Damaged skin organoid model and drug screening method using same

Abstract

The present invention relates to a damaged skin organoid model and a drug screening method using same, and is a technology that uses a technique for producing an air-liquid interface skin organoid, in which skin cells, appendages, nerve cells, adipocytes, and the like are formed as a structure similar to actual skin, and which produces damaged skin by irradiating the skin organoid with ultraviolet rays that simulate ultraviolet radiation reaching the earth's surface. In the present invention, by irradiation with simulated solar ultraviolet rays including 94.5% of UV-A and 5.5% of UV-B, the skin barrier was damaged and collagen degradation was promoted by increasing the activity of matrix metalloproteinases, and, at the same time, the characteristics of UV-damaged skin, such as increased secretion of pro-inflammatory cytokines, were successfully mimicked. Therefore, it is determined that the present invention will be effectively used as a UV-induced damage and photoaging model, and will serve as a useful technology for screening for potential therapeutic drugs.

Description

Skin injury organoid model and drug screening method using the same

[0001] This application claims the benefit of Republic of Korea Patent Application No. 10-2023-0134662, filed October 10, 2023, the entire disclosure of which is incorporated herein by reference.

[0002]

[0003] The present invention relates to a skin damage organoid model and a drug screening method using the same, and relates to a method for producing a photoaging-like skin model caused by damage from ultraviolet rays by irradiating differentiated human skin organoids with ultraviolet rays that mimic ultraviolet rays reaching the actual earth's surface, and a method for evaluating the efficacy of a potential therapeutic drug.

[0004]

[0005] This invention was completed with the support of the Ministry of Science and ICT of the Republic of Korea under project number 00215812 (1711195569).

[0006]

[0007] With recent improvements in living standards, modern people are increasingly concerned with maintaining healthy skin in addition to maintaining physical health. Consequently, there is growing interest in not only skin beauty but also the prevention of skin damage and aging. Skin is the largest organ in the human body, accounting for approximately 15% of body weight. It covers the outer surface and is a vital organ in direct contact with the outside world. Skin cells are damaged by numerous external factors that attempt to penetrate the body. Among these, exposure to ultraviolet rays is a well-known extrinsic aging factor, and exposure alone can cause significant damage to the skin. Due to the recent depletion of the ozone layer, which protects the upper atmosphere, the amount of ultraviolet rays reaching the Earth's surface is increasing. This increase has the potential to cause skin damage.

[0008] Currently, active research is being conducted on methods to improve UV-induced damage and aging-related phenomena, and in particular, efforts are ongoing to discover the main mechanisms that cause skin damage and aging and to identify substances that alleviate them.

[0009]

[0010] [Prior Art Literature]

[0011] [Patent Document]

[0012] Korean Patent Publication No. 10-2023-0115654 (published on August 3, 2023)

[0013]

[0014] An object of the present invention is to provide a method for producing skin damage or hair follicle damage organoids by irradiating them with solar-mimicking ultraviolet rays.

[0015] In addition, another object of the present invention is to provide a skin damage organoid or a hair follicle damage organoid manufactured by the above manufacturing method.

[0016] In addition, another object of the present invention is to provide a method for screening a skin damage treatment, preventive agent, or improvement agent using the skin damage organoid.

[0017] In addition, another object of the present invention is to provide a method for screening a hair growth promoter, hair loss preventer, hair loss alleviator, or hair loss treatment agent using the above hair follicle damage organoid.

[0018] In addition, another object of the present invention is to provide a biomarker composition for detecting hair follicle damage, which comprises NF-kB as an active ingredient.

[0019] In addition, another object of the present invention is to provide a kit for detecting hair follicle damage comprising a preparation capable of detecting NF-kB.

[0020] In addition, another object of the present invention is to provide a method for providing information necessary for detecting hair follicle damage by measuring the expression level of NF-kB.

[0021] In addition, another object of the present invention is to provide a pharmaceutical composition for hair growth promotion, hair loss prevention, hair loss relief, or hair loss treatment, which contains an NF-kB activation or expression inhibitor as an active ingredient.

[0022] In addition, another object of the present invention is to provide a pharmaceutical composition or cosmetic composition for hair removal containing an NF-kB activation or expression promoter as an active ingredient.

[0023] In addition, another object of the present invention is to provide a method for promoting hair growth, preventing hair loss, alleviating hair loss, or treating hair loss, which comprises a step of administering an effective amount of an NF-kB activity or expression inhibitor to a subject in need thereof.

[0024] In addition, another object of the present invention is to provide a hair removal method comprising a step of administering an effective amount of an NF-kB activation or expression promoter to a subject in need thereof.

[0025]

[0026] A method for producing an organoid, comprising: (1) culturing an organoid derived from pluripotent stem cells in the presence of a Wnt agonist; (2) culturing the culture of step (1) in a medium for maturation of skin organoids; (3) cutting the culture of step (2) and culturing it at an air-liquid interface to produce a skin organoid; and (4) irradiating the skin organoid with solar-mimetic ultraviolet light, wherein the Wnt agonist is added at the start of inducing differentiation of non-neural ectoderm into cranial neural crest cells (CNCC).

[0027] In addition, the present invention provides a skin damage organoid or a hair follicle damage organoid manufactured by the above manufacturing method.

[0028] In addition, the present invention provides a method for screening for a skin damage treatment, preventive, or improving agent, comprising a step of treating the skin damage organoid with a candidate substance for a skin damage treatment, preventive, or improving agent.

[0029] In addition, the present invention provides a method for screening a hair growth promoter, hair loss preventer, hair loss reliever, or hair loss treatment agent, comprising a step of treating the hair follicle damaged organoid with a hair growth promoter, hair loss preventer, hair loss reliever, or hair loss treatment agent candidate.

[0030] In addition, the present invention provides a biomarker composition for detecting hair follicle damage, which comprises NF-kB as an active ingredient.

[0031] In addition, the present invention provides a kit for detecting hair follicle damage, comprising a preparation capable of detecting NF-kB.

[0032] In addition, the present invention provides a method for providing information necessary for detecting hair follicle damage, comprising the steps of (1) measuring the expression level of NF-kB from a separated sample; (2) comparing the expression level of NF-kB with that of a control sample; and (3) determining that the hair follicle is damaged if the expression level of NF-kB is higher than that of the control sample.

[0033] In addition, the present invention provides a pharmaceutical composition for a hair growth promoter, hair loss preventer, hair loss alleviator, or hair loss treatment agent, which contains an NF-kB activation or expression inhibitor as an active ingredient.

[0034] In addition, the present invention provides a pharmaceutical composition for hair removal comprising an NF-kB activation or expression promoter as an active ingredient.

[0035] In addition, the present invention provides a cosmetic composition for hair removal comprising an NF-kB activation or expression promoter as an active ingredient.

[0036] In addition, the present invention provides a method for promoting hair growth, preventing hair loss, alleviating hair loss, or treating hair loss, which comprises administering an effective amount of an NF-kB activation or expression inhibitor to a subject in need thereof.

[0037] In addition, the present invention provides a hair removal method comprising a step of administering an effective amount of an NF-kB activation or expression promoter to a subject in need thereof.

[0038]

[0039] The present invention provides a method for producing skin damaged organoids, comprising the following steps: (1) culturing pluripotent stem cell-derived organoids in the presence of a Wnt agonist; (2) culturing the culture of step (1) in a medium for maturation of skin organoids; (3) cutting the culture of step (2) and culturing it at an air-liquid interface to produce skin organoids; and (4) irradiating the skin organoids with solar-mimetic ultraviolet light, wherein the Wnt agonist is added at the start of inducing differentiation of non-neural ectoderm into cranial neural crest cells (CNCC).

[0040] Preferably, the skin damage organoid may be a hair follicle damage organoid in which a hair follicle among the skin's accessory organs is damaged, but is not limited thereto.

[0041] Preferably, the solar mimicking UV light can treat 50 - 94.5% UV-A and 5.5 - 50% UV-B, but is not limited thereto.

[0042] Preferably, the solar-mimetic UV is 25 to 75 kJ / m after culturing the skin organoids under dry conditions to mature the stratum corneum. 2 It can be treated with sUV dose, but is not limited to this.

[0043] Preferably, the Wnt agonist may be selected from the group consisting of CHIR-99021, Wnt3A, WNT5A and RSPO1, but is not limited thereto.

[0044] Preferably, the Wnt agonist may be added on the 5th to 7th day of culture of pluripotent stem cells, but is not limited thereto.

[0045] Preferably, the medium for maturation of the skin organoid may include, but is not limited to, one or more components selected from the group consisting of glutamax, 2-mercaptoethanol, B-27, N2, and normoxin.

[0046] Preferably, the step (3) may be performed by cutting the culture of step (2) into four equal-sized pieces and culturing them at an air-liquid interface on a collagen-coated transwell culture insert with the dermal layer facing the collagen side and the epidermal layer exposed to the air. More preferably, the air-liquid interface culture may be performed in a medium for maturation of skin organoids, but is not limited thereto.

[0047] Preferably, the skin damaged organoid or hair follicle damaged organoid may be, but is not limited to, one in which skin or hair follicle is damaged through NF-kB activation.

[0048] Preferably, the skin damage organoid may be one in which an epidermal barrier forming protein selected from the group consisting of, but not limited to, filaggrin, loricrin and CK10 is down-regulated.

[0049] Preferably, the skin damage organoid may have decreased expression of type I collagen and increased expression of MMP-1 in the dermal layer, but is not limited thereto.

[0050] Preferably, the hair follicle damage organoid may have reduced expression of one or more genes selected from the group consisting of a-SMA, a dermal sheath marker, KRT5 or KRT15, ​​a root sheath marker, KRT15 or LHX2, a hair follicle stem cell marker, and SOX2, a dermal papilla marker, but is not limited thereto.

[0051] Preferably, the hair follicle damage organoid may have increased gene expression of inflammatory cytokines such as COX-2, TNF-α or IL-1β, but is not limited thereto.

[0052]

[0053] As used herein, “air-liquid interface culture (ALI culture)” may refer to, but is not limited to, culture in a partially open culture vessel or a culture vessel partially filled with medium. Air-liquid interface culture may refer to exposing the surface of cells or organoids to air.

[0054]

[0055] In this specification, “Filaggrin” is one of several structural proteins expressed by keratinocytes during the differentiation stage, and is involved in differentiation from the basal layer of the epidermis to the stratum corneum. It also forms the main body of natural moisturizing factor (NMF) essential for maintaining moisture in skin tissue, and is used as a major indicator of skin moisture maintenance and skin barrier function.

[0056]

[0057] In this specification, “Loricrin” is one of the skin barrier proteins expressed by keratinocytes at the differentiation stage.

[0058]

[0059] In addition, the present invention provides a skin damage organoid manufactured by the above manufacturing method.

[0060]

[0061] In addition, the present invention provides a method for screening for a skin damage treatment, preventive, or improving agent, comprising a step of treating the skin damage organoid with a candidate substance for a skin damage treatment, preventive, or improving agent.

[0062]

[0063] In addition, the present invention provides a hair follicle damage organoid manufactured by the above manufacturing method.

[0064] Preferably, the hair follicle damage organoid may have reduced expression of one or more genes selected from the group consisting of a-SMA, a dermal sheath marker, KRT5 or KRT15, ​​a root sheath marker, KRT15 or LHX2, a hair follicle stem cell marker, and SOX2, a dermal papilla marker, but is not limited thereto.

[0065]

[0066] In addition, the present invention provides a method for screening a hair growth promoter, hair loss preventer, hair loss alleviator, or hair loss treatment agent, comprising a step of treating the hair follicle damaged organoid with a hair growth promoter, hair loss preventer, hair loss alleviator, or hair loss treatment agent candidate.

[0067] Preferably, the candidate substance can inhibit activated NF-kB in hair follicle damaged organoids, but is not limited thereto.

[0068]

[0069] The term "candidate substance" used in referring to the screening method of the present invention refers to an unknown candidate substance used in screening to determine whether it affects the expression level of a gene or the expression or activity of a protein. The sample includes, but is not limited to, chemicals, nucleotides, antisense RNA, siRNA (small interference RNA), and natural product extracts.

[0070]

[0071] In the present invention, the term "organoid" refers to a three-dimensional aggregate of one or more cell types that mimics the superficial appearance or actual structure or function of a tissue or organ. Furthermore, organoids can closely replicate the physiological functions of the human body, and by constructing organoids from patient tissue, they can be used for disease modeling based on the patient's genetic information, drug screening through repeated testing, and more.

[0072]

[0073] In the present invention, "organoid culture" encompasses all actions capable of producing or maintaining organoids. For example, this may involve differentiating cells isolated from a specific tissue or tissue into tissue or organ cells with a specific function, and / or allowing the organoid to survive, grow, or proliferate.

[0074]

[0075] In addition, the present invention provides a biomarker composition for detecting hair follicle damage, which comprises NF-kB as an active ingredient.

[0076]

[0077] In addition, the present invention provides a kit for detecting hair follicle damage, comprising a preparation capable of detecting NF-kB.

[0078] Preferably, the formulation may be selected from the group consisting of, but not limited to, primers, probes, antisense oligonucleotides, aptamers and antibodies specific for NF-kB.

[0079]

[0080] As used herein, the term "primer" refers to a short nucleic acid sequence having a short free 3' hydroxyl group, which can form base pairs with a complementary template and serves as a starting point for copying the template strand. The primer can initiate DNA synthesis in the presence of a polymerization reagent (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. PCR conditions and the lengths of the sense and antisense primers can be appropriately selected according to techniques known in the art.

[0081]

[0082] As used herein, the term "probe" refers to a nucleic acid fragment, such as RNA or DNA, ranging from a few bases to several hundred bases in length, capable of specifically binding to mRNA, and is labeled so that the presence or absence of a specific mRNA and its expression level can be confirmed. The probe can be produced in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, etc. The selection of an appropriate probe and hybridization conditions can be appropriately selected according to techniques known in the art.

[0083]

[0084] The term "antisense oligonucleotide" as used herein refers to an oligomer having a sequence of nucleotide bases and an intersubunit backbone that allows the antisense oligomer to hybridize with a target sequence within RNA by Watson-Crick base pairing, typically forming an mRNA and RNA:oligomer heteroduplex within the target sequence. The oligomer may have exact sequence complementarity or near complementarity to the target sequence. The antisense oligomer may block or inhibit translation of the mRNA and alter the processing of the mRNA to produce splice variants of the mRNA.

[0085]

[0086] As used herein, the term "aptamer" refers to a type of polynucleotide composed of a special type of single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure in itself and the characteristic of being able to bind to a target molecule with high affinity and specificity. As described above, aptamers can specifically bind to an antigenic substance in the same way as antibodies, but are composed of polynucleotides that are more stable than proteins, have a simpler structure, and are easy to synthesize, and therefore can be used as a substitute for antibodies.

[0087]

[0088] As used herein, the term "antibody" is a term known in the art and refers to a specific immunoglobulin directed against an antigenic site. The antibody in the present invention refers to an antibody that specifically binds to the biomarker of the present invention, and the antibody can be prepared according to a conventional method in the art. The form of the antibody includes a polyclonal antibody or a monoclonal antibody, and all immunoglobulin antibodies are included. The antibody refers to a complete form having two full-length light chains and two full-length heavy chains. The antibody also includes specialized antibodies such as humanized antibodies.

[0089]

[0090] In addition, the present invention provides a method for providing information necessary for detecting hair follicle damage, comprising the steps of (1) measuring the expression level of NF-kB from a separated sample; (2) comparing the expression level of NF-kB with that of a control sample; and (3) determining that the hair follicle is damaged if the expression level of NF-kB is higher than that of the control sample.

[0091]

[0092] The term "isolated sample" as used herein includes, but is not limited to, a sample such as tissue, cell, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine that differs from a control group in the expression level of NF-kB, a biomarker of the present invention.

[0093]

[0094] In addition, the present invention provides a pharmaceutical composition for a hair growth promoter, hair loss preventer, hair loss alleviator, or hair loss treatment agent, which comprises an NF-kB activation or expression inhibitor as an active ingredient.

[0095] Preferably, the NF-kB activity inhibitor may be selected from the group consisting of small molecule compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to the NF-kB protein, and the NF-kB expression inhibitor may be selected from the group consisting of antisense nucleotides, small interfering RNA (siRNA), and short hairpin RNA (shRNA) that complementarily bind to the mRNA of the NF-kB gene, but is not limited thereto.

[0096]

[0097] In addition, the present invention provides a pharmaceutical composition for hair removal comprising an NF-kB activation or expression promoter as an active ingredient.

[0098] Preferably, the NF-kB activity promoter may be selected from the group consisting of low-molecular-weight compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to the NF-kB protein, and the NF-kB expression promoter may be selected from the group consisting of adeno-associated viruses (AAV), adenoviruses, lentiviruses, and retroviruses that include an NF-kB coding gene that increases the expression of mRNA of the NF-kB gene.

[0099]

[0100] The pharmaceutical composition of the present invention may include chemicals, nucleotides, antisense, siRNA oligonucleotides, and natural product extracts as active ingredients. The pharmaceutical composition or complex preparation of the present invention may be prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the active ingredients, and the adjuvants may include solubilizing agents such as excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, glidants, or flavoring agents. The pharmaceutical composition of the present invention may be preferably formulated as a pharmaceutical composition by additionally including one or more pharmaceutically acceptable carriers in addition to the active ingredients for administration. In the composition to be formulated as a liquid solution, acceptable pharmaceutical carriers include those that are sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

[0101] The pharmaceutical formulation form of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions, and sustained-release formulations of the active compound, etc. The pharmaceutical composition of the present invention may be administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. The effective amount of the active ingredient of the pharmaceutical composition of the present invention refers to the amount required for the prevention or treatment of a disease. Therefore, it can be adjusted according to various factors including the type of disease, the severity of the disease, the types and contents of the active ingredient and other ingredients contained in the composition, the type of formulation, and the patient's age, body weight, general health condition, sex and diet, administration time, administration route and secretion rate of the composition, treatment period, and concurrently used drugs. Although not limited thereto, for example, in the case of adults, when administered once to several times a day, the composition of the present invention may be administered at a dose of 0.1 ng / kg to 10 g / kg in the case of a compound, 0.1 ng / kg to 10 g / kg in the case of a polypeptide, protein or antibody, and 0.01 ng / kg to 10 g / kg in the case of an antisense nucleotide, siRNA, shRNAi or miRNA, when administered once to several times a day.

[0102]

[0103] In addition, the present invention provides a cosmetic composition for hair removal comprising an NF-kB activation or expression promoter as an active ingredient.

[0104]

[0105] The cosmetic composition of the present invention may include, in addition to the active ingredient, conventional auxiliary agents such as stabilizers, solubilizers, vitamins, pigments, and fragrances, and carriers.

[0106] The formulation of the above cosmetic composition may be manufactured into any formulation commonly manufactured in the art, and may have a formulation selected from the group consisting of external skin ointment, cream, emollient toner, nourishing toner, pack, essence, hair tonic, shampoo, rinse, hair conditioner, hair treatment, gel, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, eye cream, moisture cream, hand cream, foundation, nourishing essence, sunscreen, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, and body cleanser, but is not limited thereto. The composition of each of these formulations may contain various bases and additives necessary and appropriate for the formulation of the formulation, and the types and amounts of these ingredients may be easily selected by those skilled in the art.

[0107] When the above formulation is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.

[0108] When the above formulation is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, when it is a spray, it may additionally include a propellant such as chlorofluorohydrocarbon, propane / butane or dimethyl ether.

[0109] When the above formulation is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

[0110] When the above formulation is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth may be used as a carrier component.

[0111]

[0112] In addition, the present invention provides a method for promoting hair growth, preventing hair loss, alleviating hair loss, or treating hair loss, which comprises administering an effective amount of an NF-kB activation or expression inhibitor to a subject in need thereof.

[0113] Preferably, the NF-kB activity inhibitor may be selected from the group consisting of small molecule compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to the NF-kB protein, and the NF-kB expression inhibitor may be selected from the group consisting of antisense nucleotides, small interfering RNA (siRNA), and short hairpin RNA (shRNA) that complementarily bind to the mRNA of the NF-kB gene, but is not limited thereto.

[0114]

[0115] In addition, the present invention provides a hair removal method comprising a step of administering an effective amount of an NF-kB activation or expression promoter to a subject in need thereof.

[0116] Preferably, the NF-kB activity promoter may be selected from the group consisting of low-molecular-weight compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to the NF-kB protein, and the NF-kB expression promoter may be selected from the group consisting of adeno-associated viruses (AAV), adenoviruses, lentiviruses, and retroviruses that include an NF-kB coding gene that increases the expression of mRNA of the NF-kB gene.

[0117]

[0118] The present invention relates to a skin damage organoid model and a drug screening method using the same. Human-induced pluripotent stem cell-derived skin organoids were used to model skin damaged by an external environment (ultraviolet radiation). By utilizing organoid technology that mimics actual human development, skin organoids containing intact skin cells, hair follicle cells, and all microstructures were created. By irradiating the organoids with ultraviolet radiation, a technique was established to model damaged skin in vitro by mimicking external stimuli in a real-world environment. Therefore, this technology enables the identification of the mechanisms that cause ultraviolet radiation-induced damage and aging in vitro, and a platform for identifying candidate substances capable of alleviating these effects was developed. The platform established through the present invention can be applied in the future to evaluate and derive the mechanisms of disease development and the efficacy of various drug candidates.

[0119]

[0120] Figure 1 shows a skin organoid differentiation method using a human induced pluripotent stem cell line.

[0121] Figure 2 shows the results of comparing the structure of the skin appearing in the fabricated skin organoid with the structure of the skin of a human adult.

[0122] Figure 3a shows the results of investigating the effects of various sUV doses on SkOs.

[0123] Figure 3b shows the results of examining the effects of SkOs on the epidermal layer, dermal layer, or hair follicle after exposure to sUV.

[0124] Figure 3c shows the results confirming that the influence of sUV extends to the deep dermal layer of SkOs.

[0125] Figure 4a shows the results of investigating the effects of sUV exposure on skin appendages in the dermis of SkOs.

[0126] Figure 4b shows the results of an investigation into whether sUV exposure can also affect hair follicles and induce an inflammatory response in the dermis of SkOs.

[0127] Figure 5a shows the results of investigating the effect of UCB-Exos on photodamage using a SkOs model exposed to sUV.

[0128] Figure 5b shows the results confirming that UCB-Exos treatment effectively reduced epidermal thickness and alleviated the destruction of skin barrier proteins (Filaggrin and Loricrin), thereby improving epidermal damage caused by sUV exposure in SkOs.

[0129] Figure 6 shows the results of investigating the effect of UCB-Exos on hair follicles in SkOs.

[0130] Figure 7a shows the results confirming that sUV induces phosphorylation of IκBα in the cytosolic fraction of SkOs and subsequently causes degradation of IκBα.

[0131] Figure 7b shows the results confirming that sUV induces phosphorylation of IκBα in the cytosolic fraction of SkOs and subsequently causes degradation of IκBα.

[0132] Figure 7c shows the results confirming that UCB-Exos alleviated the inflammatory response in fibroblasts and keratinocytes in the hair follicle by inhibiting IκB degradation and NF-κB activation induced by sUV exposure.

[0133] Figure 8a shows the results confirming that inhibition of NF-κB activation by UCB-Exos has the potential to alleviate the detrimental effects of SASP, thereby creating a more favorable environment for tissue regeneration.

[0134] Figure 8b shows the results confirming that inhibition of NF-κB activation by UCB-Exos has the potential to alleviate the detrimental effects of SASP, thereby creating a more favorable environment for tissue regeneration.

[0135] Figure 8c shows the results confirming that UCB-Exos effectively restored hair follicles damaged by sUV through NF-κB inhibition.

[0136] Figure 9 shows the results of confirming the hair follicle growth promotion effect by an NF-κB signal inhibitor in normal hair follicle organoids.

[0137]

[0138] Hereinafter, to aid understanding of the present invention, examples and other embodiments will be described in detail. However, the following examples and other embodiments merely illustrate the content of the present invention and are not intended to limit the scope of the present invention. The examples and other embodiments of the present invention are provided to more fully explain the present invention to those of average skill in the art.

[0139]

[0140] <Example 1> Production and verification of human induced pluripotent stem cell-derived skin organoids

[0141] Culture of human induced pluripotent stem cells: Human induced pluripotent stem cell lines (iPSCs; CMC3, CMC11) were cultured on Vitronectin (ThermoFisher)-coated culture dishes in Essential 8 (Gibo) medium supplemented with Y-27632 (10 μM). The medium was changed daily, and passages were performed using ReLeSR (Stem Cell Technology) every 4 days.

[0142] Skin organoid differentiation method using human induced pluripotent stem cell lines: Induced pluripotent stem cell colonies were dissociated into single cells using Acuutase (Gibco). To form embryoid bodies (EBs), 1,500 single cells were seeded into each well of a U-bottom low-attachment 96-well plate (SPL). The embryoid body culture medium used was Stemflex (Gibco) containing Y-27632 (20 μM). The embryoid bodies were cultured until they reached a size of 250–300 μm. To induce non-neural ectoderm differentiation, the formed embryoid bodies were transferred to Essential 6 (Gibco) containing 2% Matrigel (Corning), 10 μM SB431542 (Tocris), 4 ng / ml FGF2 (PeproTech), and 2.5–15 ng / ml BMP4 (PeproTech). From day 3 or 4, 200 nM LDN193189 (Torcris), 50 ng / ml FGF2, and 3 μM CHIR-99021 were added to the culture medium to induce cranial neural crest cells. In addition, on day 12, the organoids were replaced with skin organoid maturation medium. The medium was changed every 3 days. On day 20, organoids were transferred to 6-well plates (low-attachment 6-well plates; SPL) containing 3 ml of skin organoid maturation medium and cultured in an orbital shaker (65 rpm; Thermo Fisher). The medium was changed every 3 days. Skin organoid maturation medium consisted of Advanced DMEM / F12 (Gibco) and Neurobasal (Gibco) in a 1:1 ratio, supplemented with 1X GlutaMax (Gibco), 0.5X B-27 minus Vitamin A (Gibco), 0.5X N2 (Gibco), and 0.Contains 1 mM 2-mercaptoethanol (Gibco) and 100 μg / ml Primocin (Invivogen). After culturing the skin organoids for approximately 40 to 100 days, the organoids were cut into 4-12 equal-sized pieces using Dumount #3 forceps (Fine Science Tools) and Spring Scissors (Fine Science Tools) and cultured on collagen (Corning)-coated transwell culture inserts (0.4 μm pore size, 12-well plates (Corning)) with the dermal layer facing the collagen and the epidermal layer exposed to the air. The ALI-skin organoids were cultured in an air-liquid interface (ALI) condition with skin maturation medium in a 100% humidity incubator for an additional 3 to 4 weeks, and then cultured under dry conditions (0% humidity condition) for 2 to 6 days to allow maturation of the stratified epithelium (Fig. 1A and Fig. 1B).

[0143] Characterization of Skin Organoids: The skin structure of the developed skin organoids was compared with that of adult human skin. H&E and immunofluorescence staining were performed to identify the basal layer (KRT5) and squamous epithelium (KRT10) of the epidermis. Furthermore, expression of skin barrier markers Filaggrin (superior layer, squamous epithelium) and Loricrin (keratinized layer) confirmed the similarity with the epidermal layer of actual skin.

[0144] In addition, the similarity to actual human skin was verified by confirming the expression of both Collagen 3, a major structural protein mainly expressed in the dermal layer, and Vimentin, one of the intermediate filaments of the skin (Fig. 2A and Fig. 2B).

[0145] To verify the structural characteristics of the skin organoid hair follicles, H&E and immunofluorescence staining were performed. The microstructure of the hair follicles, including the dermal sheath (α-SMA), outer root sheath (KRT17), inner root sheath (KRT71), and follicular cortex (AE13), were confirmed. In addition, dermal papilla cells (SOX2), hair follicle cells (Ki67, P63), and bulge zone (NFATc1), which are responsible for the growth and function of the hair follicles, were also confirmed (Fig. 2C-F).

[0146]

[0147] <Example 2> Establishment of a skin damage model using solar-mimetic ultraviolet irradiation.

[0148] There are two types of sUV that reach the skin: UV-A and UV-B. UV-A can penetrate from the epidermis to the deepest layer of the dermis, whereas UV-B can only penetrate the epidermis and the upper part of the dermis. Exposure to sUV can cause signs of aging, such as sunburn and hyperpigmentation, by damaging ECM (extracellular matrix) components and key proteins. To mimic this and reproduce sUV-induced skin damage in vitro, SkOs were irradiated with a combination of UV-A and UV-B (94.5% UV-A and 5.5% UV-B) three times, two days apart.

[0149] First, the effects of various sUV doses on SkOs were investigated. As a result, a high dose of 75 kJ / m² sUV peeled off the entire epidermal layer of SkOs, significantly damaging the structural integrity of the skin (Fig. 3A, A). Immunofluorescence staining results showed that the degree of dermal damage induced by 50 kJ / m² sUV was similar to that induced by 75 kJ / m² sUV, whereas 25 kJ / m² sUV did not cause significant damage to SkOs (Fig. 3A, B, C). Therefore, 50 kJ / m² sUV was determined to be the optimal dose for inducing photodamage in SkOs (Fig. 3A, D).

[0150] After sUV exposure, the number of sunburned cells and epidermal thickness, characterized by fragmentation or condensation of cell nuclei, significantly increased in both the epidermis and dermis of SkOs (Fig. 3B, E-G). Furthermore, the number of melanocytes in the epidermis and hair follicles increased compared to the control SkOs, suggesting skin pigmentation (Fig. 3B, H, I). Notably, sUV also affected the outer layer of the epidermis of SkOs, which serves as a protective skin barrier. The mRNA expression of skin barrier-related genes, Filaggrin and Loricrin, was significantly decreased in sUV-exposed SkOs, suggesting impaired skin barrier function after irradiation (Fig. 3B, J). Consistently, epidermal barrier proteins, such as Filaggrin, Loricrin, and CK10, were significantly downregulated in the epidermis of sUV-exposed SkOs compared to the control SkOs (Fig. 3B, K, L).

[0151] Furthermore, the effects of sUV extended to the deep dermal layer of SkOs. The density of collagen fibers was significantly reduced in the dermal layer of SkOs exposed to sUV (Fig. 3c, M, N). qPCR analysis showed a significant decrease in COL1A1 expression and an increase in matrix metalloproteinase-1 (MMP-1), an enzyme responsible for the direct degradation of ECM fibers (Fig. 3c, O). Notably, immunofluorescence staining observed a significant decrease in type I collagen expression and an increase in MMP-1 expression in the dermal layer of SkOs exposed to sUV (Fig. 3c, P, Q). Based on these results, sUV reaching the epidermis and dermis induced complete skin damage in SkOs. Therefore, we successfully established in vitro conditions to reproduce sUV-induced photodamage in SkOs.

[0152]

[0153] <Example 3> Evaluation of hair follicle damage caused by solar-mimetic ultraviolet irradiation

[0154] We investigated the effects of sUV exposure on skin appendages in the dermis of SkOs. As a result, we confirmed that the expression of the KRT5 marker, which constitutes the outer layer of hair follicles, was reduced in SkOs. Moreover, apoptosis was significantly increased in SkOs, particularly in cells colocalized with KRT5-expressing cells in hair follicles (Fig. 4A, A, B, and C). Macroscopically, the DS (dermal sheath) and ORS (outer root sheath) of hair follicles were significantly destroyed, and the hair shaft was relatively thinner in the sUV group, indicating that the hair follicles had transitioned from an anagen phase to a catagen-like morphology (Fig. 4A, D, E). Therefore, to thoroughly compare the degree of hair follicle damage, hair follicles were isolated from each skin organoid and subjected to qRT-PCR analysis. Gene expression of DS marker (a-SMA), ORS marker (KRT5, KRT15), hair follicle stem cell marker (KRT15, ​​LHX2), and DP marker (SOX2) was significantly decreased in the sUV group, indicating that sUV could penetrate the dermal layer and adversely affect hair follicles (Fig. 4F).

[0155] It is known that sUV irradiation induces the secretion of proinflammatory cytokines in skin tissue, leading to skin inflammation and subsequent skin damage. Therefore, we investigated whether sUV exposure could also affect hair follicles and induce an inflammatory response in the dermis of SkOs. First, gene expression of the proinflammatory cytokines COX-2, TNF-α, and IL-1β significantly increased in the hair follicles of SkOs exposed to sUV (Fig. 4G).

[0156] Next, immunofluorescence analysis was performed to confirm the local inflammatory response in SkOs. Results showed that increased production of proinflammatory cytokines was expressed throughout the skin layer, but was particularly evident within the hair follicles of SkOs exposed to sUV (Fig. 4B, H, I). These results suggest that sUV irradiation induces structural damage and an inflammatory response in the hair follicles of SkOs.

[0157] In summary, the present invention demonstrates that sUV exposure can sensitize and destroy not only skin tissue but also hair follicles responsible for hair growth.

[0158]

[0159] <Example 4> Derivation and Evaluation of Candidate Therapeutic Substances Using a Skin Organoid Model Damaged by UV Rays

[0160] We explored the potential use of sUV-damaged SkOs as a model for studying the photodamage mechanisms of potential drugs. Currently, exosomes derived from hUCB-MSCs are considered as potential therapeutic agents for various skin conditions due to their ability to promote skin regeneration and reduce skin inflammation. In this context, we investigated the effect of UCB-Exosomes on photodamage using a sUV-exposed SkOs model.

[0161] To determine the optimal concentration of UCB-Exos to alleviate SkOs damage induced by sUV, SkOs were treated with various concentrations of UCB-Exos 2 h after sUV exposure, and this process was repeated three times (Fig. 5A). As a result, UCB-Exos treatment effectively reduced SkO damage in a dose-dependent manner.

[0162] In particular, treatment with UCB-Exos at a concentration of 1 × 10^8 particles or more effectively alleviated sUV-induced SkOs-induced apoptosis in the dermal layer, including hair follicles (Fig. 5a, B, C). However, a more significant effect on alleviating inflammatory responses was observed at a concentration of 1 × 10^9 particles of UCB-Exos (Fig. 5a, D). Based on these results, we confirmed that a minimum of 1 × 10^9 particles of UCB-Exos was required to fully exert the beneficial effects of apoptosis inhibition, skin barrier function restoration, and modulation of inflammatory cytokine secretion.

[0163] Treatment with UCB-Exos (1 × 10^9) effectively reduced epidermal thickness (Fig. 5B, E, F) and alleviated the destruction of skin barrier proteins (Filaggrin and Loricrin), indicating that sUV exposure-induced epidermal damage was improved in SkOs (Fig. 5B, G, H, I).

[0164] After sUV exposure, the reduced collagen fiber density was restored (Fig. 5B, J, K). Consistently, MMP-1 expression was reduced and COL1A1 expression was increased in UCB-Exos-treated SkOs (Fig. 5B, L). Immunofluorescence analysis also confirmed that the dermis of UCB-Exos-treated SkOs maintained a higher level of collagen fibers and decreased MMP-1 expression, suggesting that collagen degradation was inhibited in sUV-damaged SkOs (Fig. 5B, M, N).

[0165] The effects of UCB-Exos on hair follicles in SkOs were investigated. Apoptosis (Cleaved Caspase-3) was induced in the hair follicles. + ) is reduced and regenerative cells (Ki67 + ) increased in number (Fig. 6A, B). In addition, keratinocytes and fibroblasts in hair follicles aged by sUV are known to secrete matrix metalloproteinases (MMPs) and inflammatory cytokines, known as senescence-associated secretory phenotype (SASP). We confirmed that genes related to SASP were significantly downregulated in the hair follicles of the UCB-Exos group, indicating a younger and healthier state (Fig. 6C). These results suggest that UCB-Exos effectively alleviates sUV-induced hair follicle cell death and senescence, while promoting hair follicle regeneration.

[0166] UV-induced hair follicle damage, such as shrunken hair bulbs and destruction of the outer wall, such as the DS and ORS, was significantly restored in the UCB-Exos-treated group. Furthermore, abnormal hair shaft entry into the catagen phase was restored after UCB-Exos treatment (Figs. 6D and 6E). Consistently, gene expression related to hair follicle structural components was upregulated in the UCB-Exos group (Fig. 6F). Furthermore, hair growth-related genes (β-catenin and LEF1) were upregulated, while hair loss-related genes (DKK1) were downregulated, suggesting that UCB-Exos restored the hair formation ability of SkOs (Fig. 6G).

[0167] Melanocytes were also affected by UCB-Exos treatment. The number of melanocytes was reduced in both the epidermis and hair follicles (Figs. 6H and 6I), and the expression of genes associated with melanocyte development and pigmentation (KIT and MITF) was also reduced (Fig. 6J).

[0168] In summary, the present invention demonstrated the therapeutic potential of UCB-Exos to improve skin damage and promote hair follicle regeneration in sUV-exposed SkOs.

[0169]

[0170] <Example 5> Identification of IκB / NF-κB-mediated damage and healing mechanisms using a UV-damaged skin organoid model.

[0171] Numerous studies have suggested that exposure to sUV radiation activates an inflammatory cascade in the skin, leading to cell death. NF-κB is known to play a crucial role in regulating the expression of immune mediators. In its inactive state, NF-κB binds to an inhibitory protein called IκBα, forming the NF-κB-IκB complex in the cytoplasm. Upon activation by external stimuli such as inflammatory cytokines or cellular stress, IκBα protein is phosphorylated and subsequently degraded, releasing NF-κB from the NF-κB-IκB complex. The released NF-κB translocates to the cell nucleus, where it promotes the transcription of genes involved in inflammation and induces cytokine production. Considering this, we investigated whether IκB-dependent NF-κB activation mediates the inflammatory response in hair follicles.

[0172] In the present invention, we found that sUV induced phosphorylation of IκBα in the cytosolic fraction of SkOs and subsequent degradation of IκBα. This resulted in the disruption of the NF-κB-IκB complex, resulting in the activation and nuclear translocation of NF-κB, as evidenced by increased NF-κB expression in the nuclear region of SkOs exposed to sUV. After treatment of SkOs exposed to sUV with UCB-Exos, we observed a decrease in both IκBα phosphorylation and degradation, and inhibition of NF-κB nuclear translocation (Fig. 7A, B). Immunofluorescence staining also confirmed localized expression of IκBα and active NF-κB, i.e., phosphorylated NF-κB (p-NF-κB), in cells constituting the hair follicle structure. Hair follicles are generally composed of keratinocytes and fibroblasts, both of which play important roles in skin inflammatory responses. In particular, in the sUV-treated group, we observed a significant suppression of IkBα expression throughout the hair follicle, including keratinocytes of the ORS. Accordingly, sUV exposure significantly increased p-NF-κB expression in the ORS and the stroma of the hair follicle (Fig. 7a, C, D). In sUV-induced SkOs, the nuclear translocation of p-NF-κB in these structures was also increased, indicating NF-κB activation after sUV exposure. Notably, the NF-κB-mediated inflammatory response occurred primarily in the hair follicle rather than in dermal fibroblasts (Fig. 7b, E, F). Meanwhile, the epidermis of the SkOs, the uppermost layer exposed to sUV, also developed inflammation, which may have extended into the dermis and indirectly damaged the hair follicle (Fig. 7b, G, H). This expression pattern of IkBα and NF-κB was effectively reversed by UCB-Exos treatment (Fig. 7b, E-H).

[0173] Furthermore, mRNA expression of inflammatory genes TNF-α and IL-6 was significantly reduced in isolated hair follicles after UCB-Exos treatment (Fig. 7c, I). TNF-α and IL-6 protein levels, which were high throughout the hair bulb in the vehicle control group, were significantly reduced in the stromal region after UCB-Exos treatment (Fig. 7c, J, K). This was consistently reflected in the secretion of TNF-α and IL-6 by SkOs confirmed by ELISA (Fig. 7c, L). Overall, these results suggest that UCB-Exos alleviated the inflammatory response in fibroblasts and keratinocytes in the hair follicle by inhibiting IκB degradation and NF-κB activation induced by sUV exposure.

[0174]

[0175] <Example 6> Inhibition of NF-κB signaling mediates the therapeutic efficacy of exosomes against hair follicle photodamage.

[0176] To assess the direct effect of UCB-Exos in mitigating sUV-induced damage through NF-κB activation, we treated sUV-damaged organoids with the NF-κB inhibitor BAY11-7082. BAY11-7082 is known to inhibit the nuclear translocation of NF-κB by inhibiting the degradation of the IκBα subunit, thereby preventing further activation of NF-κB signaling.

[0177] Western blot analysis confirmed that after BAY11-7082 treatment, sUV-damaged SkOs showed a decrease in phosphorylated IκBα, which inhibited IκBα degradation and, consequently, reduced nuclear translocation of NF-κB. Similarly, sUV-exposed SkOs supplemented with UCB-Exos also showed a significant decrease in IκBα phosphorylation, resulting in decreased IκBα degradation and NF-κB activation. Notably, co-treatment with BAY11-7082 and UCB-Exos synergistically reduced NF-κB and phosphorylated IκBα levels. As expected, IL-6 expression was positively correlated with NF-κB expression levels (Fig. 8A, B).

[0178] Furthermore, the secretion of IL-6, TNF-α, and IL-1β, which are inflammatory cytokines increased by sUV exposure, was evaluated using ELISA. There was no significant difference in the secretion of IL-6 and TNF-α between SkOs treated with BAY11-7082 and SkOs treated with UCB-Exos, but the levels of TNF-α and IL-1β were significantly reduced in the co-treated group, indicating a synergistic effect of BAY11-7082 and UCB-Exos (Fig. 8C).

[0179] In the context of inflammatory responses, NF-κB signaling is known as a key regulator of components of the senescence-associated secretory phenotype (SASP), which delays tissue regeneration through accumulation of senescent cells, abnormalities in matrix remodeling, and chronic inflammation.

[0180] We explored whether UCB-Exos treatment, which inhibits NF-κB activation, could modulate SASP within hair follicles. Results indicated that mRNA expression of inflammatory cytokines and ECM components associated with SASP was dramatically reduced after treatment with UCB-Exos or BAY11-7082 (Fig. 8B, D). These results suggest that inhibition of NF-κB activation by UCB-Exos has the potential to alleviate the detrimental effects of SASP and thereby create a more favorable environment for tissue regeneration.

[0181] Immunofluorescence staining confirmed a significant decrease in both nuclear NF-κB and cytoplasmic IL-6 in hair follicles after treatment with BAY11-7082 or UCB-Exos (Fig. 8c, E, F, G). Consequently, cell death was reduced in these hair follicles after treatment with BAY11-7082 or UCB-Exos, with the lowest apoptosis rate observed after co-treatment (Fig. 8c, H, I). Ultimately, NF-κB inhibition restored the structural integrity of hair follicles, including the DS, ORS, and hair bulb, and protected hair follicles from transition to catagen (Fig. 8c, J). Taken together, these results suggest that UCB-Exos in the present invention effectively repaired sUV-damaged hair follicles through NF-κB inhibition.

[0182]

[0183] <Example 7> Confirmation of the hair follicle growth promotion effect by NF-κB signaling inhibitor in normal hair follicle organoids.

[0184] To evaluate the effectiveness of NF-κB signaling inhibition in normal hair follicles, skin organoids with hair follicles were treated with the NF-κB inhibitor BAY11-7082, and hair follicle growth was observed. Minoxidil, known to promote hair follicle growth, and dihydrotestosterone (DHT), an endogenous hormone that induces hair loss, were also treated for comparison.

[0185] Observation of hair follicle morphology on the 14th day after drug treatment revealed that hair follicle length in the BAY11-7082-treated group increased approximately 1.4-fold compared to the control group, and in the Minoxidil-treated group, it increased approximately 1.3-fold. Conversely, hair follicle length in the DHT-treated group decreased approximately 0.7-fold compared to the control group. This suggests that the NF-κB inhibitor BAY11-7082 promotes hair follicle growth similarly to Minoxidil (Figs. 9A and 9B).

[0186] Additionally, the expression of genes associated with hair growth promotion (IGF-1, WNT3A) was upregulated compared to the control group, while hair loss-related genes (DKK1, TGFb-2) were downregulated. This suggests that the NF-κB inhibitor BAY11-7082 has the effect of promoting hair follicle growth (Fig. 9C).

[0187] The present invention demonstrates that BAY11-7082 can promote hair follicle growth through inhibition of NF-κB signaling even in normal hair follicles, confirming its potential as a new therapeutic agent for promoting hair growth.

[0188]

[0189] In conclusion, based on the above evidence, the present invention demonstrated that skin damage caused by actual external environmental factors can be mimicked in an in vitro model using human-induced pluripotent stem cell-derived skin organoids through mechanisms that occur in actual skin. Furthermore, when evaluating the efficacy of candidate substances that could potentially be used as therapeutics in a UV-induced skin damage model, we verified that various disease phenotypes were alleviated, confirming its potential as a drug screening platform. The present invention will be applied to research on UV-induced skin damage and aging, contributing to improving patients' quality of life. Furthermore, the invention is expected to find wide-ranging applications in drug and toxicity evaluations, preclinical studies, regeneration, and tissue engineering research.

[0190]

[0191] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that modifications to other specific forms are possible without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention is defined by the claims that follow, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

[0192]

[0193] The present invention relates to a skin damage organoid model and a drug screening method using the same. Human-induced pluripotent stem cell-derived skin organoids were used to model skin damaged by an external environment (ultraviolet radiation). By utilizing organoid technology that mimics the actual human developmental process, skin organoids containing intact skin cells, hair follicle cells, and all microstructures were created. By irradiating the organoids with ultraviolet radiation, a technique was established to model damaged skin in vitro by mimicking external stimuli in a real-world environment. Therefore, this technology enables the identification of the mechanisms that cause ultraviolet radiation-induced damage and aging in vitro, and a platform for identifying candidate substances that can alleviate these effects has been developed. The platform established through the present invention can be applied as a platform for evaluating and identifying disease pathogenesis and the efficacy of various drug candidates, and thus has industrial applicability.

Claims

1. A method for producing skin damage organoids comprising the following steps: (1) A step of culturing pluripotent stem cell-derived organoids in the presence of a Wnt agonist; (2) A step of culturing the culture of step (1) in a medium for maturation of skin organoids; (3) a step of cutting the culture of step (2) and culturing it at an air-liquid interface to produce skin organoids; and (4) A step of irradiating the skin organoid with solar-mimicking ultraviolet rays, A method characterized in that the Wnt agonist is added at the start of differentiation of non-neural ectoderm into cranial neural crest cells (CNCC).

2. In paragraph 1, A method characterized in that the above skin damage organoid is a hair follicle damage organoid in which a hair follicle among the skin's accessory organs is damaged.

3. In paragraph 1, A method, characterized in that the Wnt agonist is selected from the group consisting of CHIR-99021, WNT3A, WNT5A, and RSPO1.

4. In paragraph 1, A method characterized in that the Wnt agonist is added on the 5th to 7th day of culturing pluripotent stem cells.

5. In paragraph 1, The above step (3) is a method characterized in that the culture of step (2) is cut into four equal-sized pieces and cultured at an air-liquid interface on a collagen-coated transwell culture insert so that the dermal layer faces the collagen side and the epidermal layer is exposed to the air.

6. In paragraph 1, The above skin damage organoid is characterized in that the skin is damaged through NF-kB activation.

7. Skin damage organoid manufactured by the manufacturing method of paragraph 1.

8. A method for screening for a skin damage treatment, preventive or improvement agent, comprising a step of treating a skin damage organoid of paragraph 7 with a candidate substance for a skin damage treatment, preventive or improvement agent.

9. In paragraph 2, The above hair follicle damaged organoid is characterized in that the hair follicle is damaged through NF-kB activation.

10. In paragraph 2, A method characterized in that the above hair follicle damaged organoid has reduced expression of one or more genes selected from the group consisting of a-SMA, a dermal sheath marker, KRT5 or KRT15, ​​an outer root sheath marker, KRT15 or LHX2, a hair follicle stem cell marker, and SOX2, a dermal papilla marker.

11. In paragraph 2, The above hair follicle damaged organoid is characterized by increased gene expression of inflammatory cytokines COX-2, TNF-α or IL-1β.

12. Hair follicle damage organoid manufactured by the manufacturing method of paragraph 2.

13. A method for screening a hair growth promoter, hair loss preventer, hair loss reliever, or hair loss treatment agent, comprising a step of treating a hair follicle damaged organoid of Article 12 with a hair growth promoter, hair loss preventer, hair loss reliever, or hair loss treatment agent candidate.

14. In paragraph 13, A method characterized in that the above candidate substance inhibits activated NF-kB in hair follicle damaged organoids.

15. A biomarker composition for detecting hair follicle damage comprising NF-kB as an active ingredient.

16. A kit for detecting hair follicle damage comprising a preparation capable of detecting NF-kB.

17. In paragraph 16, A kit characterized in that the above preparation is selected from the group consisting of primers, probes, antisense oligonucleotides, aptamers and antibodies specific for NF-kB. 18.(1) A step of measuring the expression level of NF-kB from a separated sample; (2) a step of comparing the expression level of the NF-kB with that of the control sample; and (3) A method for providing information necessary for detecting hair follicle damage, including a step of determining that the hair follicle is damaged if the expression level of the NF-kB is higher than that of the control sample.

19. A pharmaceutical composition for hair growth promotion, hair loss prevention, hair loss relief or hair loss treatment comprising an NF-kB activation or expression inhibitor as an active ingredient.

20. In paragraph 19, A pharmaceutical composition characterized in that the above NF-kB activity inhibitor is any one selected from the group consisting of low-molecular-weight compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to the NF-kB protein.

21. In paragraph 19, A pharmaceutical composition characterized in that the above NF-kB expression inhibitor is any one selected from the group consisting of an antisense nucleotide, a small interfering RNA (siRNA), and a short hairpin RNA (shRNA) that complementarily binds to the mRNA of the NF-kB gene.

22. A pharmaceutical composition for hair removal comprising an NF-kB activation or expression promoter as an active ingredient.

23. In paragraph 22, A pharmaceutical composition characterized in that the NF-kB activity promoter is any one selected from the group consisting of low-molecular-weight compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to the NF-kB protein.

24. In paragraph 22, A pharmaceutical composition characterized in that the NF-kB expression promoter is any one selected from the group consisting of adeno-associated virus (AAV), adenovirus, lentivirus, and retrovirus containing an NF-kB coding gene that increases the expression of mRNA of the NF-kB gene.

25. A cosmetic composition for hair removal comprising an NF-kB activation or expression promoter as an active ingredient.

26. A method for promoting hair growth, preventing hair loss, alleviating hair loss, or treating hair loss, comprising administering an effective amount of an NF-kB activation or expression inhibitor to a subject in need thereof.

27. A hair removal method comprising a step of administering an effective amount of an NF-kB activation or expression promoter to a subject in need thereof.

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