ml of R11e increased to 39%. Compared with the negative control group and the positive control group, the cell proliferation rate was significantly increased.

[0133]

Example 4: Confirmation of cell regeneration effect

[0134] To confirm the renewal effect of fibroblasts, skin fibroblasts were treated with the exosomes (BxC-R11e) pretreated with the hyaluronic acid produced in Example 2, and the expression of the cell NANOG gene after 24 hours was compared.

[0135] In each well of a 12-well plate, inoculate aged human dermal fibroblasts (Aged HDF) at 20,000 cells / well, and for cell attachment, culture in a 37°C and 5% CO2 environment using a basal medium (high glucose DMEM, 10% FBS, 100 U / mL penicillin-streptomycin) for more than 16 hours. In serum-free DMEM, the experimental group was treated with exosomes (BxC-R11e) pretreated with 1 - 100 μg / ml of the test drug (hyaluronic acid), the negative control group was treated with PBS, and the positive control group was treated with 100 μg / ml of hyaluronic acid (HA), and then cultured for 24 hours.

[0136] After culturing for 24 hours, recover the cells, extract total RNA using Trizol (Invitrogen), and analyze by real-time PCR to measure the mRNA expression of each gene from an equal amount of total RNA. Normalize each measured value based on GAPDH expression and perform relative quantification by the (2 -ΔΔ Ct method), and the results are shown in Figure 2 and Table 2.

[0137]

Table 2

[0138]

[0139] As Figure 2 and Table 2 show, taking the number of cells in the negative control group (PBS-treated group) after 24 hours of drug treatment as 1, the result of measuring NANOG gene expression shows that there is little difference between the positive control group (HA-treated group) and the negative control group, which is 1.01. On the other hand, it was confirmed that compared with the negative control group, the expression level in the group treated with 10 μg / ml of R11e increased by 17%, and the group treated with 100 μg / ml of R11e increased to 370%, similar to the NANOG expression level of non-aged young HDFs.

[0140]

Example 5: Confirm the reduction of SA-β-Gal activity in aged cells

[0141] To confirm whether it is possible to reduce the increased senescence-associated beta galactosidase (SA-β-Gal) activity in aged cells, aged skin fibroblasts were treated with exosomes (BxC-R11e) pretreated with the hyaluronic acid produced in Example 2, and the cell proliferation rate after 24 hours was compared.

[0142] Aged human dermal fibroblasts (Aged HDF) were seeded at 20,000 cells / well in each well of a 12-well plate, and to allow cell attachment, they were cultured for more than 16 hours in a 37 °C and 5% CO2 environment using a basal medium (high-glucose DMEM, 10% FBS, 100 U / mL penicillin-streptomycin). In serum-free DMEM, the experimental group was treated with exosomes (BxC-R11e) pretreated with 100 μg / ml of the test drug (hyaluronic acid), the negative control group was treated with PBS, and the positive control group was treated with 100 μg / ml of hyaluronic acid (HA) and then cultured for 24 hours.

[0143] SA-β-Gal assay was performed using a Senescence Detection Kit (Abcam, Cambridge, UK). After 24 hours of culture, 4% paraformaldehyde was used to fix the cells by allowing them to stand at room temperature for 20 minutes. Then, referring to the kit's catalogue, SA-β-Gal staining was performed and observed under a microscope Figure 3 ). Aged cells were stained blue, and the stained area was also measured using the ImageJ program, and the results are shown in Figure 4 and Table 3.

[0144]

Table 3

[0145] Differentiation Vector HA-iMSC-EVs(BxC-R11e) HA Young HDFs SA-β-Gal activity 4.45 1.25 8.67 1

[0146] As Figure 4 shown in and Table 3, after 24 hours of drug treatment, it was measured that the SA-β-Gal activity of the negative control group was approximately 4 times (4.45) higher than that of non-aged young HDFs. Conversely, compared with the negative control group, the activity of the group treated with R11e was reduced by approximately 3.6 times to 1.25 and showed a level similar to that of non-aged young HDFs.

[0147]

Example 6: Confirmation of increased expression of epidermal growth factor (EFG) and elastin genes

[0148] To confirm the renewal effect of the test substance on fibroblasts, the epidermal growth factor (EGF) and elastin gene expressions of cells after 24 hours were compared by treating skin fibroblasts with exosomes (BxC-R11e) pretreated with hyaluronic acid produced in Example 2.

[0149] Aged human dermal fibroblasts (Aged HDF) were seeded at 20,000 cells / well in each well of a 12-well plate and cultured for more than 16 hours in a basal medium (high glucose DMEM, 10% FBS, 100 U / mL penicillin-streptomycin) at 37 °C and 5% CO2 for cell attachment. In serum-free DMEM, the experimental group was treated with exosomes (BxC-R11e) pretreated with 100 μg / ml of the test drug (hyaluronic acid), the negative control group was treated with PBS, and the positive control group was treated with 100 μg / ml of hyaluronic acid (HA) and then cultured for 24 hours.

[0150] After culturing for 24 hours, the cells were harvested, total RNA was extracted using Trizol (Invitrogen), and the mRNA expressions of each gene were measured from an equal amount of total RNA by real-time PCR analysis. Each measured value was normalized based on GAPDH expression and relatively quantified by the (2 -ΔΔ Ct method), and the results are shown in Figures 5 to 6 and Table 4.

[0151]

Table 4

[0152] Differentiation Vector HA-iMSC-EVs(BxC-R11e) HA Young HDFs EGF expression 1 2.65 0.67 2.45 Elastin expression 1 3.39 1.51 2.14

[0153] After 24 hours of drug treatment, with the cell count of the negative control group (PBS-treated group) as 1, the results of measuring relative gene expression are shown in Figures 5 to 6As shown in Table 4, it was confirmed that, compared with the negative control group, in the experimental group treated with BxC-R11e, the EGF expression increased by 2.7 times and the elastin increased by 3.4 times.

[0154]

Example 7: Confirmation of elastin protein expression

[0155] In each well of a 12-well plate, 40,000 aged human dermal fibroblasts (Aged HDF) were seeded per well, and for cell attachment, they were cultured for more than 24 hours at 37 °C and 5% CO2 using a basal medium (high glucose DMEM, 10% FBS, 100 U / mL penicillin-streptomycin). In DMEM containing 5% FBS (serum-free DMEM), the experimental group was treated with exosomes (BxC-R11e) pretreated with 100 μg / ml of the test drug (hyaluronic acid), the negative control group was treated with PBS, and the positive control group was treated with 100 μg / ml of hyaluronic acid (HA) and then cultured for 48 hours.

[0156] After culturing for 48 hours, the cells were fixed with 100% methanol and washed with PBS, and then reacted with a primary antibody (anti-tropoelastin antibody, Elastin Products, Owensville, MI). After washing again, they were reacted with an FITC-labeled secondary antibody and then observed under a fluorescence microscope ( Figure 7 ). The ImageJ program was used to measure the stained area, which is shown in Figure 8 and Table 5.

[0157]

Table 5

[0158] Differentiation Vector HA-iMSC-EVs(BxC-R11e) HA Young HDFs EGF protein expression 1 2.65 0.67 2.45

[0159] Generally, although the increase in elastin protein expression is important, elastin fibers need to assemble to perform their inherent functions, and in an in vitro environment, the assembly of elastin protein into elastin fibers takes time.

[0160] For these reasons, as Figures 7 to 8As shown in Table 4, in young HDFs, only the protein expression of elastin increased, and no elastin fibers were observed. In contrast, in the experimental group treated with BxC-R11e, not only did the protein expression of elastin increase, but elastin fibers were also observed. Thus, it was confirmed that exosomes pretreated with hyaluronic acid (BxC-R11e) also accelerated the assembly of elastin fibers.

[0161]

Example 8: Confirmation of stretch mark prevention effect

[0162] 【8-1. Confirmation of inhibition of collagen and elastin expression by dexamethasone treatment】

[0163] Skin fibroblasts (HDFs) were seeded at 20,000 cells / well in each well of a 12-well plate, and to promote cell attachment, they were cultured for more than 24 hours at 37 °C in a 5% CO2 environment using a basal medium (high-glucose DMEM, 10% FBS, 100 U / mL penicillin-streptomycin).

[0164] Then, dexamethasone, one of the corticosteroid drugs, was mixed at different concentrations in a serum-free DMEM medium and used to treat the skin fibroblasts. After 24 hours of treatment, the skin fibroblasts were obtained, and the expression levels of collagen and elastin genes were confirmed and shown in Figures 9 to 11 as follows.

[0165] As Figures 9 to 10 shown, the experimental results indicated that the expression of collagen and elastin could be inhibited by dexamethasone treatment. On the other hand, as Figure 11 shown, it was confirmed that dexamethasone treatment did not affect the cell viability of skin fibroblasts, and the concentration of dexamethasone used for treatment was non-toxic to the cells.

[0166] 【8-2. Confirmation of stretch mark prevention effect by treatment with exosomes pretreated with hyaluronic acid】

[0167] To confirm the stretch mark prevention effect, after treating skin fibroblasts treated with dexamethasone, a glucocorticoid steroid, with the test substance for 24 hours, the changes in the gene expression of collagen and elastin were confirmed.

[0168] Human dermal fibroblasts (HDF) were seeded at 20,000 cells / well in a 12-well plate and cultured for more than 16 hours in a basal medium (high-glucose DMEM, 10% FBS, 100 U / mL penicillin-streptomycin) at 37 °C and 5% CO2 for cell attachment. After mixing dexamethasone in serum-free DMEM, the test drug or control drug was mixed and treated, and then cultured for 24 hours. At this time, the normal control group was set as human dermal fibroblasts cultured in serum-free DMEM medium, the negative control group was set as human dermal fibroblasts treated with vehicle (PBS), the positive control group was set as the group treated with hyaluronic acid (HA) at a concentration of 100 μg / ml, and the test group was set as the group pretreated with exosomes pretreated with 100 μg / ml hyaluronic acid (HA-iMSC-EVs, BxC-R11e).

[0169] After 24 hours of culture, the cells were harvested, total RNA was extracted using Trizol (Invitrogen), and mRNA expression of each gene was measured from an equal amount of total RNA by real-time PCR analysis. Each measured value was normalized based on GAPDH expression and relatively quantified by the (2 -ΔΔ -ΔΔCt method).

[0170] [Table 6]

[0171] Gene Normal control group Dexamethasone only Dexamethasone + PBS Dexamethasone + R11e Dexamethasone + HA COL1a 3.55 1.53 1.00 3.30 1.73 ELN 5.96 1.44 1.00 4.59 1.65

[0172] As Figures 12 to 13 shown in Table 6, the experimental results showed that in the group treated only with dexamethasone, the gene expressions of collagen (COL1a) and elastin (ELN) were decreased by 57% and 75%, respectively, compared with the normal control group. On the other hand, after 24 hours of drug treatment, when the cell number of the negative control group (PBS-treated group) was set as 1, the relative proliferation rate was measured. The expressions of COL1a and ELN in the positive control group (HA-treated group) were increased by about 73% (1.73) and 65% (1.65), respectively. In the group treated with exosomes pretreated with the test substance (hyaluronic acid) (HA-iMSC-EVs, BxC-R11e), the COL1a gene expression was increased by 330% and the ELN gene expression was increased by 459% compared with the vehicle group.

[0173] Clinically, striae distensae skin lesions are characterized by reduced expression of collagen and elastin. Currently, to treat striae distensae, drugs that increase collagen in the dermis and laser therapy are commonly used. It has been confirmed that the hyaluronic acid-pretreated exosomes (HA-iMSC-EVs, BxC-R11e) of the present invention can significantly increase the expression of collagen and elastin genes in a dexamethasone-treated skin fibroblast model, and thus effectively prevent striae distensae.

[0174]

Industrial Applicability

[0175] The present invention relates to a skin aging inhibitory composition containing exosomes derived from stem cells and a method for preparing the same. More specifically, the present invention relates to a composition containing exosomes isolated from mesenchymal stem cells or their cultures, which are excellent in preventing skin striae distensae, increasing skin elasticity, preventing skin aging, or promoting skin regeneration.

Claims

1. A cosmetic material composition for preventing striae cutis distensae, increasing skin elasticity, preventing skin aging, or promoting skin regeneration, which contains, as an active ingredient, exosomes isolated from mesenchymal stem cells derived from induced pluripotent stem cells.

2. The cosmetic material composition according to claim 1, wherein the mesenchymal stem cells derived from induced pluripotent stem cells are differentiated from precursor cells of mesenchymal stem cells derived from induced pluripotent stem cells that do not express stage-specific embryonic antigen 4 protein.

3. The cosmetic material composition according to claim 1, wherein the induced pluripotent stem cells are human-derived induced pluripotent stem cells.

4. The cosmetic material composition according to claim 1, wherein the mesenchymal stem cells derived from induced pluripotent stem cells are stem cells pretreated with a pretreatment substance.

5. The cosmetic material composition according to claim 4, wherein the pretreatment substance is hyaluronic acid.

6. The cosmetic material composition according to claim 1, wherein the dosage form of the cosmetic material composition is selected from the group consisting of solution, topical ointment, cream, foam, nutritional lotion, soft lotion, facial mask, skin softener, emulsion, makeup primer, essence, soap, liquid detergent, bath agent, sunscreen, sunscreen oil, suspension, emulsion, paste, gel, emollient lotion, powder, soap, surfactant-containing facial cleanser, oil, foundation, powdery emulsion foundation, waxy foundation, patch, and spray.

7. An external preparation for pharmaceuticals for preventing striae cutis distensae, increasing skin elasticity, preventing skin aging, or promoting skin regeneration, which contains, as an active ingredient, exosomes isolated from mesenchymal stem cells derived from induced pluripotent stem cells.

8. The external preparation for pharmaceuticals according to claim 7, wherein the mesenchymal stem cells derived from induced pluripotent stem cells are stem cells pretreated with a pretreatment substance.

9. The external preparation for pharmaceuticals according to claim 8, wherein the pretreatment substance is hyaluronic acid.

10. An external preparation for pharmaceuticals for preventing striae cutis distensae, increasing skin elasticity, preventing skin aging, or promoting skin regeneration, which contains, as an active ingredient, exosomes isolated from mesenchymal stem cells derived from induced pluripotent stem cells.

11. The topical skin preparation according to claim 10, wherein the mesenchymal stem cells derived from induced pluripotent stem cells are stem cells pretreated with a pretreatment substance.

12. The topical skin preparation according to claim 8, wherein the pretreatment substance is hyaluronic acid.

Images