High-efficiency technology for increasing stem cell-derived extracellular vesicles and proteins by using nucleic acid fragment mixture-based culture method and culture medium

Abstract

A medium comprising a nucleic acid fragment mixture, and a stem cell culture technique based on same for high-efficiency production of stem cell-derived extracellular vesicles and proteins, of the present invention, can significantly increase the expression level of proteins such as growth factors and the production of extracellular vesicles of stem cells. Stem cells cultured by the method or extracellular vesicles obtained therefrom have a high protein amount, and thus can be effectively used in the cosmetic field for applications such as skin whitening, skin wrinkle reduction, hair color change, hair loss alleviation or prevention, acne alleviation, ultraviolet protection, skin inflammation alleviation, cell regeneration, antioxidant activity, anti-aging, skin moisturizing, skin condition improvement, skin soothing and stress relief, or skin texture refining and elasticity enhancement, and in the medical field for applications such as hair loss, inflammatory diseases, acne and folliculitis management, and scar prevention.

Description

High-efficiency stem cell-derived extracellular vesicle and protein amplification technology using nucleic acid fragment mixture-based culture methods and culture media

[0001] The present invention relates to a technology for producing high-efficiency stem cell-derived extracellular vesicles and proteins through stem cell culture using a nucleic acid fragment mixture base.

[0002]

[0003] Stem cells are cells capable of differentiating into various types of cells that constitute biological tissues; they are undifferentiated or intermediately differentiated cells obtained from the tissues of embryos, fetuses, and adults, representing a stage prior to final differentiation. Stem cells undergo differentiation into specific cell types in response to differentiation stimuli (environmental factors). Unlike cells that have completed differentiation and ceased cell division, stem cells possess the characteristic of proliferation (expansion) as they can self-renewal by dividing to produce identical cells. Furthermore, they exhibit plasticity in differentiation, as they can differentiate into other cell types under different environments or different differentiation stimuli. In particular, stem cells are reported to produce and secrete biological substances, such as various growth factors and cytokines, at high levels; consequently, their applications are being researched in various fields, including medicine and cosmetics.

[0004] Recently, extracellular vesicles, particularly exosomes, have garnered attention as a novel modality capable of efficiently delivering various payloads to all types of cells within living organisms. While the precise nature and mechanisms of this action are still under investigation, methods to produce exosomes at high titers have been required to exert desired biological and clinical effects (Cheng and Schorey, 2016, Biotech Bioeng 113(6): 1315-1324; Kalluri, 2016, J Clin Invest 126(4): 1208-1215). Although various methods for the mass production of exosomes have been reported, they generally utilize media supplemented with the blood of cancer patients or animal serum (Chaput and Thery, 2011, Semin Immunopathol (2011) 33:419-440; Tarte K et al., 2010, Blood 115, 1549-1553, et al.).

[0005] Generally, culture methods used for the culture of stem cells and the production of exosomes utilize basal culture media (e.g., DMEM, alpha-MEM) supplemented with 5 to 20% fetal bovine serum (FBS) and additional growth agents. Animal-derived components, such as fetal bovine serum, are known to cause problems such as transmissible spongiform encephalopathy (TSE). Including a process to remove animal-derived components can significantly reduce the yield of the final product (stem cells or exosomes) (Cell Prolif. 2013 Sep 30;46(6):608-627). Furthermore, trace components within fetal bovine serum are not only very difficult to analyze but can also vary depending on the time or location of production; this variability makes it difficult to trust the consistency of experimental results and acts as a limiting factor in establishing reproducible testing and production processes (Stem Cell Research & Therapy volume 1, Article number: 8 (2010)). Therefore, there is a strong demand for the development of serum-free media that do not contain heterologous serum, such as bovine fetal serum.

[0006] Meanwhile, polydeoxyribonucleotide (PDRN®) is a mixture of DNA fragments extracted from animals or plants with specific specifications (e.g., defined by molecular weight range or average molecular weight) that is known to have anti-inflammation, cell proliferation, and tissue regeneration effects by stimulating adenosine A2 receptors (Kim, YH, et al., 2010). In Europe and Korea, a mixture of DNA fragments obtained from the sperm of fish such as salmon or trout was named PDRN®. PDRN has been used in the medical field, particularly in dermatology, as an injectable composition for tissue regeneration to accelerate the body's natural wound healing process. Recently, the anti-inflammatory effects of PDRN on scar formation were investigated, and it was discovered that PDRN prevents scar formation by exerting anti-inflammatory and collagen synthesis effects through the inhibition of HMGB-1 (Jeong W et al Int J Mol Sci. 2017 Aug 3;18(8):1698.).

[0007] Polynucleotide (PN) is a mixture of DNA fragments similar to PDRN, but has a longer nucleic acid chain length and a larger average molecular weight than PDRN. These polynucleotides are used as raw materials for medical devices to provide cell adhesion, lubrication, and buffering effects by acting as a physical support.

[0008]

[0009] Under these background technologies, the inventors made diligent efforts to develop a novel serum-free culture process for stem cells. As a result, they confirmed that when stem cells are cultured using a serum-free culture medium containing a mixture of nucleic acid fragments such as PDRN or PN, the amount of protein and the number of exosome particles in the stem cell culture medium increase significantly compared to conventional culture media, and thus completed the present invention.

[0010]

[0011] Summary of the Invention

[0012] The object of the present invention is to provide a method for producing extracellular vesicles comprising the step of culturing stem cells in a culture medium containing a mixture of nucleic acid fragments.

[0013] Another objective of the present invention is to provide a culture medium composition for the production of stem cell-derived extracellular vesicles comprising a mixture of nucleic acid fragments.

[0014] The present invention provides a method for culturing stem cells, comprising the step of culturing stem cells in a culture medium containing a mixture of nucleic acid fragments.

[0015] Another objective of the present invention is to provide a culture medium composition for stem cells comprising a mixture of nucleic acid fragments.

[0016] Another objective of the present invention is to provide stem cells cultured by the above-described culture method, a population thereof, or a culture medium.

[0017] Another objective of the present invention is to provide extracellular vesicles produced by the above-described culture method.

[0018] Another objective of the present invention is to provide a use for the cultured stem cells or a population thereof, the culture medium, or the extracellular vesicles.

[0019]

[0020] To achieve the above-mentioned objective of the present invention, the present invention provides a method for producing extracellular vesicles comprising the steps of: culturing stem cells in a culture medium containing a nucleic acid fragment mixture; and recovering extracellular vesicles from the culture.

[0021] The present invention also provides a method for culturing stem cells, comprising the step of culturing stem cells in a culture medium containing a mixture of nucleic acid fragments.

[0022] The present invention also provides stem cells cultured by the above-described culture method, a population thereof, or a culture medium.

[0023] The present invention also provides an extracellular vesicle produced by the above-described manufacturing method.

[0024] The present invention also provides a pharmaceutical composition comprising the cultured stem cells or a population thereof, a culture medium, or the extracellular vesicles.

[0025] The present invention also provides a cosmetic composition comprising the cultured stem cells or a population thereof, a culture medium, or the extracellular vesicles.

[0026] The present invention also provides the use of the cultured stem cells or a population thereof, a culture medium, or extracellular vesicles for whitening, wrinkle improvement, skin regeneration, antioxidant, wound healing, skin soothing and stress relief, or skin texture improvement and elasticity enhancement.

[0027] The present invention also provides the use of the cultured stem cells or a population thereof, the culture medium, or the extracellular vesicles for the management of hair loss, inflammatory diseases, acne and folliculitis, and for the prevention or treatment of scars.

[0028] The present invention also provides a use for the preparation of a pharmaceutical composition or a cosmetic composition of the cultured stem cells or a population thereof, a culture medium, or extracellular vesicles.

[0029] The present invention also provides a culture medium composition for stem cells comprising a mixture of nucleic acid fragments.

[0030] The present invention also provides a culture medium composition for the production of stem cell-derived extracellular vesicles comprising a mixture of nucleic acid fragments.

[0031]

[0032] Figure 1 shows the results of determining the number of stem cells according to the concentration of PDRN and oxygen conditions (Normoxia or Hypoxia) after 24 or 48 hours of culture.

[0033] Figure 2 shows the results of confirming the total protein amount in the stem cell culture medium according to the concentration of PDRN and oxygen conditions (Normoxia or Hypoxia) after 24 or 48 hours of culture.

[0034] Figure 3 shows the results of determining the number of exosome particles in the cell culture medium according to the concentration of PDRN and oxygen conditions (Normoxia or Hypoxia) after 24 or 48 hours of culture.

[0035] Figure 4 is the result of identifying exosome-specific markers in particles present in a cell culture medium prepared by the method of the present invention.

[0036] Figure 5 is the result of confirming the growth factor content in the cell culture medium prepared by the method of the present invention.

[0037]

[0038] Detailed Description of the Invention and Preferred Embodiments

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled expert in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0040]

[0041] Clinical-grade stem cell therapies or exosomes are typically characterized by culturing cells in media supplemented with the blood of cancer patients or animal serum. Since animal serum contains various impurities and has been reported to cause various side effects due to the aggregation of modified proteins when administered to humans, there is a strong demand for the development of serum-free media for stem cell culture or exosome production (Escudier et al., 2005, J Transl Med 3(1): 10; Morse et al., 2005, J Transl Med 3(l):9). However, serum-free media generally have the problem of showing insufficient effects in terms of cell growth, protein production, and exosome count compared to media containing animal serum.

[0042]

[0043] In one embodiment of the present invention, the inventors confirmed that when stem cells are cultured in a serum-free medium containing PDRN, the amount of protein expression and the number of exosome particles in the culture medium are significantly increased compared to a medium without PDRN.

[0044]

[0045] Accordingly, in one aspect, the present invention relates to a method for producing extracellular vesicles comprising the steps of: culturing stem cells in a culture medium containing a mixture of nucleic acid fragments; and recovering extracellular vesicles from the culture.

[0046] In another aspect, the present invention relates to a culture medium composition for the production of stem cell-derived extracellular vesicles comprising a mixture of nucleic acid fragments.

[0047] In another aspect, the present invention relates to a method for culturing stem cells comprising the step of culturing stem cells in a culture medium containing a mixture of nucleic acid fragments.

[0048] In another aspect, the present invention relates to a culture medium composition for stem cells comprising a mixture of nucleic acid fragments.

[0049]

[0050] Culture medium containing a mixture of nucleic acid fragments (medium composition)

[0051] The term “nucleic acid” in the present invention refers to a polynucleotide of any length, which may include DNA, RNA, or a combination of DNA and RNA. In the present invention, the nucleic acid may be a deoxyribonucleotide, a ribonucleotide, as well as a modified nucleotide or base and / or an analog thereof, or any substrate that may be incorporated by DNA or RNA polymerase. In the present invention, the nucleic acid may be RNA, DNA, TNA (Threose nucleic acids), GNA (glycol nucleic acids), PNA (peptide nucleic acids), LNA (locked nucleic acids, e.g., LNA having a β-D-ribo sequence, α-LNA having an α-ribo sequence (a diastereomer of LNA), 2'-amino-LNA or 2'-amino-α-LNA having 2'-amino functionalization), ENA (ethylene nucleic acid), CeNA (cyclohexenyl nucleic acids), or a hybrid or combination thereof, but is not limited thereto.

[0052] In the present invention, preferably, the nucleic acid may be DNA, RNA, or a combination of DNA and RNA, and more preferably, the nucleic acid may be characterized as being DNA.

[0053] The term “nucleic acid fragment mixture” of the present invention refers to a mixture of nucleic acid polymers in which nucleotide monomers (e.g., DNA or RNA monomers) are linked by covalent bonds such as phosphodiester bonds, and may contain a mixture of nucleic acid fragments of various molecular weights.

[0054] In the present invention, the nucleic acid fragment may be in a hydrolyzed form.

[0055] In the present invention, preferably, the nucleic acid fragment mixture may be a DNA fragment mixture. For example, the DNA fragment mixture may be PDRN® (polydeoxyribonucleotide) or PN (polynucleotide), but is not limited thereto.

[0056] The term polydeoxyribonucleotide (PDRN) in the present invention is a mixture of short deoxyribonucleotides and is a type of low molecular weight DNA complex produced by reducing DNA chains to a molecular weight within a certain range.

[0057] In some embodiments of the present invention, the mixture of nucleic acid fragments may be defined by the molecular weight of the nucleic acid fragments included therein. For example, in the present invention, the molecular weight of the nucleic acid fragments included in the mixture of nucleic acid fragments may be about 1 to 100,000 kDa, about 5 to 50,000 kDa, about 50 to 50,000 kDa, about 50 to 10,000 kDa, about 50 to 5,000 kDa, about 50 to 3,000 kDa, about 100 to 3,000 kDa, about 50 to 1,500 kDa, about 1,000 to 10,000 kDa, about 1,000 to 5,000 kDa, or about 5,000 to 10,000 kDa, but is not limited thereto.

[0058] In the present invention, as a preferred example, the nucleic acid fragment mixture may be PDRN or PN. For example, in the case of PDRN, it may include DNA fragments having a molecular weight of about 50 to 1500 kDa or about 100 to 3000 kDa, and in the case of PN, it may include DNA fragments having a molecular weight of about 50 to 10,000 kDa, but is not limited thereto.

[0059] In the present invention, as another preferred example, the molecular weight of the nucleic acid fragment included in the nucleic acid fragment mixture may be 1,000 to 2,700 kDa, but is not limited thereto.

[0060] In the present invention, the mixture of nucleic acid fragments may be characterized by having an average molecular weight of about 10 to 5000 kDa, an average molecular weight of about 50 to 4000 kDa, an average molecular weight of about 100 to 3000 kDa, an average molecular weight of about 200 kDa to 2000 kDa, an average molecular weight of about 300 kDa to 1500 kDa, or an average molecular weight of about 1500 to 2700 kDa, but is not limited thereto. For example, PDRN may include a DNA fragment having an average molecular weight of about 350 kDa, and PN may include a DNA fragment having an average molecular weight of about 1000 kDa, but is not limited thereto.

[0061]

[0062] In addition, the above polynucleotide (PN) refers to a polymer of nucleotides in which nucleotide monomers are linked in a chain shape by covalent bonds, and represents a DNA or RNA strand of a certain length or longer. For example, the above polynucleotide may have a relatively longer nucleic acid length or a larger molecular weight compared to polydeoxyribonucleotide.

[0063] In the present invention, the nucleic acid fragment mixture may be derived from various organisms. For example, it may be derived from microbiomes, animals, plants, or seaweed, and more specifically, from fish such as salmon, trout, cod, mackerel, halibut, carp, sturgeon, etc., mammals such as rabbits, chimpanzees, monkeys, humans, etc., flowers (e.g., Rosaceae, Camellia, Liliaceae, Houttuynia cordata, hibiscus, etc.), ginseng family (e.g., ginseng, red ginseng, wild ginseng, etc.), tea trees (e.g., green tea, black tea, etc.), fruit fruits (e.g., jujube, blueberry, etc.), Angelica (e.g., Apiaceae), seaweed, kelp, sea tangle, Sargassum, laver, and Ecklonia cava, but is not limited thereto. In the embodiments of the present invention, a DNA fragment mixture derived from salmon testes or semen (PDRN, 100403-24-5, HTL biotechnology) was used, but is not limited thereto. In the present invention, the nucleic acid fragment mixture may preferably be a DNA fragment mixture derived from the testes or semen of a fish, more preferably a DNA fragment mixture derived from the testes or semen of a salmon or trout.

[0064] In the technical field of the present invention, various methods for preparing nucleic acid fragment mixtures from various biological sources are well known (e.g., Korean Registered Patents No. 10-0986603, No. 10-2569532, No. 102249241, etc.), and a person skilled in the art may use commercially available nucleic acid fragment mixtures or fragment mixtures extracted using known methods in the present invention.

[0065]

[0066] The method for manufacturing extracellular vesicles and the method for culturing stem cells according to the present invention are characterized by including the step of culturing stem cells in a culture medium containing a mixture of nucleic acid fragments.

[0067] The above medium composition and culture medium may contain a nucleic acid fragment mixture at a concentration of about 50 μg / ml to 1000 μg / ml, about 100 μg / ml to 800 μg / ml, or about 200 μg / ml to 600 μg / ml, preferably about 300 μg / ml to 600 μg / ml, but are not limited thereto.

[0068] In the present invention, the culture medium composition and the culture medium may include, without limitation, components necessary for cell growth such as carbon sources, amino acids, inorganic salts, and vitamins.

[0069] In the present invention, the medium composition and culture medium may be prepared, for example, by adding a mixture of nucleic acid fragments to basal media. The term "basal media" in the present invention refers to a medium containing a minimum amount of components essential for cell culture, and commercially available basal media are known in the art, and any medium usable for the culture of stem cells may be selected and used without limitation. For example, the above basic medium may be selected from the group consisting of DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, DMEM / F12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), MacCoy's 5A medium, AmnioMax complete medium, AminoMax ± complete medium, EBM (Endothelial Basal Medium) medium, Chang's Medium, MesenCult-XF, DMEM / HG (Dulbecco's Modified Eagle's Medium high glucose) medium, DMEM / F12 medium, and MCDB+DMEM / LG (MCDB +Dulbecco's Modified Eagle's Medium low glucose) medium, but is not limited thereto.

[0070] In the present invention, the medium composition or culture medium may be characterized by not containing animal-derived components. More specifically, the medium composition or culture medium may be characterized as a serum-free medium that does not contain animal-derived serum.

[0071] The term “serum-free medium” in this invention refers to a medium that does not contain serum or contains only an effective amount of serum sufficient to allow its biological and physiological functions to be exerted within the culture environment. Serum is a general term for serums commonly used in cell culture, such as FBS (fetal bovine serum), FCS (fetal calf serum), dialyzed fetal bovine serum, and newborn calf serum (NCS).

[0072]

[0073] Method of culturing stem cells using a culture medium containing a mixture of nucleic acid fragments

[0074] The present invention is characterized by culturing stem cells using a culture composition or culture medium comprising a mixture of nucleic acid fragments.

[0075] The step of culturing stem cells in a culture medium containing the nucleic acid fragment mixture of the present invention may be performed by various methods known in the art. For example, it may be performed by adding the nucleic acid fragment mixture to a stem cell culture medium and then culturing, or by exchanging the medium to a culture medium containing the nucleic acid fragment mixture, or by inoculating isolated stem cells into a culture medium containing the nucleic acid fragment mixture, but is not limited thereto.

[0076] In the present invention, the step of culturing stem cells in the culture medium may be characterized by culturing the stem cells under normal oxygen (Normoxia) or hypoxia conditions. The term "Normoxia" in the present invention refers to a condition with a normal atmospheric oxygen concentration of about 20%. The term "Hypoxia" in the present invention refers to a condition with an oxygen concentration lower than that of the atmosphere, and preferably may be an oxygen condition of about 15% or less, about 10% or less, or about 5% or less, but is not limited thereto.

[0077] In the embodiments of the present invention, culture was performed under normal oxygen conditions of about 21% and hypoxic conditions of 2%, and it was confirmed that when stem cells are cultured in a medium containing a nucleic acid fragment mixture, high protein expression rates and a number of exosome particles are exhibited regardless of oxygen conditions. Furthermore, as it is well known in the art that characteristics such as growth factors or cytokines expressed by stem cells change depending on the oxygen conditions of the stem cells, a person skilled in the art can culture stem cells in a medium containing a nucleic acid fragment mixture under appropriate oxygen conditions according to the purpose.

[0078]

[0079] The term "stem cell" in the present invention refers to a cell capable of differentiating into various cells constituting biological tissues, and collectively refers to undifferentiated cells in a pre-differentiation stage that can be obtained from the tissues of embryos, fetuses, and adults. Stem cells undergo differentiation into specific cells in response to differentiation stimuli (environment), and unlike cells whose differentiation is complete and cell division has ceased, they have the characteristic of proliferating (expansion) by producing cells identical to themselves through cell division (self-renewal), and are characterized by having plasticity in differentiation, as they can differentiate into other cells in response to different environments or different differentiation stimuli.

[0080] The types of stem cells that can be cultured by the culture method of the present invention may be selected and used without limitation. For example, in the present invention, the stem cells include pluripotent stem cells, multipotent stem cells, and unipotent stem cells depending on their differentiation potential, but are not limited thereto. Specific examples may include embryonic stem cells, induced pluripotent stem cells (iPSCs), adult stem cells, mesenchymal stem cells derived from embryonic stem cells, or mesenchymal stem cells derived from induced pluripotent stem cells, but are not limited thereto.

[0081] In the present invention, as an example that does not limit the invention, the adult stem cells may be one or more adult stem cells selected from the group consisting of mesenchymal stem cells, mesenchymal stromal cells derived from human tissue, mesenchymal stem cells derived from human tissue, and pluripotent stem cells. The mesenchymal stem cells may be stem cells derived from one or more tissues selected from the group consisting of the umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, Wharton's jelly, and placenta, preferably mesenchymal stem cells derived from said tissues, but are not limited thereto. In the present invention, the type of stem cells is not limited as long as there is no risk of infection by pathogens and no immune rejection reaction, but preferably may be human-derived stem cells, and more preferably may be human amniotic stem cells (AMSC).

[0082]

[0083] Method for manufacturing extracellular vesicles (e.g., exosomes)

[0084] Stem cells cultured with the culture composition or culture method of the present invention exhibit high protein expression, and in particular, are characterized by a significant increase in the number of exosome particles in the culture medium.

[0085] In the technical field of the present invention, various methods for isolating and purifying extracellular vesicles from cell culture medium are well known, and a person skilled in the art can isolate and recover extracellular vesicles by selecting an appropriate method or a commercially available kit depending on the type of cell and the desired size.

[0086] In the present invention, the extracellular vesicle may be recovered into a culture medium containing the same, or may be recovered as an extracellular vesicle isolated through further separation and purification. For example,

[0087] Step of recovering the cell culture supernatant;

[0088] A step of centrifuging the recovered cell culture supernatant; and

[0089] One or more steps of isolating and purifying extracellular vesicles (e.g., exosomes) may be performed to produce a culture medium containing extracellular vesicles or isolated extracellular vesicles, but is not limited thereto.

[0090]

[0091] In the present invention, the extracellular vesicle (EV) refers to a cell-derived vesicle comprising a membrane surrounding an internal space. The extracellular vesicle includes any membrane-bound vesicle having a diameter smaller than that of the cell from which it originated. Generally, the extracellular vesicle has a diameter in the range of 20 nm to 1000 nm, but is not limited thereto. The extracellular vesicle includes, for example, microvesicles (e.g., any vesicle detached from the plasma membrane of a cell), exosomes (e.g., any vesicle derived from the endo-lysosome pathway), apoptotic bodies (e.g., may be obtained from apoptotic cells), microparticles (e.g., may be derived from platelets), ectosomes (e.g., may be derived from neutrophils and monocytes in serum), prostatosomes (e.g., may be obtained from prostate cancer cells), and cardiosomes (e.g., may be derived from heart cells), but is not limited thereto. In the present invention, the extracellular vesicle may contain nucleic acids, proteins, carbohydrate lipids, small molecules and / or combinations thereof internally or externally.

[0092] In the present invention, most preferably, the extracellular vesicle may be an exosome.

[0093] The term "exosome" of the present invention refers to a small (diameter 20-300 nm, more preferably 40-200 nm) vesicle derived from a cell, comprising a membrane surrounding an internal space, and is generated in the cell by the fusion of a direct cell membrane bud or late endosome with a cell membrane.

[0094] In the present invention, the exosome may be characterized by expressing an exosome-specific marker.

[0095] In the present invention, as a non-limiting example, the exosome-specific marker may be one or more selected from the group consisting of CD9, CD81, CD63, CD82, TSG101 and ALIX.

[0096]

[0097] Stem cells or a population thereof cultured by the culture method of the present invention, a culture medium, and extracellular vesicles (e.g., exosomes) prepared by the manufacturing method of the present invention

[0098] In another aspect, the present invention relates to stem cells cultured by the culture method of the present invention.

[0099] In another aspect, the present invention relates to a population of stem cells cultured by the culture method of the present invention or a culture medium thereof.

[0100] In the present invention, stem cells or a population thereof cultured by the method of the present invention may be characterized by exhibiting a higher level of protein expression compared to stem cells cultured in a medium that does not contain a mixture of nucleic acid fragments.

[0101] Stem cells or a population thereof cultured by the method of the present invention may be characterized by exhibiting a protein expression level that is at least 1.5 times, at least 2 times, at least 3 times, or at least 4 times higher than that of stem cells cultured in a medium not containing a mixture of nucleic acid fragments.

[0102] In the present invention, the culture medium of stem cells cultured by the method of the present invention may be characterized by containing a total protein amount that is at least 1.5 times, at least 2 times, at least 3 times, or at least 4 times higher than that of stem cell culture medium cultured in a medium that does not contain a mixture of nucleic acid fragments.

[0103] For example, a culture medium of stem cells cultured by the method of the present invention may be characterized by having a protein content that is at least 120%, at least 150%, at least 200%, at least 250%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, or at least 410% higher than that of a culture medium of stem cells cultured in a medium that does not contain a mixture of nucleic acid fragments. For example, a culture medium of stem cells cultured by the method of the present invention may be characterized by having a protein content up to 300%, up to 310%, up to 320%, up to 330%, up to 340%, up to 350%, up to 360%, up to 370%, up to 380%, up to 390%, up to 400%, or up to 410% higher than a culture medium of stem cells cultured in a medium not containing a mixture of nucleic acid fragments. The protein content of the cultured stem cells or the cell culture medium containing them may be measured after at least 6 hours, at least 12 hours, at least 24 hours, or at least 48 hours of culture using the culture medium or culture method of the present invention, but is not limited thereto.

[0104]

[0105] In the present invention, stem cells or a population thereof cultured by the method of the present invention may be characterized by exhibiting high exosome production compared to stem cells cultured in a medium not containing a mixture of nucleic acid fragments. Stem cells or a population thereof cultured by the method of the present invention may be characterized by exhibiting exosome production that is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, or at least 5 times higher than stem cells cultured in a medium not containing a mixture of nucleic acid fragments.

[0106] In the present invention, the culture medium of stem cells cultured by the method of the present invention may be characterized by having at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, or at least 5 times higher number of exosome particles compared to the culture medium of stem cells cultured in a medium that does not contain a mixture of nucleic acid fragments.

[0107] For example, a culture medium of stem cells cultured by the method of the present invention may be characterized by having at least 120%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, or at least 470% of the number of exosome particles compared to a culture medium of stem cells cultured in a medium that does not contain a mixture of nucleic acid fragments. For example, a culture medium of stem cells cultured by the method of the present invention may be characterized by having an exosome particle number of up to 300%, up to 350%, up to 360%, up to 370%, up to 380%, up to 390%, up to 400%, up to 410%, up to 420%, up to 430%, up to 440%, up to 450%, up to 460%, up to 463%, or up to 470% compared to a stem cell culture medium cultured in a medium not containing a nucleic acid fragment mixture. The number of exosome particles of the cultured stem cells or a cell culture medium containing them may be measured after at least 6 hours, at least 12 hours, at least 24 hours, or at least 48 hours of culture using the culture medium or culture method of the present invention, but is not limited thereto.

[0108] In the present invention, stem cells or a population thereof cultured by the method of the present invention may be characterized by containing a higher growth factor compared to stem cells cultured in a medium that does not contain a mixture of nucleic acid fragments.

[0109] 본 발명에 있어서, 상기 성장인자는 예를 들어, bFGF (basic Fibroblast Growth Factor), bNGF (beta Nerve Growth Factor), EGF (Epidermal Growth Factor), FGF-4 (Fibroblast Growth Factor 4), FGF-6 (Fibroblast Growth Factor 6), HB-EGF (Heparin-binding Epidermal Growth Factor-like Growth Factor), HGF (Hepatocyte Growth Factor), IGFBP-1 (Insulin-like Growth Factor Binding Protein 1), IGFBP-2 (Insulin-like Growth Factor Binding Protein 2), IGFBP-3 (Insulin-like Growth Factor Binding Protein 3), IGFBP-4 (Insulin-like Growth Factor Binding Protein 4), IGFBP-6 (Insulin-like Growth Factor Binding Protein 6), IGF-I (Insulin-like Growth Factor I), IGF-I SR (Insulin-like Growth Factor I Signal Receptor), IGF-II (Insulin-like Growth Factor II), PDGF-AA (Platelet-Derived Growth Factor-AA), PDGF-AB (Platelet-Derived Growth Factor-AB), PDGF-BB (Platelet-Derived Growth Factor-BB), TGF-β (Transforming Growth Factor beta), TGF-β3 (Transforming Growth Factor beta 3), PIGF (Placenta Growth Factor),VEGF (Vascular Endothelial Growth Factor), VEGF R2 (Vascular Endothelial Growth Factor Receptor 2), VEGF R3 (Vascular Endothelial Growth Factor Receptor 3), VEGF D (Vascular Endothelial Growth Factor D), AR (Androgen Receptor), G-CSF (Granulocyte Colony-Stimulating Factor), GDNF (Glial cell line-Derived) Neurotrophic Factor), M-CSF (Macrophage Colony-Stimulating Factor), MCSF (Macrophage Colony-Stimulating Factor), NT-3 (Neurotrophin-3), NT-4 (Neurotrophin-4), SCF (Stem Cell Factor), and SCF R (Stem Cell Factor Receptor, also known as c-Kit or CD117), but is not limited thereto. The above growth factor may preferably be selected from the group consisting of FGF-6 (fibroblast growth factor-6), HB-EGF (heparin-binding epidermal growth factor), IGF family (insulin-like growth factor), PDGF family (platelet-derived growth factor), VEGF (vascular endothelial growth factor), and GDNF (glial-derived neurotrophic factor).

[0110] For example, a culture medium of stem cells cultured by the method of the present invention may be characterized by containing growth factors that are at least 105%, 110%, 120%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, or at least 400% higher than a culture medium of stem cells cultured in a medium not containing a nucleic acid fragment mixture. For example, a culture medium of stem cells cultured by the method of the present invention may be characterized by containing growth factors that are up to 250%, up to 300%, up to 350%, up to 360%, up to 370%, up to 380%, up to 390%, or up to 400% higher than a culture medium of stem cells cultured in a medium not containing a nucleic acid fragment mixture. It may be a comparison of each growth factor contained in the culture medium containing the growth factor, or a comparison of the total growth factor content. The growth factor content of the cultured stem cells or the cell culture medium containing them may be measured after culturing each growth factor for at least 6 hours, at least 12 hours, at least 24 hours, or at least 48 hours using the culture medium or culture method of the present invention, but is not limited thereto.

[0111] For specific examples, the culture medium of stem cells cultured by the method of the present invention may contain FGF-6 at least 150%, at least 200%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, or at least 360% higher than that of stem cells cultured in a medium not containing a nucleic acid fragment mixture, and the culture medium of stem cells prepared in the example contained FGF-6 at 356% higher after 24 hours of culture, but is not limited thereto.

[0112] For specific examples, the culture medium of stem cells cultured by the method of the present invention may contain HB-EGF at least 150%, at least 200%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, or at least 300% higher than the culture medium of stem cells cultured in a medium not containing a nucleic acid fragment mixture, and the culture medium of stem cells prepared in the example contained HB-EGF at 283% higher after 24 hours of culture, but is not limited thereto.

[0113] For a specific example, the culture medium of stem cells cultured by the method of the present invention may contain at least 150%, at least 200%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 345%, at least 350%, or at least 360% higher IGF family compared to the culture medium of stem cells cultured in a medium not containing a nucleic acid fragment mixture, and the culture medium of stem cells prepared in the example contained 345% higher IGF family after 24 hours of culture, but is not limited thereto.

[0114] For a specific example, the culture medium of stem cells cultured by the method of the present invention may contain at least 100%, at least 200%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 345%, or at least 350% higher PDGF family compared to the culture medium of stem cells cultured in a medium not containing a nucleic acid fragment mixture, and the culture medium of stem cells prepared in the example contained 194% higher PDGF family after 24 hours of culture, but is not limited thereto.

[0115] For a specific example, the culture medium of stem cells cultured by the method of the present invention may contain at least 150%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 265%, or at least 270% higher VEGF family compared to the culture medium of stem cells cultured in a medium not containing a nucleic acid fragment mixture, and the culture medium of stem cells prepared in the example contained 270% higher VEGF family after 24 hours of culture, but is not limited thereto.

[0116] For specific examples, the culture medium of stem cells cultured by the method of the present invention may contain GDNF that is at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200% higher than that of stem cell culture medium cultured in a medium not containing a mixture of nucleic acid fragments, and the culture medium of stem cells prepared in the examples contained GDNF that was 176% higher after 24 hours of culture, but is not limited thereto. In the examples of the present invention, the change in the amount of each growth factor as shown in Table 1 was confirmed, but is not limited thereto. In the present invention, the increase in the content of the growth factor or specific examples of growth factors may be calculated by comparing the sum of all growth factors or by comparing each growth factor individually. When described as a family of specific growth factors, it may be calculated by comparing the content of the entire family or the content of each growth factor belonging thereto.

[0117]

[0118] In another aspect, the present invention relates to an extracellular vesicle produced by the manufacturing method of the present invention.

[0119] In the present invention, most preferably, the extracellular vesicle may be an exosome.

[0120] In the present invention, the exosome may be characterized by expressing an exosome-specific marker.

[0121] In the present invention, as a non-limiting example, the exosome-specific marker may be one or more selected from the group consisting of CD9, CD81, CD63, CD82, TSG101 and ALIX.

[0122]

[0123] Uses of stem cells or populations thereof, culture media, or extracellular vesicles

[0124] The uses of stem cells, stem cell culture media, or extracellular vesicles produced from stem cells in various fields such as medicine and cosmetics are well known and are continuously being researched and reported. Since the stem cells cultured by the method of the present invention and the extracellular vesicles isolated therefrom are characterized by an increase in the amount of proteins such as growth factors and cytokines without altering the characteristics of existing stem cells, they can exhibit superior efficacy compared to stem cells or extracellular vesicles produced by conventional methods.

[0125] In another aspect, the present invention relates to a composition comprising the cultured stem cells or a population thereof, a culture medium, or the extracellular vesicles as an active ingredient.

[0126] In another aspect, the present invention relates to a cosmetic composition comprising the cultured stem cells or a population thereof, a culture medium, or the extracellular vesicles as an active ingredient.

[0127] In another aspect, the present invention relates to the use of the cultured stem cells or a population thereof, the culture medium, or the extracellular vesicles for the preparation of cosmetic compositions.

[0128] In the present invention, the cosmetic composition can be any formulation commonly manufactured, for example, skin lotion, skin toner, pack, nourishing cream, moisturizing cream, essence, body cream, body lotion, body oil, cleansing foam, cleansing lotion, soap, patch, foundation, lipstick, makeup base, lipstick, shampoo, rinse, hair essence, hair pack, etc., but is not limited thereto.

[0129] The cultured stem cells or a population thereof, a culture medium, or extracellular vesicles, which are active ingredients of the above cosmetic composition, may be characterized by being contained in a cosmetically acceptable amount, and preferably, may be contained within the scope of regulations set by each country.

[0130] In the present invention, the cosmetic composition may be contained in a functional cosmetic. The standards for the functional cosmetic may comply with the relevant regulations of each country. For example, it may comply with the Korean "Notification of the Ministry of Food and Drug Safety's Standards and Test Methods for Functional Cosmetics," and may have functions such as skin whitening, improvement of skin wrinkles, change in hair color, alleviation or prevention of hair loss, alleviation of acne, UV protection, alleviation of skin inflammation, cell regeneration, antioxidant, anti-aging, skin moisturization, improvement of skin condition, skin soothing and stress relief, or refinement of skin texture and enhancement of elasticity, and may be characterized by being used for such purposes, but is not limited thereto. In the present invention, the improvement of skin condition is used in a comprehensive sense that includes not only the improvement of observed skin condition but also factors that directly and indirectly affect skin health.

[0131]

[0132] In the technical field of the present invention, it is well known that stem cells and exosomes derived therefrom can also be used for the prevention and treatment of various diseases. Stem cells and exosomes are reported to have excellent anti-inflammatory and tissue regeneration effects.

[0133] Accordingly, in another aspect, the present invention relates to a pharmaceutical composition comprising the cultured stem cells or a population thereof, a culture medium, or the extracellular vesicles as an active ingredient.

[0134] In another aspect, the present invention relates to the use of the cultured stem cells or a population thereof, the culture medium, or the extracellular vesicles for the preparation of a pharmaceutical composition.

[0135] In the present invention, the pharmaceutical composition may be used for the prevention or treatment of inflammatory diseases, neurodegenerative diseases, alopecia, acne, folliculitis, or scars.

[0136] In the present invention, the inflammatory disease may be characterized as being selected from the group consisting of, for example, atopic dermatitis, arthritis, seborrheic dermatitis, and folliculitis, but is not limited thereto.

[0137] In the present invention, the pharmaceutical composition may also be used for medical cosmetic purposes to achieve effects such as those described in the cosmetic composition.

[0138] In the present invention, the term “medical beauty” comprehensively includes a series of medical acts and purposes treated using pharmaceuticals and medical devices at medical institutions such as hospitals. The distinction between pharmaceuticals, medical devices, and cosmetics (cosmetic compositions) may be defined by the notifications issued by relevant agencies in each country (Korea: MFDS, US FDA, European EMA).

[0139] In the present invention, the pharmaceutical composition may be characterized as being used for purposes such as, for example, skin whitening, improvement of skin wrinkles, change of hair color, alleviation or prevention of hair loss, alleviation of acne, protection against ultraviolet rays, alleviation of skin inflammation, cell regeneration, antioxidant, anti-aging, skin moisturization, improvement of skin condition, skin soothing and stress relief, or skin texture refinement and elasticity enhancement.

[0140] In the cosmetic and pharmaceutical compositions of the present invention, as another example, the active ingredients, stem cells and exosomes derived therefrom, may be provided in a freeze-drying formulation or in a powder formulation by freeze-drying, heat drying, etc., and in this case, they may be characterized by being rehydrated by a suitable solvent before use.

[0141] In the technical field of the present invention, methods for freeze-drying stem cells, their culture medium, and exosomes are well known in the art, and a person skilled in the art can prepare freeze-dried formulations by selecting and using appropriate methods.

[0142]

[0143] Examples

[0144] The present invention will be described in more detail below through examples. These examples are intended solely to illustrate the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0145]

[0146] Example 1: Stem cell culture using PDRN

[0147] Amniotic stem cells (AMSCs) were isolated from human amniotic tissue, which was harvested from cadavers donated by a tissue bank for non-profit patient treatment. The donated amniotic membrane was cut into small pieces using scissors and then lysed using Collagenase Type II (purchased from ThermoFisher Scientific). Subsequently, the extracellular matrix and cells were separated using a cell strainer and centrifugation. Red blood cells were removed from the separated cells by treating them with RBC lysis buffer (purchased from ThermoFisher Scientific). The cells were prepared by subculturing in Alpha-MEM (purchased from Hyclone) medium containing 10% fetal bovine serum (Gibco) and 10 µg / mL Gentamicin (purchased from ThermoFisher Scientific) under conditions of 5% CO2 and 37°C.

[0148] For stem cell culture using a nucleic acid fragment mixture, PDRN (100403-24-5, HTL biotechnology) was diluted to concentrations of 0, 200, 400, and 600 μg / mL in DMEM / F12 medium (LM002-02; Welgene) without added FBS to prepare a PDRN-containing medium.

[0149] After washing the cultured cells with phosphate-buffered saline (purchased from Welgene), the cells were isolated using TrypLE™ Express Enzyme reagent (12604-021; Gibco). Subsequently, amniotic stem cells were inoculated into 25T flasks at a rate of 4 mL / cell using the medium containing the PDRN diluted as described above. The cells were then cultured for 24 and 48 hours under Normoxia (21% oxygen) and 24 and 48 hours under Hypoxia (2% oxygen), respectively, for each sample condition.

[0150] After culture, the culture sample was filtered once through a 0.22 μm filter (S6534-FMGUK; Millipore) to obtain the final culture solution.

[0151]

[0152] Example 2: Evaluation of characteristics of cell culture medium produced using PDRN

[0153] Example 2-1: Confirmation of changes in cell number according to the culture process supplemented with PDRN

[0154] To confirm the effect of the stem cell culture process using PDRN on cell growth, cells were counted after the process. Specifically, the cells remaining after obtaining the cell culture medium in Example 1 were separated using TrypLE™ Express Enzyme reagent (12604-021; Gibco), stained with Trypan blue (15250061; Gibco), and the cell count was measured using a Countess™ automate cell counter (C10227; Thermo Fisher).

[0155] As shown in Figure 1, it was found that the addition of PDRN did not have a significant effect on cell growth.

[0156]

[0157] Example 2-2: Measurement of protein content in cell culture medium according to PDRN-added culture process

[0158] To determine the effect of the culture process using PDRN on the amount of protein produced by cells, the amount of protein in the cell culture medium was measured using Bio-Rad's Bradford assay reagent.

[0159] As a result of measurement, it was confirmed that the protein content in the cell culture medium produced by the addition of PDRN, as shown in Figure 2, increased by up to approximately 340% (24-hour group) and 410% (48-hour group) compared to the group without PDRN addition. This means that the culture process using PDRN of the present invention can induce a significant improvement in protein production.

[0160]

[0161] Example 2-3: Confirmation of the number of exosome particles in cell culture medium according to the culture process supplemented with PDRN

[0162] To confirm the exosome concentration in the cell culture medium prepared in Example 2-1 above, the number of particles was measured using an NTA (NS300, red laser) from Malvern Panalytical. The experimental group was configured according to Example 2-1. However, samples cultured with the addition of PDRN 600 μg / mL were excluded from the experimental group because NTA measurement was impossible due to the high viscosity of the sample.

[0163] As shown in Figure 3, in the case of the particle number of the sample cultured with PDRN (400 μg / mL) added during 24-hour serum-free culture, it was found that the exosome concentration was high, reaching up to 463% compared to the negative control without PDRN added. This means that cell culture medium containing a high concentration of exosomes can be harvested by the process of adding PDRN during serum-free culture.

[0164]

[0165] Example 3: Evaluation of the efficacy and characteristics of exosomes in cell culture medium produced using PDRN-supplemented culture technology

[0166] In order to verify whether the particles in the cell culture medium produced in Example 2 were exosomes, a marker specifically expressed in exosomes was identified.

[0167] Specifically, the produced cell culture medium was concentrated using an Amicon Ultra Centrifugal Filter, 30kDa MWCO (UFC803024, Milipore), and then exosomes were separated using CD9 Exosome Capture Beads (ab239685, ABCam) and CD63 Exosome Capture Beads (ab239686, ABCam). Afterward, the separated exosomes were stained with FITC anti-human CD9 Antibody (312104, BioLegend), APC anti-human CD63 Antibody (353008, BioLegend), and Pacific Blue™ anti-human CD81 (TAPA-1) Antibody (349516, BioLegend), respectively. At this time, FITC Mouse IgG1, κ Isotype Ctrl Antibody (400108, BioLegend), APC Mouse IgG1, κ Isotype Ctrl (FC) Antibody (400122, BioLegend), and Pacific Blue™ Mouse IgG1, κ Isotype Ctrl Antibody (400151, BioLegend) were used as negative controls for staining. After staining was completed, measurements were taken using an Aria / fusion FACS (BD) instrument. As shown in Figure 4, exosome markers were expressed in the particles within the cell culture medium, confirming that they were exosomes.

[0168]

[0169] Example 4: Confirmation of changes in growth factor content in cell culture medium according to the culture process supplemented with PDRN

[0170] To confirm the amounts of various growth factors in the cell culture medium prepared in Example 2 above, growth factors in the culture medium were measured using the Human Growth Factor array kit (AAH-GF-1-2) from Ray Biotech. The experimental group consisted of a group untreated with PDRN and samples cultured with 300 μg / mL of PDRN added.

[0171] As shown in Figure 5 and Table 1, it was confirmed that the expression levels of growth factors in samples cultured with PDRN (300 μg / mL) added during 24-hour serum-free culture increased significantly compared to the negative control group without PDRN. In particular, factors such as FGF-6 (fibroblast growth factor-6; 356%), HB-EGF (heparin-bound epidermal growth factor; 283%), IGF Family (insulin-like factor; up to 345%), PDGF Family (platelet-derived growth factor), VEGF Family (angiogenic factor; up to 270%), and GDNF (glial-derived neurotrophic factor; 176%) increased significantly.

[0172] This means that cell culture medium containing high concentrations of growth factors can be harvested through a process of adding PDRN during serum-free culture.

[0173]

[0174] The medium containing the nucleic acid fragment mixture of the present invention and the high-efficiency stem cell-derived extracellular vesicle and protein production technology based thereon can significantly increase the expression levels of proteins such as growth factors of stem cells and the production of extracellular vesicles. Stem cells cultured by the above method or extracellular vesicles obtained therefrom can be usefully applied in the beauty field, such as skin whitening, improvement of skin wrinkles, change of hair color, alleviation or prevention of hair loss, alleviation of acne, UV protection, alleviation of skin inflammation, cell regeneration, antioxidant, anti-aging, skin moisturization, improvement of skin condition, skin soothing and stress relief, or skin texture refinement and elasticity enhancement, due to their high protein content, and in the medical field, such as the management of hair loss, inflammatory diseases, acne and folliculitis, and prevention of scars.

[0175]

[0176] The description of the invention set forth above is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

A step of culturing stem cells in a culture medium containing a mixture of nucleic acid fragments; and A method for producing extracellular vesicles comprising the step of recovering extracellular vesicles from a culture. A method for preparing an extracellular vesicle according to claim 1, characterized in that the nucleic acid fragment mixture has an average molecular weight of 100 kDa to 3000 kDa. A method for preparing an extracellular vesicle according to claim 1, characterized in that the nucleic acid fragment mixture is a mixture of nucleic acid fragments having a molecular weight of 1 to 100,000 kDa. A method for preparing an extracellular vesicle according to claim 1, wherein the nucleic acid fragment mixture is a DNA fragment mixture. A method for preparing an extracellular vesicle according to claim 4, characterized in that the DNA fragment mixture is PDRN (polydeoxyribonucleotide) or PN (polynucleotide). A method for preparing an extracellular vesicle according to claim 1, wherein the nucleic acid fragment mixture is derived from an organism selected from the group consisting of microbiome, animals, plants, and algae. A method for preparing an extracellular vesicle according to claim 6, characterized in that the nucleic acid fragment mixture is a mixture of DNA fragments derived from fish. A method for producing extracellular vesicles according to claim 1, characterized in that the culture medium is a serum-free or serum-containing culture medium. A method for manufacturing an extracellular vesicle according to claim 1, characterized in that the extracellular vesicle is an exosome. A method for preparing an extracellular vesicle according to claim 9, characterized in that the exosome expresses one or more exosome markers selected from the group consisting of CD9, CD81, CD63, CD82, TSG101, and ALIX. A method for preparing an extracellular vesicle according to claim 1, characterized in that the concentration of the nucleic acid fragment mixture is 50 μg / ml to 1000 μg / ml. A culture medium composition for the culture of stem cells or the production of extracellular vesicles comprising a mixture of nucleic acid fragments. A method for culturing stem cells comprising the step of culturing stem cells in a culture medium containing a mixture of nucleic acid fragments. Stem cells cultured by the culture method of paragraph 13 or a culture medium thereof. Extracellular vesicles manufactured by the manufacturing method of claim 1. A cosmetic composition comprising as an active ingredient any one or more of the stem cells or culture medium thereof of claim 14 and the extracellular vesicles of claim 15. A cosmetic composition according to claim 16 for skin whitening, improvement of skin wrinkles, change of hair color, alleviation or prevention of hair loss, alleviation of acne, protection against UV rays, alleviation of skin inflammation, cell regeneration, antioxidant, anti-aging, skin moisturization, improvement of skin condition, skin soothing and stress relief, or skin texture refinement and elasticity enhancement. A pharmaceutical composition for the prevention or treatment of hair loss, inflammatory diseases, neurodegenerative diseases, acne and folliculitis or scars comprising any one or more of the stem cells or culture medium thereof of claim 14 and the extracellular vesicles of claim 15.

Images