Composition for treating or preventing hair loss containing stem cell-derived exosomes and method for producing the same

Abstract

The present invention relates to a composition for treating hair loss containing stem cell-derived exosomes and a method for producing the same. The exosomes of the present invention have excellent effects of promoting the growth and recovery of human dermal papilla cells and can be used in pharmaceutical compositions and various cosmetics for treating hair loss.

Description

[Technical Field]

[0001] The present invention relates to a composition for treating or preventing hair loss, which contains stem cell-derived exosomes, and a method for producing the same. More specifically, the present invention relates to a composition containing exosomes isolated from mesenchymal stem cells or cultures thereof, which have an excellent effect of promoting the growth and recovery of human dermal papilla cells, and are thereby capable of treating or preventing hair loss. [Background technology]

[0002] Extracellular vesicles are vesicles composed of spherical lipid bilayers of 30 to 1000 nm in size, including exosomes.

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

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

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

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

[0007] Even after transplantation, stem cells are known to exert trophic effects on the paracrine factors secreted by the cells, even if they do not survive for long periods. These factors include small molecules such as growth factors, chemokines, and cytokines, secreted by extracellular vesicles such as exosomes, which are derived from stem cells. Therefore, exosomes are being used to characterize stem cells and evaluate their therapeutic efficacy. Recently, active research has been conducted into the therapeutic effects of exosomes secreted by mesenchymal stem cells (MSCs) on various diseases, rather than using the MSCs themselves. This research is expected to provide a new alternative that overcomes the limitations of existing stem cell therapies in both academia and industry. Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, the present inventors developed a composition containing exosomes isolated from stem cells or their cultures, and confirmed that the composition according to the present invention has an excellent effect of promoting the growth and recovery of human hair papilla cells, and is capable of treating or preventing hair loss.

[0009] Therefore, an object of the present invention is to provide a composition comprising stem cell-derived exosomes.

[0010] Another object of the present invention is to provide a pharmaceutical composition for treating or preventing hair loss, comprising stem cell-derived exosomes.

[0011] Yet another object of the present invention is to provide a method for treating or preventing hair loss using stem cell-derived exosomes.

[0012] Yet another object of the present invention is to provide a use of a composition containing stem cell-derived exosomes for the treatment or prevention of hair loss.

[0013] Yet another object of the present invention is to provide a cosmetic composition for preventing hair loss, strengthening hair roots, restoring hair follicles, or promoting hair growth, which comprises stem cell-derived exosomes.

[0014] Yet another object of the present invention is to provide a method for preventing hair loss, strengthening hair roots, restoring hair follicles, or promoting hair growth using stem cell-derived exosomes.

[0015] Yet another object of the present invention is to provide a use of a composition containing stem cell-derived exosomes for preventing hair loss, strengthening hair roots, restoring hair follicles, or promoting hair growth.

[0016] Yet another object of the present invention is to provide a method for producing a composition containing stem cell-derived exosomes. [Means for solving the problem]

[0017] The present invention relates to a composition for treating or preventing hair loss, which contains stem cell-derived exosomes, and a method for producing the same, and hair loss can be treated or prevented by the composition of the present invention.

[0018] The present invention will now be described in more detail.

[0019] One aspect of the present invention is a composition comprising stem cell-derived exosomes.

[0020] The term "exosome" as used herein refers to a cellular endoplasmic reticulum present in the body fluids of almost all eukaryotes, with a diameter of approximately 30 to 100 nm, which is larger than LDL protein but much smaller than red blood cells. Exosomes can be released from cells when multivesicular bodies (MVDs) fuse with the cell membrane, or can be released immediately from the cell membrane, and are well known to have important yet specialized functions such as coagulation and intercellular signaling.

[0021] As used herein, the term "stem cell" refers to an undifferentiated cell that has the ability to self-renew and differentiate into two or more different types of cells.

[0022] In one embodiment of the present invention, the stem cells may be autologous or allogeneic stem cells, may be derived from any type of animal, including humans and non-human mammals, may be adult-derived stem cells, or may be embryo-derived stem cells, such as, but not limited to, adult stem cells, embryonic stem cells, induced pluripotent stem cells (iPSCs), induced pluripotent stem cell-derived mesenchymal stem cells, BxC stem cells, hyaluronic acid (HA)-pretreated induced pluripotent stem cell-derived mesenchymal stem cells, and BxC-HA stem cells.

[0023] As used herein, the term "adult stem cell" refers to a cell extracted from umbilical cord blood, adult bone marrow, blood, etc., which is just before differentiating into a specific organ cell, and refers to an undifferentiated cell that retains the ability to develop into a tissue within the body when needed.

[0024] In one embodiment of the present invention, the adult stem cells may be selected from the group consisting of adult stem cells of human, animal or animal tissue origin, mesenchymal stem cells derived from human, animal or animal tissue origin, and mesenchymal stem cells derived from induced pluripotent stem cells of human, animal or animal tissue origin, but are not limited thereto.

[0025] In the present invention, the human, animal or animal tissue may be selected from the group consisting of, but is not limited to, umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta.

[0026] In the present invention, stem cells of various human or animal tissue origins may be selected from the group consisting of hematopoietic stem cells, mammary stem cells, intestinal stem cells, vascular endothelial stem cells, neural stem cells, olfactory neural stem cells, and testicular stem cells, but are not limited thereto.

[0027] As used herein, the term "embryonic stem cell" refers to a cell extracted during the developmental process of an embryo, which is the inner cell mass extracted from an embryo at the blastula stage, just before the fertilized egg implants in the mother's uterus, and cultured in vitro.

[0028] Embryonic stem cells are cells that have the ability to self-renew and are either pluripotent (able to differentiate into cells of all tissues of an individual) or totipotent (self-renewing), and in a broad sense also include embryoid bodies (EBs) derived from embryonic stem cells.

[0029] In the present invention, stem cells may include, but are not limited to, embryonic stem cells of any origin, such as human, monkey, pig, horse, cow, sheep, dog, cat, mouse, rabbit, etc.

[0030] As used herein, the term "induced pluripotent stem cells (iPSCs)" refers to cells that have been induced to have pluripotent differentiation ability from differentiated cells through an artificial reverse differentiation process, and may be used interchangeably with "reverse differentiated stem cells."

[0031] The artificial reverse differentiation process may be carried out by the use of viral or non-viral vectors using retroviruses, lentiviruses, and Sendai viruses, or by the introduction of non-viral reverse differentiation factors using proteins and cell extracts, or may include the reverse differentiation process using stem cell extracts, compounds, etc.

[0032] Induced pluripotent stem cells have almost identical properties to embryonic stem cells; specifically, they have a similar cell shape, similar gene and protein expression, and full differentiation potential in vitro and in vivo, can form teratomas, and when inserted into mouse blastocysts, can form chimera mice and are capable of germline transmission of genes.

[0033] In one embodiment of the present invention, the stem cells may be induced pluripotent stem cell-derived mesenchymal stem cells.

[0034] As used herein, the term "mesenchymal stem cells (MSCs)" refers to stem cells derived from mesenchyme. Mesenchymal stem cells may differentiate into one or more cells selected from the group consisting of osteoblasts, chondrocytes, adipocytes, or myocytes. Mesenchymal stem cells may be isolated from any type of adult tissue, such as bone marrow, adipose tissue, umbilical cord, or peripheral blood. A mesenchymal stem cell population may be defined as one that exhibits a specific phenotype. A mesenchymal stem cell population differentiated from induced pluripotent stem cells may exhibit the same phenotypic characteristics as a normal mesenchymal stem cell population. A mesenchymal stem cell population may be understood as a stem cell population in which 95% or more of the cells express CD105, CD73, and CD90 markers, and 2% or less express CD45, CD34, and SSEA-4 markers. In this case, cells that express CD45, CD34, or SSEA-4 at 2% or less can be conveniently described as "not expressing" CD45, CD34, or SSEA-4.

[0035] In one embodiment of the present invention, the composition may comprise mesenchymal stem cell-derived exosomes derived from induced pluripotent stem cells.

[0036] In one embodiment of the present invention, the stem cells may be BxC stem cells.

[0037] As used herein, the term "BxC stem cells" refers to stem cells produced by culturing induced pluripotent stem cells (iPSCs), isolating a population of induced pluripotent stem cells that do not express stage-specific embryonic antigen 4 (SSEA-4) protein, and then further culturing the isolated population. BxC stem cells are cells at a stage immediately prior to full differentiation from induced pluripotent stem cells into mesenchymal stem cells, and further culturing can result in the development of fully differentiated mesenchymal stem cells. Therefore, the phenotype of a BxC stem cell population may not be completely identical to that of a mesenchymal stem cell population, but may be 96% to 99.9% similar to that of a mesenchymal stem cell population. For example, while an induced pluripotent stem cell population expresses CD90 protein at 0.3%, a mesenchymal stem cell population expresses CD90 protein at 99.7%, and a BxC stem cell population expresses CD90 protein at 96.9%, approximately 98% of that of mesenchymal stem cells. Therefore, BxC stem cells may be defined as stem cells that are 96% to 99.9% differentiated without completely differentiating into mesenchymal stem cells, obtained by further culturing induced pluripotent stem cells that do not express SSEA-4 protein after culturing induced pluripotent stem cells. Compared to mesenchymal stem cells differentiated from the same induced pluripotent stem cells, BxC stem cells have superior stemness and can secrete large amounts of functional proteins. Specifically, when passaged nine or more times, the BxC stem cells of the present invention exhibit a proliferation potential that is at least 10-fold greater than that of mesenchymal stem cells derived from the same tissue, and no decrease in proliferation potential is observed even after passaged 12 or more times. Furthermore, BxC stem cells also exhibit at least two-fold higher expression of Ki67, a marker associated with cell proliferation potential, compared to general mesenchymal stem cells.Furthermore, compared to mesenchymal stem cells differentiated from the same induced pluripotent stem cells, BxC stem cells can express at higher levels 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 at lower levels one or more genes selected from the group consisting of DHRS3, BMPER, IFI6, PRSS12, RDH10, and KCNE4.

[0038] As used herein, the term "stem cell potential" refers to pluripotency, the ability to generate any type of cell, and self-renewal, the ability to generate cells similar to oneself indefinitely. For example, it refers to increasing the proliferation of undifferentiated cells while maintaining their undifferentiated state, increasing telomerase activity, increasing the expression of stemness-acting signals, or increasing cell migration activity, and may include the presence of one or more of these characteristics.

[0039] In one embodiment of the present invention, the composition may comprise exosomes derived from BxC stem cells.

[0040] In one embodiment of the present invention, the stem cells may be induced pluripotent stem cell-derived mesenchymal stem cells pretreated with hyaluronic acid.

[0041] As used herein, the term "pretreatment" refers to a process of culturing mesenchymal stem cells by adding a specific substance to a medium for mesenchymal stem cells. For example, pretreatment refers to a process of further culturing mesenchymal stem cells that have completed differentiation from induced pluripotent stem cells in a medium supplemented with hyaluronic acid.

[0042] In the present invention, hyaluronic acid can increase the stem cell potential and proliferation potential of stem cells, and increase the number of stem cell-derived exosomes and the protein and RNA contents in exosomes.

[0043] In one embodiment of the present invention, the composition may contain exosomes derived from induced pluripotent stem cells-derived mesenchymal stem cells pretreated with hyaluronic acid.

[0044] In one embodiment of the present invention, the stem cells may be BxC-HA stem cells.

[0045] As used herein, the term "BxC-HA stem cells" refers to mesenchymal stem cells obtained by culturing BxC stem cells according to the present invention until they are fully differentiated into mesenchymal stem cells, and then culturing (pre-treating) them by adding hyaluronic acid to the medium. For example, BxC-HA stem cells may be produced by further culturing BxC stem cells until they are fully differentiated into mesenchymal stem cells, and then adding 0.1 to 1000 μg / mL, e.g., 40 μg / mL, of hyaluronic acid to the medium and culturing them for 12 to 48 hours. Compared to mesenchymal stem cells that have not been pre-treated with any substance, BxC-HA stem cells exhibit an approximately 360% increase in cell proliferation rate, an approximately 5-fold increase in exosome production efficiency, and a 5-fold or greater increase in the amount of exosome-derived proteins.

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

[0047] Another aspect of the present invention is a pharmaceutical composition for treating, preventing, alleviating, or suppressing hair loss, comprising exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells as an active ingredient.

[0048] The term "hair loss" as used herein refers to a condition in which hair is missing or decreases at an abnormal rate in a specific individual at a site where hair would normally be present, and more specifically refers to a condition in which hair that should be present on the scalp is missing or decreases. Hair loss can occur or progress due to various internal or external factors. In the case of male pattern baldness, a typical hair loss symptom, it can be caused by the male hormone dihydrotestosterone (DHT) or testosterone binding to the androgen receptor (AR) of hair papilla cells that make up the hair tissue, which can then regulate the expression of genes related to hair growth in the hair papilla cells, such as IGF1, EGF, FGF7, VEGF, IL-6, and TGF-β1.

[0049] As used herein, the term "containing as an active ingredient" means containing a sufficient amount of exosomes isolated from stem cells or a culture thereof to achieve a specific effect, such as the activity of treating or preventing hair loss, or preventing hair loss, strengthening hair roots, restoring hair follicles, or promoting hair growth.

[0050] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier.

[0051] The term "pharmaceutically acceptable" as used herein means that it does not irritate an organism when administered and does not inhibit the biological activity and properties of the compound being administered, as commonly used in the pharmaceutical field.

[0052] In the present invention, any carrier commonly used in the art can be used, including, but not limited to, 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.

[0053] In the present invention, the pharmaceutical composition may be used by adding other pharmaceutically acceptable additives such as excipients, diluents, antioxidants, buffers, or bacteriostats, if necessary, and may also be used by additionally adding fillers, extenders, wetting agents, disintegrants, dispersants, surfactants, binders, or lubricants.

[0054] In one embodiment of the present invention, the induced pluripotent stem cell-derived mesenchymal stem cells may be differentiated from progenitor cells of the induced pluripotent stem cell-derived mesenchymal stem cells that do not express stage-specific embryonic antigen 4 (SSEA-4) protein.

[0055] In one embodiment of the present invention, the induced pluripotent stem cells may be human-derived induced pluripotent stem cells.

[0056] In one embodiment of the present invention, the induced pluripotent stem cell-derived mesenchymal stem cells may be pretreated with a pretreatment substance.

[0057] In one embodiment of the present invention, the pretreatment material may be hyaluronic acid.

[0058] Yet another aspect of the present invention is a method for treating or preventing hair loss, comprising the steps of:

[0059] A providing step in which exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells are administered to or contacted with a subject.

[0060] As used herein, the term "subject" may be a mammal, including but not limited to a human, monkey, cow, horse, sheep, pig, cat, dog, mouse, rat, rabbit, or guinea pig.

[0061] As used herein, the term "administration" refers to providing a predetermined substance to a subject by any appropriate method. The administration route of the exosome-containing composition of the present invention may be any conventional route, such as oral or parenteral, as long as it can reach the target tissue. Furthermore, the composition of the present invention may be administered using any device that can deliver an active ingredient to target cells.

[0062] As used herein, the term "contact" refers to providing a predetermined substance to a subject by any suitable method that is non-invasive to the body. For example, a composition comprising the exosomes of the present invention can be contacted with a subject so that the composition directly touches or is indirectly delivered to the epidermis or integument of the subject.

[0063] In one embodiment of the present invention, the exosomes may include BxC-HA stem cell-derived exosomes (BxC-HAe).

[0064] Yet another aspect of the present invention is use of a pharmaceutical composition comprising, as an active ingredient, exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells for the treatment or prevention of hair loss.

[0065] Yet another aspect of the present invention is a cosmetic composition for preventing hair loss, strengthening hair roots, restoring hair follicles, or promoting hair growth, which comprises exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells as an active ingredient.

[0066] As used herein, the term "preventing hair loss" refers to preventing a hair loss condition in a particular individual, thereby slowing or completely preventing hair loss in a particular area.

[0067] The term "hair root" as used herein refers to the part of the hair that is present from the inside of the epidermis to the dermis and is not exposed to the outside, and includes the lower hair bulb, the hair portion formed by keratinized epithelial cells that have proliferated from the hair bulb, and the dermal papilla that provides nutrients to the hair bulb and is composed of dermal papilla cells that express factors related to hair development and growth regulation. Therefore, the term "strengthening the hair root" as used herein refers to an increase in the depth and thickness of the hair portion contained in the hair root, an increase in the size, survival rate, and expression of hair growth-promoting factors of dermal papilla cells, etc.

[0068] The term "hair follicle" as used herein refers to a part located in the epidermis or subcutaneous tissue that surrounds and protects the hair root, and the hair follicle is connected to the capillaries and the hair root to provide nutrients to the hair root and enable continuous cell division in the hair root and the associated hair production. Therefore, the term "hair follicle restoration" as used herein refers to the restoration of hair follicle function, which allows a thinned or weakened hair follicle to provide sufficient protection and nutrition to the hair root.

[0069] The term "hair growth" as used herein means the generation of hair in a body part, and more specifically, the generation and growth of hair from the scalp. Therefore, the term "promoting hair growth" as used herein means the regeneration of lost or reduced hair in a specific body part, including the scalp, or the promotion of the growth of hair whose growth has slowed down.

[0070] In the present invention, the cosmetic composition may have a dosage form selected from the group consisting of a solution, an external ointment, a cream, a foam, a soap, a liquid cleanser, a bath additive, an external hair preparation, a suspension, an emulsion, a paste, a gel, a lotion, a powder, an oil, a patch, and a spray, but is not limited to these.

[0071] When the dosage form of the present invention is an ointment, paste, cream, or gel, the carrier component may be, but is not limited to, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. These may be used alone or in combination of two or more.

[0072] When the formulation of the present invention is a powder or spray, carrier components that can be used include lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc., and particularly when the formulation is a spray, it may further contain a propellant such as, but not limited to, chlorofluorohydrocarbon, propane / butane, or dimethyl ether, which may be used alone or in combination of two or more.

[0073] When the dosage form of the present invention is a solution or emulsion, a solvent, solubilizer, or emulsifier may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, etc., and particularly, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, sesame oil, glycerol aliphatic esters, polyethylene glycol, or sorbitan fatty acid esters, but are not limited to these. These may be used alone or in combination of two or more.

[0074] When the dosage form of the present invention is a suspension, the carrier component may be, but is not limited to, a liquid diluent such as water, ethanol, or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, or polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant, etc. These may be used alone or in combination of two or more.

[0075] When the formulation of the present invention is an external preparation for hair, the cosmetic composition may specifically have the form of a hair tonic, hair conditioner, hair essence, hair lotion, hair nourishing lotion, hair shampoo, hair rinse, hair treatment, hair cream, hair nourishing cream, hair moisture cream, hair massage cream, hair wax, hair aerosol, hair pack, hair nourishing pack, hair soap, hair cleansing foam, hair oil, hair drying agent, hair preservative, hair dye, hair waving agent, hair bleach, hair gel, hair glaze, hair dressing, hair lacquer, hair moisturizer, hair mousse, or hair spray.

[0076] In one embodiment of the present invention, the induced pluripotent stem cell-derived mesenchymal stem cells may be differentiated from progenitor cells of the induced pluripotent stem cell-derived mesenchymal stem cells that do not express stage-specific embryonic antigen 4 (SSEA-4) protein.

[0077] In one embodiment of the present invention, the induced pluripotent stem cells may be human-derived induced pluripotent stem cells.

[0078] In one embodiment of the present invention, the induced pluripotent stem cell-derived mesenchymal stem cells may be pretreated with a pretreatment substance.

[0079] In one embodiment of the present invention, the pretreatment material may be hyaluronic acid.

[0080] Yet another aspect of the present invention is a method for preventing hair loss, strengthening hair roots, restoring hair follicles or promoting hair growth, comprising the steps of:

[0081] A providing step in which exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells are administered to or contacted with a subject.

[0082] Yet another aspect of the present invention is use of a cosmetic composition containing exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells as an active ingredient for preventing hair loss, strengthening hair roots, restoring hair follicles, or promoting hair growth.

[0083] Yet another aspect of the present invention is a method for preparing a composition comprising the steps of:

[0084] A separation step of isolating exosomes from stem cells or their cultures.

[0085] In one embodiment of the present invention, the separating step may involve separating exosomes from induced pluripotent stem cell-derived mesenchymal stem cells or a culture thereof.

[0086] In the isolation step, the stem cell culture medium is centrifuged at 200-400xg for 5-20 minutes to remove any remaining cells and cell debris, and the supernatant is then taken and centrifuged at high speed at 9,000-12,000xg for 60-80 minutes. The supernatant is then taken again and centrifuged at 90,000-120,000xg for 80-100 minutes, and the supernatant is then removed to obtain the exosomes remaining in the lower layer.

[0087] In one embodiment of the present invention, the stem cells may be autologous or allogeneic stem cells, may be derived from any type of animal, including humans and non-human mammals, and may be adult-derived stem cells or embryo-derived stem cells, such as, but not limited to, adult stem cells, embryonic stem cells, induced pluripotent stem cells, induced pluripotent stem cell-derived mesenchymal stem cells, BxC stem cells, and BxC-HA stem cells.

[0088] In one embodiment of the present invention, the method may further include a pretreatment step of pretreating the induced pluripotent stem cell-derived mesenchymal stem cells with hyaluronic acid.

[0089] In one embodiment of the present invention, the method may further include a selective culture step in which SSEA-4(-) cells that do not express SSEA-4 protein are separated from the cultured induced pluripotent stem cells, cultured, and differentiated into BxC stem cells.

[0090] In one embodiment of the present invention, the method may further include a production step of culturing stem cells in a cell culture medium.

[0091] The production step according to 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 any stem cell culture medium commonly used in the art, such as, for example, commercially produced media such as DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, DMEM / F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10), DMEM / F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), α-MEM (α-Minimal essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Isocove's Modified Dulbecco's Medium), KnockOut DMEM, and E8 (Essential 8 Medium), or artificially synthesized media, but is not limited to these.

[0092] In an embodiment of the present invention, the cell culture medium may further include components such as a carbon source, a nitrogen source, trace elements, amino acids, and antibiotics.

[0093] In one embodiment of the present invention, the production step may include an additional culturing step of culturing stem cells in exosome-depleted fetal bovine serum (FBS). Unlike general FBS, which contains a large amount of bovine serum-derived exosomes, exosome-depleted FBS is exosome-depleted, which can prevent bovine serum-derived exosomes, which are not exosomes secreted from stem cells, from being mixed into the culture medium.

[0094] Yet another aspect of the present invention is a method for preparing a pharmaceutical composition for treating hair loss, comprising the steps of:

[0095] The first stage involves culturing induced pluripotent stem cells in a culture medium;

[0096] A selective culture step in which SSEA-4(-) cells that do not express SSEA-4 protein are isolated from the cultured induced pluripotent stem cells and cultured to differentiate into BxC stem cells;

[0097] A second culture step in which BxC stem cells are cultured and differentiated into mesenchymal stem cells;

[0098] a pretreatment step in which mesenchymal stem cells are pretreated with hyaluronic acid;

[0099] A production step in which the pretreated mesenchymal stem cells are cultured to produce exosomes; and

[0100] A step of obtaining a culture supernatant from the mesenchymal stem cells or a culture thereof.

[0101] In the present invention, the method may further include a separation step of separating exosomes from the culture supernatant.

[0102] In one embodiment of the present invention, the first culturing step may involve culturing the induced pluripotent stem cells in a medium containing FBS and basic fibroblast growth factor (bFGF) for 1 to 10 days.

[0103] In one embodiment of the present invention, the selective culture step may involve isolating SSEA-4(-) cells, which do not express SSEA-4 protein, from the induced pluripotent stem cells and culturing them in a medium containing FBS and bFGF for 1 to 10 days to differentiate them into BxC stem cells.

[0104] In one embodiment of the present invention, the pretreatment step may include culturing mesenchymal stem cells in a medium containing hyaluronic acid at a concentration of 0.1 to 1000 μg / ml, 0.5 to 1000 μg / ml, 1 to 500 μg / ml, 1 to 200 μg / ml, 1 to 100 μg / ml, 1 to 80 μg / ml, 1 to 60 μg / ml, 10 to 60 μg / ml, for example, 40 μg / ml.

[0105] In one embodiment of the present invention, the production step may include an additional culturing step of culturing mesenchymal stem cells in exosome-depleted FBS.

[0106] In one embodiment of the present invention, the obtaining step may include further culturing the pretreated mesenchymal stem cells, and then recovering the culture medium to remove cells and cell debris, thereby obtaining a culture supernatant.

[0107] In one embodiment of the present invention, the separation step involves collecting the supernatant and centrifuging it at high speed at 9,000 to 12,000 x g for 60 to 80 minutes, and then collecting the supernatant again and centrifuging it at 90,000 to 120,000 x g for 80 to 100 minutes, and removing the supernatant to obtain exosomes remaining in the lower layer. [Effects of the Invention]

[0108] The present invention relates to a composition for treating hair loss containing stem cell-derived exosomes and a method for producing the same. The exosomes of the present invention have excellent effects of promoting the growth and recovery of human dermal papilla cells and can be used in pharmaceutical compositions and various cosmetics for treating hair loss. [Brief explanation of the drawings]

[0109] [Figure 1] 1 is a graph showing the results of a comparative experiment on whether or not cell viability of human dermal papilla cells (treated with DHT) is restored by treatment with exosomes (BxC-HAe) derived from hyaluronic acid-pretreated induced pluripotent stem cells (BxC-HA) according to one example of the present invention.

[0110] [Figure 2]1 is a graph showing the results of a comparative experiment on whether or not cell viability of human dermal papilla cells (testosterone-treated) was restored by treatment with exosomes (BxC-HAe) derived from hyaluronic acid-pretreated induced pluripotent stem cells (MES) derived from mesenchymal stem cells (BxC-HA) according to one embodiment of the present invention.

[0111] [Figure 3] 1 shows photographs of the degree of wound healing immediately after and 24 hours after treatment of a wounded human dermal papilla cell monolayer (DHT-treated) with exosomes (BxC-HAe) derived from hyaluronic acid-pretreated induced pluripotent stem cell-derived mesenchymal stem cells (BxC-HA) according to one embodiment of the present invention, compared with a control group.

[0112] [Figure 4] 1 is a graph showing the results of a comparative experiment on whether or not the wound recovery rate of human dermal papilla cells (treated with DHT) is restored by treatment with exosomes (BxC-HAe) derived from hyaluronic acid-pretreated induced pluripotent stem cells (BxC-HA) according to one embodiment of the present invention.

[0113] [Figure 5] 1 is a graph showing the results of a comparative experiment on changes in IGF1 gene expression levels in human dermal papilla cells (treated with DHT) following treatment with exosomes (BxC-HAe) derived from hyaluronic acid-pretreated induced pluripotent stem cells (MES) derived from BxC-HA according to one embodiment of the present invention.

[0114] [Figure 6] 1 is a graph showing the results of a comparative experiment on changes in EGF gene expression levels in human dermal papilla cells (treated with DHT) following treatment with exosomes (BxC-HAe) derived from hyaluronic acid-pretreated induced pluripotent stem cells (MES) derived from mesenchymal stem cells (BxC-HA) according to one example of the present invention.

[0115] [Figure 7]1 is a graph showing the results of a comparative experiment on changes in IGF1 gene expression levels in human dermal papilla cells (testosterone-treated) treated with exosomes (BxC-HAe) derived from hyaluronic acid-pretreated induced pluripotent stem cells (MES) (BxC-HA) according to one example of the present invention.

[0116] [Figure 8] 1 is a graph showing the results of a comparative experiment on changes in FGF7 gene expression levels in human dermal papilla cells (testosterone-treated) treated with exosomes (BxC-HAe) derived from hyaluronic acid-pretreated induced pluripotent stem cells (MES) (BxC-HA) according to one example of the present invention.

[0117] [Figure 9] 1 is a graph showing the results of a comparative experiment on changes in VEGF gene expression levels in human dermal papilla cells (testosterone-treated) treated with exosomes (BxC-HAe) derived from hyaluronic acid-pretreated induced pluripotent stem cells (MES) derived from mesenchymal stem cells (BxC-HA) according to one embodiment of the present invention.

[0118] [Figure 10] 1 is a graph showing the results of a comparative experiment on changes in TGF-β1 gene expression levels in human dermal papilla cells (treated with DHT) following treatment with exosomes (BxC-HAe) derived from hyaluronic acid-pretreated induced pluripotent stem cells (MES) derived from mesenchymal stem cells (BxC-HA) according to one embodiment of the present invention.

[0119] [Figure 11] 1 is a graph showing the results of a comparative experiment on changes in IL-6 gene expression levels in human dermal papilla cells (treated with DHT) following treatment with exosomes (BxC-HAe) derived from hyaluronic acid-pretreated induced pluripotent stem cells (MES) derived from mesenchymal stem cells (BxC-HA) according to one example of the present invention.

[0120] [Figure 12]1 is a graph showing the results of a comparative experiment on changes in TGF-β1 gene expression levels in human dermal papilla cells (testosterone-treated) treated with exosomes (BxC-HAe) derived from hyaluronic acid-pretreated induced pluripotent stem cells (MES) (BxC-HA) according to one embodiment of the present invention.

[0121] [Figure 13] 1 is a graph showing the results of a comparative experiment on changes in IL-6 gene expression levels in human dermal papilla cells (testosterone-treated) treated with exosomes (BxC-HAe) derived from hyaluronic acid-pretreated induced pluripotent stem cells (MES) (BxC-HA) according to one example of the present invention.

[0122] [Figure 14] 1 is a graph showing the results of a comparative experiment on changes in AR gene expression levels in human dermal papilla cells (treated with DHT) treated with exosomes (BxC-HAe) derived from hyaluronic acid-pretreated induced pluripotent stem cells (MES) (BxC-HA) according to one example of the present invention.

[0123] [Figure 15] 1 is a graph showing the results of a comparative experiment on changes in AR gene expression levels in human dermal papilla cells (testosterone-treated) treated with exosomes (BxC-HAe) derived from hyaluronic acid-pretreated induced pluripotent stem cells (MES) (BxC-HA) according to one example of the present invention.

[0124] [Figure 16] 1 is a graph showing the results of a comparative experiment on changes in body weight of mice (testosterone-treated) treated with exosomes (BxC-HAe) derived from hyaluronic acid-pretreated induced pluripotent stem cells (MES) (BxC-HA) according to one example of the present invention.

[0125] [Figure 17] 1 is a graph showing the results of a comparative experiment on the change in hair growth area in mice (testosterone-treated) treated with exosomes (BxC-HAe) derived from hyaluronic acid-pretreated induced pluripotent stem cells (MES) (BxC-HA) according to one embodiment of the present invention. [Figure 18] 1 is a graph showing the results of a comparative experiment on the change in hair growth area in mice (testosterone-treated) treated with exosomes (BxC-HAe) derived from hyaluronic acid-pretreated induced pluripotent stem cells (MES) (BxC-HA) according to one embodiment of the present invention.

[0126] [Figure 19] Photographs showing the change in hair growth area in mice (testosterone-treated) treated with exosomes (BxC-HAe) derived from hyaluronic acid-pretreated induced pluripotent stem cells-derived mesenchymal stem cells (BxC-HA) according to one example of the present invention, compared with a control group.

[0127] [Figure 20] 1 shows photographs of H&E stained skin tissue from mice (testosterone-treated) treated with exosomes (BxC-HAe) derived from hyaluronic acid-pretreated induced pluripotent stem cells-derived mesenchymal stem cells (BxC-HA) according to one example of the present invention, compared with a control group.

[0128] [Figure 21] 1 is a graph showing the results of a comparative experiment on the change in the ratio of anagen hair follicles (Anagen) in the skin tissue of mice (testosterone-treated) treated with exosomes (BxC-HAe) derived from hyaluronic acid-pretreated induced pluripotent stem cells (BxC-HA) according to one embodiment of the present invention.

[0129] [Figure 22] 1 shows comparative photographs of immunostained skin tissues from mice (testosterone-treated) treated with exosomes (BxC-HAe) derived from hyaluronic acid-pretreated induced pluripotent stem cells-derived mesenchymal stem cells (BxC-HA) according to one example of the present invention.

[0130] [Figure 23]1 is a graph showing the results of a comparative experiment on the fluorescent signal intensity of AR obtained by immunostaining skin tissues of mice (testosterone-treated) treated with exosomes (BxC-HAe) derived from hyaluronic acid-pretreated induced pluripotent stem cells (BxC-HA) according to one embodiment of the present invention.

[0131] [Figure 24] 1 is a graph showing the results of a comparative experiment on the fluorescent signal intensity of β-catenin, obtained by immunostaining skin tissues of mice (testosterone-treated) treated with exosomes (BxC-HAe) derived from hyaluronic acid-pretreated induced pluripotent stem cells (BxC-HA) according to one embodiment of the present invention.

[0132] [Figure 25] 1 shows photographs of the degree of wound healing immediately after and 24 hours after treatment of a wounded human dermal papilla cell monolayer (testosterone-treated) with culture supernatant (CM) of hyaluronic acid-pretreated induced pluripotent stem cells-derived mesenchymal stem cells according to one embodiment of the present invention, compared with a control group.

[0133] [Figure 26] 1 is a graph showing the results of a comparative experiment on whether or not the wound recovery rate of human dermal papilla cells (testosterone-treated) is restored by treatment with culture supernatant (CM) of mesenchymal stem cells derived from hyaluronic acid-pretreated induced pluripotent stem cells according to one embodiment of the present invention.

[0134] [Figure 27] 1 is a graph showing the results of a comparative experiment on changes in IGF1 gene expression levels in human hair papilla cells (testosterone-treated) treated with culture supernatant (CM) of mesenchymal stem cells derived from hyaluronic acid-pretreated induced pluripotent stem cells according to one embodiment of the present invention.

[0135] [Figure 28] 1 is a graph showing the results of a comparative experiment on changes in EGF gene expression levels in human hair papilla cells (testosterone-treated) treated with culture supernatant (CM) of mesenchymal stem cells derived from hyaluronic acid-pretreated induced pluripotent stem cells according to one embodiment of the present invention.

[0136] [Figure 29] 1 is a graph showing the results of a comparative experiment on the change in TGF-β1 gene expression levels in human hair papilla cells (testosterone-treated) treated with culture supernatant (CM) of mesenchymal stem cells derived from hyaluronic acid-pretreated induced pluripotent stem cells according to one embodiment of the present invention.

[0137] [Figure 30] 1 is a graph showing the results of a comparative experiment on the change in IL-6 gene expression levels in human hair papilla cells (testosterone-treated) treated with culture supernatant (CM) of mesenchymal stem cells derived from hyaluronic acid-pretreated induced pluripotent stem cells according to one example of the present invention.

[0138] In Figures 1 to 30, p-value analysis was performed using one-way ANOVA on the experimental results of the control group and the experimental group compared to the untreated group. In each figure, "* or #" means p<0.05, "** or ##" means p<0.01, "*** or ###" means p<0.001, and "**** or ####" means p<0.0001. DETAILED DESCRIPTION OF THE INVENTION

[0139] A pharmaceutical composition for treating, preventing, alleviating, or suppressing hair loss, comprising exosomes isolated from mesenchymal stem cells (MSCs) derived from induced pluripotent stem cells (iPSCs) as an active ingredient. [Example]

[0140] The present invention will be described in more detail below with reference to the following examples, but these examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

[0141] Example 1: Induced pluripotent stem cell-derived mesenchymal stem cell culture

[0142] Induced pluripotent stem cells (iPSCs) were cultured for 7 days in DMEM supplemented with 10% FBS (Fetal Bovine Serum) and 10 ng / ml bFGF. Subsequently, SSEA-4(-) cells, which do not express stage-specific embryonic antigen 4 (SSEA-4) on the cell surface, were isolated by FACS analysis and used to identify progenitor cells for induced pluripotent stem cells (iPSCs). The isolated SSEA-4(-) cells were then passaged and further cultured for 7 days in DMEM supplemented with 10% FBS and 10 ng / ml bFGF to generate BxC stem cells.

[0143] The BxC stem cells were then further cultured in a culture 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 induced pluripotent stem cell-derived mesenchymal stem cells.

[0144] Example 2: Hyaluronic acid pre-treated exosome (BxC-HAe) isolation

[0145] The induced pluripotent stem cell-derived mesenchymal stem cells prepared in Example 1 were cultured for 24 hours in a high-glucose DMEM culture medium containing 10% fetal bovine serum, 1% MEM non-essential amino acid solution, and 40 μg / ml hyaluronic acid to prepare hyaluronic acid-pretreated induced pluripotent stem cell-derived mesenchymal stem cells (BxC-HA stem cells).

[0146] After completing the culture, the BxC-HA stem cells were washed and cultured for an additional 72 hours in culture medium supplemented with 10% exosome-depleted FBS.

[0147] After 72 hours of incubation, the pretreatment-treated culture medium was collected and centrifuged at 300 x g for 10 minutes to remove remaining cells and cell debris. The supernatant was then filtered through a 0.22 μm filter and centrifuged at 10,000 x g for 70 minutes at 4°C in a high-speed centrifuge. The supernatant was then collected again and centrifuged at 100,000 x g for 90 minutes at 4°C in an ultracentrifuge. The supernatant was then removed, and the remaining exosomes were diluted with PBS to isolate hyaluronic acid-pretreated exosomes (hereafter referred to as BxC-HAe) for the following experiments.

[0148] Example 3: Confirmation of recovery of decreased cell viability of human dermal papilla cells (treated with DHT)

[0149] To determine whether the composition of the present invention can restore the decreased cell viability of human dermal papilla cells, human dermal papilla cells were treated with BxC-HAe isolated in Example 2 and the cell proliferation rates were compared 24 hours later.

[0150] Specifically, 1,000 human dermal papilla cells were inoculated per well of a 96-well plate and cultured in a basal medium at 37°C and 5% CO for 24 hours. The composition of the basal medium is shown in Table 1 below.

[0151] [Table 1]

[0152] To set up the control and experimental groups, the DHT (dihydrotestosterone; hereafter referred to as DHT)-untreated group was treated with 0.05% (v / v) DMSO (dimethyl sulfoxide) in basal medium, the control group was treated with 500 nM DHT in basal medium, the first experimental group was treated with 500 nM DHT in basal medium mixed with 1-100 μg / ml of the first test drug (BxC-e), and the second experimental group was treated with 500 nM DHT in basal medium mixed with 1-100 μg / ml of the second test drug (BxC-HAe). After each treatment, the cultures were cultured for 24 hours.

[0153] After incubation, overall cell viability was measured using a Cell Count Kit-8 (CCK-8) assay (Enzo, New York, NY, USA). Specifically, after 24 hours of incubation in the experimental and control groups, CCK-8 solution was added to each medium, and after incubation at 37°C for 2 hours, the optical density (OD) was measured at 450 nm using a microplate reader. The results are shown in Figure 1 and Table 2. The values ​​for each well (n = 5 / group) were averaged and expressed relative to the average value for the control group, which was set at 100%.

[0154] [Table 2]

[0155] The results showed that cell viability, which had been reduced by approximately 15% by DHT treatment compared to the untreated group, increased by 2.4% with 100μg / ml BxC-e treatment compared to the untreated group. Meanwhile, with BxC-HAe treatment, cell viability increased by 4.0% at 1μg / ml or 10μg / ml compared to the untreated group, and by 6.5% at 100μg / ml compared to the untreated group, demonstrating a significant increase in cell viability compared to the control group.

[0156] Example 4: Confirmation of recovery of decreased cell viability of human dermal papilla cells (testosterone treatment)

[0157] To determine whether the composition of the present invention can restore the decreased cell viability of human dermal papilla cells, human dermal papilla cells were treated with BxC-HAe isolated in Example 2 and the cell proliferation rates were compared 24 hours later.

[0158] Specifically, 1,000 human dermal papilla cells were inoculated per well of a 96-well plate and cultured in a basal medium at 37°C and 5% CO for 24 hours. The composition of the basal medium is shown in Table 3 below.

[0159] [Table 3]

[0160] To establish the control and experimental groups, the testosterone-free group was treated with 0.05% (v / v) DMSO (dimethyl sulfoxide) in basal medium. The control group was treated with 50 μM testosterone in basal medium. The first experimental group was treated with 50 μM testosterone in basal medium mixed with 10–50 μg / ml of the first test drug (BxC-e). The second experimental group was treated with 50 μM testosterone in basal medium mixed with 10–50 μg / ml of the second test drug (BxC-HAe). After each treatment, the cultures were cultured for 24 hours.

[0161] After incubation, overall cell viability was measured using a Cell Count Kit-8 (CCK-8) assay (Enzo, New York, NY, USA). Specifically, after 48 hours of incubation, CCK-8 solution was added to each medium for the experimental and control groups, and the cells were incubated at 37°C for 2 hours. The optical density (OD) was measured at 450 nm using a microplate reader, and the results are shown in Figure 2 and Table 4. The values ​​for each well (n = 6 / group) were averaged and expressed relative to the control group average value, which was set at 100%.

[0162] [Table 4]

[0163] The results showed that testosterone treatment reduced cell viability by approximately 15% compared to the untreated group, and that 50μg / ml BxC-e treatment reduced cell viability by approximately 6% compared to the untreated group. Furthermore, treatment with BxC-HAe at 10μg / ml or 25μg / ml reduced cell viability by approximately 4% or 2% compared to the untreated group, whereas treatment with 50μg / ml increased cell viability by 1.5% compared to the untreated group, demonstrating a significant increase in cell viability compared to the control group.

[0164] Example 5: Confirmation of recovery of the decreased wound healing rate of human dermal papilla cells

[0165] To determine whether the composition of the present invention can restore the reduced wound healing rate of human dermal papilla cells, human dermal papilla cells were treated with BxC-HAe isolated in Example 2 and the wound healing rates were compared after 24 hours.

[0166] Specifically, 25,000 human dermal papilla cells were seeded per well in a 12-well plate and cultured in basal medium at 37°C and 5% CO for 24 hours. The composition of the basal medium was the same as in Table 1 above. A cross-shaped wound was made in the cell monolayer formed by the culture using a 1 ml pipette tip, and the cells were washed three times with 1X D-PBS to remove any cellular debris.

[0167] To set up the control and experimental groups, the DHT-untreated group was treated with 0.05% (v / v) DMSO in basal medium, the control group was treated with 500 nM DHT in basal medium, the first experimental group was treated with 500 nM DHT and 25 μg / ml of the first test drug (BxC-e) in basal medium, and the second experimental group was treated with 500 nM DHT and 25 μg / ml of the second test drug (BxC-HAe) in basal medium. After treatment with each substance, each medium was cultured for 24 hours.

[0168] To examine the recovery of the cell layer in each medium, the changes in the cell layer at the time of drug treatment and after 24 hours were photographed using a camera (Dixi eXcope system). The photographs taken in each medium are shown in Figure 3.

[0169] The photographs were quantitatively analyzed using a program (ImageJ), and the experiment was repeated five times for each control and experimental group to calculate the average analytical values, which are shown in Figure 4 and Table 5. The average analytical values ​​for each group were expressed relative to the average analytical value for the DHT-untreated group, which was set at 100%.

[0170] [Table 5]

[0171] The analysis showed that when DHT was applied to the culture medium in which the wounds were formed, the wound healing rate decreased by approximately 34% compared to the DHT-untreated group; when the first test drug, BxC-e, was applied, the wound healing rate decreased by approximately 15% compared to the DHT-untreated group; and when the second test drug, BxC-HAe, was applied, the wound healing rate actually increased by 7% compared to the DHT-untreated group.

[0172] Meanwhile, the wound healing rate of the first experimental group did not show a statistically significant increase compared to the control group, whereas the wound healing rate of the second experimental group showed a statistically significant increase compared to the control group. This confirmed that BxC-HAe can significantly promote the wound healing of human dermal papilla cells.

[0173] Example 6: Confirmation of growth factor expression promotion in human dermal papilla cells (treated with DHT)

[0174] To confirm whether the composition of the present invention promotes the expression of IGF1 (Insulin-like Growth Factor 1) and EGF (Epidermal Growth Factor), which are known to be important growth factors for hair growth, from human dermal papilla cells, human dermal papilla cells were treated with BxC-HAe isolated in Example 2 and the expression levels of IGF1 and EGF genes were compared 24 hours later.

[0175] Specifically, 25,000 human dermal papilla cells were seeded per well of a 12-well plate and cultured in a basal medium at 37°C and 5% CO for 24 hours. The composition of the basal medium was the same as that shown in Table 1 above.

[0176] To set up the control and experimental groups, the DHT-untreated group was treated with 0.05% (v / v) DMSO in basal medium, the control group was treated with 500 nM DHT in basal medium, the first experimental group was treated with 500 nM DHT and 10 μg / ml of the first test drug (BxC-e) in basal medium, and the second experimental group was treated with 500 nM DHT and 10 μg / ml of the second test drug (BxC-HAe) in basal medium. After treatment with each substance, each medium was cultured for 24 hours.

[0177] After the culture was completed, cells were collected from each medium, and total RNA was extracted from each medium using TRIzol® (Invitrogen). The mRNA expression levels of IGF1 and EGF genes were measured for the same amount of total RNA extracted from each medium using real-time PCR (Applied Biosystems). Each measurement was calculated based on the GAPDH expression level. -ΔΔCt After normalization using a technique, the quantitative value in the DHT-untreated group was set at 100% for relative quantification, and the results of quantifying the expression levels of the IGF1 gene and EGF gene are shown in Figure 5 and Table 6, and Figure 6 and Table 7, respectively.

[0178] [Table 6]

[0179] [Table 7]

[0180] Quantitative results showed that when human dermal papilla cells were treated with DHT, the expression level of the IGF1 gene increased by 126.8% and the expression level of the EGF gene increased by 114.0% compared to the DHT-untreated group, and when treated with the first test drug, BxC-e, the expression level of the IGF1 gene increased by 181.4% and the expression level of the EGF gene increased by 239.5% compared to the DHT-untreated group.

[0181] Meanwhile, treatment with BxC-HAe, the second test drug, increased IGF1 gene expression by 319.8% and EGF gene expression by 343.2% compared to the DHT-untreated group. This marked a statistically significant increase in the expression of each gene compared to the control group. Therefore, it was confirmed that BxC-HAe promotes hair growth by promoting IGF1 and EGF gene expression in human dermal papilla cells, thereby exerting the effect of treating or preventing hair loss.

[0182] Example 7: Confirmation of promotion of growth factor expression in human dermal papilla cells (testosterone treatment)

[0183] To confirm whether the composition of the present invention promotes the expression of IGF1 (Insulin-like Growth Factor 1), FGF7 (Fibroblast Growth Factor 7), and VEGF (Vascular Epidermal Growth Factor), which are known to be growth factors important for hair growth, in human dermal papilla cells, human dermal papilla cells were treated with BxC-HAe isolated in Example 2, and the expression levels of IGF1, FGF7, and VEGF genes were compared 24 hours later.

[0184] Specifically, 25,000 human dermal papilla cells were seeded per well of a 12-well plate and cultured in a basal medium at 37°C and 5% CO for 24 hours. The composition of the basal medium was the same as that shown in Table 1 above.

[0185] To establish the control and experimental groups, the testosterone-free group was treated with 0.05% (v / v) DMSO in basal medium. The control group was treated with 50 μM testosterone in basal medium. Experimental group 1 was treated with 50 μM testosterone and 100 nM of the first test drug (finasteride) in basal medium. Experimental group 2 was treated with 50 μM testosterone and 25 μg / ml of the second test drug (BxC-e) in basal medium. Experimental group 3 was treated with 50 μM testosterone and 25 μg / ml of the third test drug (BxC-HAe) in basal medium. After each treatment, the cultures were cultured for 24 hours.

[0186] In this case, finasteride is a drug that suppresses the male hormone androgen, and refers to a substance that is commonly used to treat male pattern baldness, and the same applies hereinafter.

[0187] After the culture was completed, the cells were collected from each medium, and total RNA was extracted from each medium using TRIzol® (Invitrogen). The mRNA expression levels of IGF1, FGF7, and VEGF genes were measured by real-time PCR (Applied Biosystems) analysis for the same amount of total RNA extracted from each medium. Each measurement was calculated based on the GAPDH expression level. -ΔΔCt After normalization using a technique, relative quantification was performed with the quantitative value in the 50 μM testosterone-untreated group set at 100%, and the results of quantifying the expression levels of the IGF1 gene, FGF7 gene, and VEGF gene are shown in Figure 7 and Table 8, Figure 8 and Table 9, and Figure 9 and Table 10, respectively.

[0188] [Table 8]

[0189] [Table 9]

[0190] [Table 10]

[0191] Quantitative analysis showed that testosterone treatment of human dermal papilla cells increased IGF1 gene expression by 137% and VEGF gene expression by 122% compared to the testosterone-free group, while FGF7 gene expression decreased by 99%. Treatment with finasteride, the first test drug, increased IGF1 gene expression by 162% and VEGF gene expression by 122% compared to the testosterone-free group, while FGF7 gene expression decreased by 91%. Treatment with BxC-e, the second test drug, increased IGF1 gene expression by 214%, FGF7 gene expression by 160%, and VEGF gene expression by 140% compared to the testosterone-free group.

[0192] Meanwhile, treatment with the third test drug, BxC-HAe, increased IGF1 gene expression by 287%, FGF7 gene expression by 219%, and VEGF gene expression by 279% compared to the untreated group. This indicates that the expression of each gene increased significantly to a statistically significant level compared to the control group. Therefore, it was confirmed that BxC-HAe promotes hair growth by promoting the gene expression of IGF1, FGF7, and VEGF in human dermal papilla cells, thereby exerting the effect of treating or preventing hair loss.

[0193] Example 8: Confirmation of suppression of hair follicle growth inhibitor expression in human dermal papilla cells (treated with DHT)

[0194] To confirm whether the composition of the present invention can suppress the expression of TGF-β1 (Transforming Growth Factor-beta 1) and IL-6 (Interleukin-6) in human dermal papilla cells, which shorten the anagen phase of hair follicles and induce them into the catagen and telogen phases, human dermal papilla cells were treated with BxC-HAe isolated in Example 2 and the expression levels of TGF-β1 and IL-6 genes were compared 24 hours later.

[0195] Specifically, 25,000 human dermal papilla cells were seeded per well of a 12-well plate and cultured in a basal medium at 37°C and 5% CO for 24 hours. The composition of the basal medium was the same as that shown in Table 1 above.

[0196] To set up the control and experimental groups, the DHT-untreated group was treated with 0.05% (v / v) DMSO in basal medium, the control group was treated with 500 nM DHT in basal medium, the first experimental group was treated with 500 nM DHT and 10 μg / ml of the first test drug (BxC-e) in basal medium, and the second experimental group was treated with 500 nM DHT and 10 μg / ml of the second test drug (BxC-HAe) in basal medium. After treatment with each substance, each medium was cultured for 24 hours.

[0197] After the culture was completed, cells were collected from each medium, and total RNA was extracted from each medium using TRIzol® (Invitrogen). The mRNA expression levels of TGF-β1 and IL-6 genes were measured by real-time PCR (Applied Biosystems) analysis for the same amount of total RNA extracted from each medium. Each measurement was calculated based on the GAPDH expression level. -ΔΔCt After normalization by the technique, relative quantification was performed with the quantitative value in the DHT-untreated group set at 100%, and the results of quantifying the expression levels of the TGF-β1 gene and IL-6 gene are shown in Figure 10 and Table 11, and Figure 11 and Table 12, respectively.

[0198] [Table 11]

[0199] [Table 12]

[0200] Quantitation showed that in the case of the TGF-β1 gene, expression levels increased to 132.5% when human dermal papilla cells were treated with DHT compared to the DHT-untreated group, but decreased to 92.6% when treated with the first test drug, BxC-e, compared to the DHT-untreated group. In contrast, in the case of the IL-6 gene, expression levels increased to 136.4% when human dermal papilla cells were treated with DHT compared to the DHT-untreated group, and increased to 158.9% when treated with the first test drug, BxC-e, compared to the DHT-untreated group.

[0201] Meanwhile, treatment with the second test drug, BxC-HAe, reduced TGF-β1 gene expression by 55.3% and IL-6 gene expression by 43.7% compared to the DHT-untreated group. This indicates a statistically significant reduction in the expression of each gene compared to the control group. Therefore, it was confirmed that BxC-HAe suppresses the expression of both TGF-β1 and IL-6 genes, which inhibit hair follicle growth, thereby enabling sustainable hair follicle growth.

[0202] Example 9: Confirmation of suppression of hair follicle growth inhibitor expression in human dermal papilla cells (testosterone treatment)

[0203] To confirm whether the composition of the present invention can suppress the expression of TGF-β1 (Transforming Growth Factor-beta 1) and IL-6 (Interleukin-6) in human dermal papilla cells, which shorten the anagen phase of hair follicles and induce the transition to the catagen and telogen phases, human dermal papilla cells were treated with BxC-HAe isolated in Example 2 and the expression levels of TGF-β1 and IL-6 genes were compared 24 hours later.

[0204] Specifically, 25,000 human dermal papilla cells were seeded per well of a 12-well plate and cultured in a basal medium at 37°C and 5% CO for 24 hours. The composition of the basal medium was the same as that shown in Table 1 above.

[0205] The control and experimental groups were then established: the testosterone-free group was treated with 0.05% (v / v) DMSO in basal medium; the control group was treated with 50 μM testosterone in basal medium; the first experimental group was treated with 50 μM testosterone and 100 nM of the first test drug (finasteride) in basal medium; the second experimental group was treated with 50 μM testosterone and 25 μg / ml of the second test drug (BxC-e) in basal medium; and the third experimental group was treated with 50 μM testosterone and 25 μg / ml of the third test drug (BxC-HAe) in basal medium. After each treatment, the cultures were cultured for 24 hours.

[0206] After the culture was completed, cells were collected from each medium, and total RNA was extracted from each medium using TRIzol® (Invitrogen). The mRNA expression levels of TGF-β1 and IL-6 genes were measured by real-time PCR (Applied Biosystems) analysis for the same amount of total RNA extracted from each medium. Each measurement was calculated based on the GAPDH expression level. -ΔΔCt After normalization by technique, relative quantification was performed with the quantitative value in the testosterone-untreated group set at 100%, and the results of quantifying the expression levels of the TGF-β1 gene and IL-6 gene are shown in Figure 12 and Table 13, and Figure 13 and Table 14, respectively.

[0207] [Table 13]

[0208] [Table 14]

[0209] Quantitative results showed that in the case of the TGF-β1 gene, expression levels increased by 148% when human dermal papilla cells were treated with testosterone compared to the testosterone-untreated group, but when treated with the first test drug, finasteride, expression levels increased by 107% compared to the testosterone-untreated group, when treated with the second test drug, BxC-e, expression levels increased by 119% compared to the testosterone-untreated group, and when treated with the third test drug, BxC-HAe, expression levels increased by 105% compared to the testosterone-untreated group.

[0210] In the case of the IL-6 gene, when human dermal papilla cells were treated with testosterone, the expression level increased by 456% compared to the testosterone-untreated group, and when treated with the first test drug, finasteride, the expression level decreased to 95% compared to the testosterone-untreated group. When treated with the second test drug, BxC-e, the expression level increased by 257% compared to the testosterone-untreated group, while when treated with the third test drug, BxC-HAe, the expression level decreased to 130% compared to the testosterone-untreated group.

[0211] In other words, the expression level of each gene was significantly reduced to a statistically significant level when treated with BxC-HAe compared to the control group. Therefore, it was confirmed that BxC-HAe suppresses the expression of both TGF-β1 and IL-6 genes, which inhibit hair follicle growth, thereby enabling sustainable hair follicle growth.

[0212] Example 10: Confirmation of suppression of androgen receptor expression in human dermal papilla cells (treated with DHT)

[0213] To confirm whether the composition of the present invention suppresses the expression of androgen receptor (AR), a DHT receptor, in human dermal papilla cells, human dermal papilla cells were treated with BxC-HAe isolated in Example 2 and the level of AR expression was compared 24 hours later.

[0214] Specifically, 25,000 human dermal papilla cells were seeded per well of a 12-well plate and cultured in a basal medium at 37°C and 5% CO for 24 hours. The composition of the basal medium was the same as that shown in Table 1 above.

[0215] To set up the control and experimental groups, the DHT-untreated group was treated with 0.05% (v / v) DMSO in basal medium, the control group was treated with 500 nM DHT in basal medium, the first experimental group was treated with 500 nM DHT and 10 μg / ml of the first test drug (BxC-e) in basal medium, and the second experimental group was treated with 500 nM DHT and 10 μg / ml of the second test drug (BxC-HAe) in basal medium. After treatment with each substance, each medium was cultured for 24 hours.

[0216] After the culture was completed, cells were collected from each medium and total RNA was extracted from each medium using TRIzol® (Invitrogen). The mRNA expression level of the AR gene was measured for the same amount of total RNA extracted from each medium using real-time PCR (Applied Biosystems). Each measurement was calculated based on the GAPDH expression level. -ΔΔCt After normalization by the technique, relative quantification was performed with the quantification value in the DHT-untreated group set at 100%, and the results of quantification of the expression level of the AR gene are shown in FIG. 14 and Table 15.

[0217] [Table 15]

[0218] Quantitation showed that when human dermal papilla cells were treated with DHT, the expression level of the AR gene decreased to 97.5% compared to the DHT-untreated group, whereas when treated with the first test drug, BxC-e, the expression level of the AR gene increased to 101.3% compared to the DHT-untreated group.

[0219] Meanwhile, treatment with the second test drug, BxC-HAe, reduced AR gene expression by 26.3% compared to the DHT-untreated group, demonstrating a statistically significant suppression of AR gene expression compared to the control group. This confirms that BxC-HAe reduces the sensitivity of human dermal papilla cells to DHT and can treat or prevent DHT-induced male pattern baldness.

[0220] Example 11: Confirmation of suppression of androgen receptor expression in human dermal papilla cells (testosterone treatment)

[0221] To confirm whether the composition of the present invention suppresses the expression of androgen receptor (AR), a receptor for testosterone, in human dermal papilla cells, human dermal papilla cells were treated with BxC-HAe isolated in Example 2 and the level of AR expression was compared 24 hours later.

[0222] Specifically, 25,000 human dermal papilla cells were seeded per well of a 12-well plate and cultured in a basal medium at 37°C and 5% CO for 24 hours. The composition of the basal medium was the same as that shown in Table 1 above.

[0223] The control and experimental groups were then established: the testosterone-free group was treated with 0.05% (v / v) DMSO in basal medium; the control group was treated with 50 μM testosterone in basal medium; the first experimental group was treated with 50 μM testosterone and 100 nM of the first test drug (finasteride) in basal medium; the second experimental group was treated with 50 μM testosterone and 25 μg / ml of the second test drug (BxC-e) in basal medium; and the third experimental group was treated with 50 μM testosterone and 25 μg / ml of the third test drug (BxC-HAe) in basal medium. After each treatment, the cultures were cultured for 24 hours.

[0224] After the culture was completed, cells were collected from each medium and total RNA was extracted from each medium using TRIzol® (Invitrogen). The mRNA expression level of the AR gene was measured for the same amount of total RNA extracted from each medium using real-time PCR (Applied Biosystems). Each measurement was calculated based on the GAPDH expression level. -ΔΔCt After normalization by technique, the quantitative value in the testosterone-untreated group was set as 100% and relative quantification was performed. The results of quantification of the expression level of the AR gene are shown in FIG. 15 and Table 16.

[0225] [Table 16]

[0226] Quantitation results showed that when human dermal papilla cells were treated with testosterone, AR gene expression increased by 173% compared to the testosterone-untreated group, while when treated with the first test drug, finasteride, AR gene expression increased by 146% compared to the testosterone-untreated group, and when treated with the second test drug, BxC-e, AR gene expression increased by 151% compared to the testosterone-untreated group.

[0227] Meanwhile, treatment with the third test drug, BxC-HAe, showed 104% AR gene expression compared to the testosterone-untreated group, demonstrating a statistically significant suppression of AR gene expression compared to the control group. This confirms that BxC-HAe reduces the sensitivity of human dermal papilla cells to testosterone and can treat or prevent testosterone-induced male pattern baldness.

[0228] Example 12: Confirmation of hair growth recovery in testosterone-treated animals

[0229] To confirm whether the composition of the present invention can restore hair growth, the BxC-HAe isolated in Example 2 was administered to a testosterone-treated animal model, and then weights and subsequent photographs were taken and compared.

[0230] Specifically, 6-week-old C57BL / 6 mice were kept at 25°C and 50% humidity with self-feeding and tap water for 2 weeks, and then 8-week-old mice were used for the experiment.

[0231] Then, on day 0, the hair was primarily removed using a mouse hair clipper, and Niklean (Ildong Pharmaceutical) was applied for 90 seconds for secondary hair removal, after which the drug was washed off three times with lukewarm water.

[0232] In the testosterone treatment group, testosterone was dissolved in 50% ethanol to a concentration of 0.5% (w / w), and 100 μl of the solution was applied to the shaved back of each mouse every day from the first day.

[0233] Thereafter, to set up the control and experimental groups, the control group received a subcutaneous injection of 100 μL D-PBS every other day from the first day, the first experimental group received a subcutaneous injection of 1 mg / kg of the first test drug (finasteride) dissolved in 100 μL D-PBS, the second experimental group received a subcutaneous injection of 0.2 mg / kg of the second test drug (BxC-e) dissolved in 100 μL D-PBS, and the third experimental group received a subcutaneous injection of 0.2 mg / kg of the third test drug (BxC-HAe) dissolved in 100 μL D-PBS.

[0234] In addition, in the testosterone-untreated group, from the time hair growth began, weight was measured twice a week and photographs were taken once a week.

[0235] Specifically, the hair growth area and hair removal area were measured in photographs of mice from each group using ImageJ software, and the ratio of hair growth area / hair removal area was calculated and statistically processed.

[0236] In the testosterone-untreated group, hair growth progressed until it ended (day 27). The body weight is shown in FIG. 16, and the hair growth area is shown in FIGS. 17 and 18 and Table 17.

[0237] [Table 17]

[0238] Quantitation showed that on the 27th day, the hair growth area of ​​testosterone-treated mice was reduced to 23.0% compared to the non-testosterone-treated group, whereas the hair growth area of ​​finasteride-treated mice was 68.1% compared to the non-testosterone-treated group, and the hair growth area of ​​BxC-e-treated mice was 41.8% compared to the non-testosterone-treated group.

[0239] Meanwhile, treatment with BxC-HAe, the third test drug, showed a 54.7% increase in hair growth area compared to the testosterone-untreated group, and the hair growth area increased significantly to a statistically significant level compared to the control group. This indicates that BxC-HAe has a significantly superior hair growth recovery effect despite testosterone treatment, and can treat or prevent testosterone-induced male pattern baldness.

[0240] Example 13: Confirmation of suppression of hair follicle loss in testosterone-treated animals

[0241] To confirm whether the composition of the present invention can inhibit hair follicle loss, the BxC-HAe isolated in Example 2 was administered to a testosterone-treated animal model, and the proportion of anagen hair follicles was measured and compared.

[0242] Specifically, dorsal skin tissues were collected from each mouse in the experiment up to day 27 in Example 8, and paraffin blocks were prepared. The tissues were then sectioned to 5 μm and stained with H&E.

[0243] Then, hair follicles randomly selected from the dorsal skin tissue of five mice per group were classified into anagen hair follicles (Anagen), catagen hair follicles (Catagen), and telogen hair follicles (Telogen) based on their morphology, and the ratio of anagen hair follicles (Anagen) was calculated using the formula anagen hair follicles / (catagen hair follicles + telogen hair follicles). The results are shown in Figure 20, Figure 21, and Table 18.

[0244] [Table 18]

[0245] Quantitative analysis revealed that the proportion of anagen hair follicles was 1.55% in the testosterone-untreated group, but decreased to 0.53% with testosterone treatment. The proportion of anagen hair follicles increased compared to the testosterone-treated group, increasing to 1.29% with finasteride (the first drug), 0.65% with BxC-e (the second drug), and 1.20% with BxC-HAe (the third drug).

[0246] In particular, BxC-HAe treatment showed a significantly greater inhibitory effect on hair follicle loss than the control group despite testosterone treatment, demonstrating that BxC-HAe can treat or prevent testosterone-induced male pattern baldness.

[0247] Example 14: Hair loss in testosterone-treated animals and identification of cells labeled with hair growth factors

[0248] To confirm whether the composition of the present invention regulates the expression of hair loss and hair growth-related factors, the BxC-HAe isolated in Example 2 was treated in a testosterone-treated animal model, and the fluorescent signal intensities of cells labeled with androgen receptor (AR) and β-catenin were measured using antibodies and compared.

[0249] Specifically, dorsal skin tissues were collected from each mouse in the experiment up to day 27 in Example 8, and paraffin blocks were prepared. The tissues were then sectioned to 5 μm and immunostained.

[0250] The primary antibodies used were rabbit anti-β-catenin (Cell Signaling #9562) and mouse anti-AR (Santa Cruz, SC-7305). The secondary antibodies conjugated to fluorescent substances were goat anti-rabbit IgG conjugated Alexa Fluor 488 (Abcam, AB150077) and goat anti-mouse IgG conjugated cyanine 5 (Invitrogen, A10524).

[0251] After the antibody reaction was completed, DAPI staining was performed, and the fluorescent signal patterns were analyzed using a fluorescence microscope (Nikon ECLIPSE Ti2) and the NIS-Elements program (Nikon). The results are shown in Figures 22, 23, 24, Tables 19 and 20.

[0252] [Table 19]

[0253] [Table 20]

[0254] As a result of the analysis, the AR fluorescent signal intensity increased to 2.32 when mice were treated with testosterone compared to the testosterone-free group, while the fluorescent signal intensity increased to 1.56 when mice were treated with the first test drug, finasteride, compared to the testosterone-free group, and the fluorescent signal intensity increased to 1.85 when mice were treated with the second test drug, BxC-e, compared to the testosterone-free group.

[0255] The fluorescence signal intensity of β-catenin decreased to 0.57 when mice were treated with testosterone compared to the testosterone-free group, whereas the fluorescence signal intensity increased to 1.05 when mice were treated with the first test drug, finasteride, compared to the testosterone-free group, and increased to 0.60 when mice were treated with the second test drug, BxC-e, compared to the testosterone-free group.

[0256] Meanwhile, when treated with the third test drug, BxC-HAe, the fluorescent signal intensities were 1.01 for AR and 1.09 for β-catenin compared to the testosterone-untreated group. The fluorescent signal intensity of AR was reduced to a statistically significant level, while the fluorescent signal intensity of β-catenin was restored, compared to the control group. These results confirmed that BxC-HAe can treat or prevent testosterone-induced male pattern baldness.

[0257] Example 15: Confirmation of the effect of culture supernatant (CM) of mesenchymal stem cells derived from hyaluronic acid pretreated induced pluripotent stem cells

[0258] 15-1.Establishment of culture supernatant (CM) for BxC-HA stem cells

[0259] The induced pluripotent stem cell-derived mesenchymal stem cells (BxC-HA stem cells) prepared in Example 1 were cultured for 24 hours in a high-glucose DMEM culture medium containing 10% fetal bovine serum, 1% MEM non-essential amino acid solution, and 40 μg / ml hyaluronic acid.

[0260] After completing the culture, the BxC-HA stem cells were washed with D-PBS and cultured for an additional 72 hours in culture medium supplemented with 15% exosome-depleted FBS.

[0261] After 72 hours of incubation, the culture medium containing the pretreatment agent was collected and centrifuged at 300 × g for 10 minutes to remove any remaining cells and cell debris. The culture supernatant was then collected and used in the following experiments.

[0262] 15-2. Confirmation of reduced wound recovery rate of human dermal papilla cells

[0263] To confirm whether the composition of the present invention can restore the reduced wound healing rate of human dermal papilla cells, human dermal papilla cells were treated with the culture supernatant (CM) of BxC-HA stem cells obtained in Example 15-1 above, and the wound healing rate was compared after 24 hours.

[0264] Specifically, 25,000 human dermal papilla cells were seeded per well in a 12-well plate and cultured in basal medium at 37°C and 5% CO for 24 hours. The composition of the basal medium was the same as in Table 1 above. A cross-shaped wound was made in the cell monolayer formed by the culture using a 1 ml pipette tip, and the cells were washed three times with 1X D-PBS to remove any cellular debris.

[0265] To set up the control and experimental groups, the testosterone-free group was treated with 0.05% (v / v) DMSO in basal medium, the control group was treated with 50 μM testosterone in basal medium, and the experimental group was treated with a mixture of 50 μM testosterone and 50% (v / v) BxC-HA stem cell culture supernatant (CM) in basal medium. After treatment with each substance, each medium was cultured for 24 hours.

[0266] To examine the recovery of the cell layer in each medium, the changes in the cell layer at the time of drug treatment and after 24 hours were photographed using a camera (Dixi eXcope system). The photographs taken in each medium are shown in Figure 25.

[0267] The photographs were quantitatively analyzed using a program (ImageJ), and the experiment was repeated three times for each control group and experimental group to calculate the average analytical values, which are shown in Figure 26 and Table 21. The average analytical values ​​for each group were expressed as relative values, with the average analytical value for the testosterone-untreated group set at 100%.

[0268] [Table 21]

[0269] The analysis showed that when testosterone was added to the culture medium in which the wound was formed, the wound healing rate decreased by approximately 45% compared to the testosterone-untreated group, and when the test drug, BxC-HA stem cell culture supernatant (CM), was added, the wound healing rate decreased by approximately 24% compared to the testosterone-untreated group.

[0270] Therefore, it was analyzed that the wound healing rate increased significantly to a statistically significant level when treated with BxC-HA stem cell CM compared to the control group, confirming that BxC-HA stem cell CM can significantly promote wound healing of human dermal papilla cells despite the presence of testosterone.

[0271] 15-3. Confirmation of growth factor expression promotion in human dermal papilla cells

[0272] To confirm whether the composition of the present invention promotes the expression of IGF1 (Insulin-like Growth Factor 1) and EGF (Epidermal Growth Factor), which are known to be growth factors important for hair growth, from human dermal papilla cells, human dermal papilla cells were treated with the culture supernatant (CM) of BxC-HA stem cells obtained in Example 15-1 above, and the levels of IGF1 and EGF gene expression were compared 24 hours later.

[0273] Specifically, 25,000 human dermal papilla cells were seeded per well of a 12-well plate and cultured in a basal medium at 37°C and 5% CO for 24 hours. The composition of the basal medium was the same as that shown in Table 1 above.

[0274] To set up the control and experimental groups, the testosterone-free group was treated with 0.05% (v / v) DMSO in basal medium, the control group was treated with 50 μM testosterone in basal medium, and the experimental group was treated with a mixture of 50 μM testosterone and 50% (v / v) BxC-HA stem cell culture supernatant (CM) in basal medium. After each treatment, the medium was cultured for 24 hours.

[0275] After the culture was completed, cells were collected from each medium, and total RNA was extracted from each medium using TRIzol® (Invitrogen). The mRNA expression levels of IGF1 and EGF genes were measured for the same amount of total RNA extracted from each medium using real-time PCR (Applied Biosystems). Each measurement was calculated based on the GAPDH expression level. -ΔΔCt After normalization using a technique, the quantitative value in the 50 μM testosterone-untreated group was set as 100%, and relative quantification was performed. The results of quantifying the expression levels of the IGF1 gene and EGF gene are shown in Figure 27 and Table 22, and Figure 28 and Table 23, respectively.

[0276] [Table 22]

[0277] [Table 23]

[0278] Quantitative analysis revealed that the expression level of the IGF1 gene was 112.9% when human dermal papilla cells were treated with testosterone compared to the untreated group, and 98.2% when treated with the test drug, BxC-HA stem cell culture supernatant (CM), compared to the untreated group, with no statistically significant changes.

[0279] Meanwhile, the expression level of the EGF gene decreased by 70.6% when human dermal papilla cells were treated with testosterone compared to the untreated group, and increased by 118.6% when treated with the test drug, BxC-HA stem cell culture supernatant (CM), compared to the untreated group. This is a statistically significant increase in the expression level of the EGF gene compared to the control group. This confirms that BxC-HA stem cell culture supernatant (CM) can promote hair growth by promoting EGF gene expression in human dermal papilla cells, and can be effective in treating or preventing hair loss.

[0280] 15-4. Confirmation of suppression of hair follicle growth inhibitor expression in human dermal papilla cells

[0281] To confirm whether the composition of the present invention can suppress the expression of TGF-β1 (Transforming Growth Factor-beta 1) and IL-6 (Interleukin-6) in human dermal papilla cells, which shorten the anagen phase of hair follicles and induce them into the catagen and telogen phases, human dermal papilla cells were treated with the culture supernatant (CM) of BxC-HA stem cells obtained in Example 15-1 above, and the levels of TGF-β1 and IL-6 gene expression were compared 24 hours later.

[0282] Specifically, 25,000 human dermal papilla cells were seeded per well of a 12-well plate and cultured in a basal medium at 37°C and 5% CO for 24 hours. The composition of the basal medium was the same as that shown in Table 1 above.

[0283] To set up the control and experimental groups, the testosterone-free group was treated with 0.05% (v / v) DMSO in basal medium, the control group was treated with 50 μM testosterone in basal medium, and the experimental group was treated with a mixture of 50 μM testosterone and 50% (v / v) BxC-HA stem cell culture supernatant (CM) in basal medium. After each treatment, the medium was cultured for 24 hours.

[0284] After the culture was completed, cells were collected from each medium, and total RNA was extracted from each medium using TRIzol® (Invitrogen). The mRNA expression levels of TGF-β1 and IL-6 genes were measured by real-time PCR (Applied Biosystems) analysis for the same amount of total RNA extracted from each medium. Each measurement was calculated based on the GAPDH expression level. -ΔΔCt After normalization by technique, relative quantification was performed with the quantitative value in the testosterone-untreated group set at 100%, and the results of quantifying the expression levels of the TGF-β1 gene and IL-6 gene are shown in Figure 29 and Table 24, and Figure 30 and Table 25, respectively.

[0285] [Table 24]

[0286] [Table 25]

[0287] Quantitative analysis showed that in the case of the TGF-β1 gene, expression levels increased by 306.2% when human dermal papilla cells were treated with testosterone compared to the testosterone-untreated group, but when treated with the test drug, BxC-HA stem cell culture supernatant (CM), expression levels were significantly reduced to 83.5% compared to the testosterone-untreated group.

[0288] In the case of the IL-6 gene, when human dermal papilla cells were treated with testosterone, the expression level increased by 205.2% compared to the testosterone-untreated group, but when treated with the test drug, BxC-HA stem cell culture supernatant (CM), the expression level significantly decreased to 76.8% compared to the testosterone-untreated group.

[0289] Therefore, it was confirmed that the culture supernatant (CM) of BxC-HA stem cells suppressed the expression of both TGF-β1 and IL-6 genes, which inhibit hair follicle growth, to a statistically significant level compared to the testosterone-treated group, thereby enabling sustainable hair follicle growth. [Industrial Applicability]

[0290] The present invention relates to a composition for treating or preventing hair loss, which contains stem cell-derived exosomes, and a method for producing the same. More specifically, the present invention relates to a composition containing exosomes that are isolated from mesenchymal stem cells or cultures thereof and have excellent effects of promoting the growth and recovery of human dermal papilla cells, and are capable of treating or preventing hair loss.

Claims

1. A pharmaceutical composition for treating, preventing, alleviating, or suppressing hair loss, comprising exosomes isolated from mesenchymal stem cells (MSCs) derived from induced pluripotent stem cells (iPSCs) as an active ingredient.

2. 2. The pharmaceutical composition for treating, preventing, alleviating, or suppressing hair loss according to claim 1, wherein the induced pluripotent stem cell-derived mesenchymal stem cells are differentiated from progenitor cells of the induced pluripotent stem cell-derived mesenchymal stem cells that do not express SSEA-4 (stage-specific embryonic antigen 4) protein.

3. The pharmaceutical composition for treating, preventing, alleviating, or suppressing hair loss according to claim 1 , wherein the induced pluripotent stem cells are human-derived induced pluripotent stem cells.

4. The pharmaceutical composition for treating, preventing, alleviating, or suppressing hair loss according to claim 1 , wherein the induced pluripotent stem cell-derived mesenchymal stem cells are pretreated with a pretreatment substance.

5. The pharmaceutical composition for treating, preventing, alleviating or suppressing hair loss according to claim 4, wherein the pre-treatment substance is hyaluronic acid.

6. A cosmetic composition for preventing hair loss, strengthening hair roots, restoring hair follicles, or promoting hair growth, comprising exosomes isolated from mesenchymal stem cells (MSCs) derived from induced pluripotent stem cells (iPSCs) as an active ingredient.

7. The cosmetic composition for preventing hair loss, strengthening hair roots, restoring hair follicles or promoting hair growth according to claim 6, wherein the induced pluripotent stem cell-derived mesenchymal stem cells are differentiated from progenitor cells of induced pluripotent stem cell-derived mesenchymal stem cells that do not express SSEA-4 (stage-specific embryonic antigen 4) protein.

8. The cosmetic composition for preventing hair loss, strengthening hair roots, restoring hair follicles or promoting hair growth according to claim 6 , wherein the induced pluripotent stem cells are human-derived induced pluripotent stem cells.

9. The cosmetic composition for preventing hair loss, strengthening hair roots, restoring hair follicles or promoting hair growth according to claim 6 , wherein the induced pluripotent stem cell-derived mesenchymal stem cells have been pretreated with a pretreatment substance.

10. The cosmetic composition for preventing hair loss, strengthening hair roots, restoring hair follicles or promoting hair growth according to claim 9 , wherein the pretreatment substance is hyaluronic acid.

11. The following steps: a first culture step of culturing the induced pluripotent stem cells in a medium; a selective culture step of isolating and culturing SSEA-4(-) cells, which do not express SSEA-4 protein, from the cultured induced pluripotent stem cells and differentiating them into BxC stem cells; a second culturing step in which the BxC stem cells are cultured to differentiate into mesenchymal stem cells; a pretreatment step of pretreating mesenchymal stem cells with hyaluronic acid; A production step of culturing the pretreated mesenchymal stem cells to produce exosomes; and an obtaining step of obtaining a culture supernatant from mesenchymal stem cells or a culture thereof; A method for producing a pharmaceutical composition for treating hair loss, comprising:

12. The method for producing a pharmaceutical composition for treating hair loss according to claim 11, further comprising a separation step of separating exosomes from the culture supernatant.

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