Method for creating an atopic dermatitis model using skin organoids derived from pluripotent stem cells

Abstract

We provide a treatment for atopic dermatitis. [Solution] A use is provided for manufacturing a pharmaceutical composition for the prevention or treatment of atopic dermatitis, comprising Cutibacterium acnes or its culture medium as an active ingredient.

Description

Technical Field

[0001] The present invention relates to a method for preparing an atopic dermatitis disease model using skin organoids derived from human pluripotent stem cells, etc.

[0002] This application claims priority based on Korean Patent Application No. 10-2020-0136977 filed on October 21, 2020 and Korean Patent Application No. 10-2021-0140422 filed on October 20, 2021, and all the contents disclosed in the specifications and drawings of the said applications are incorporated herein by reference.

Background Art

[0003] Human skin has functions such as maintaining body fluids, regulating body temperature, protecting against external stress, or external harmful bacteria, and plays an important role in adjusting touch and pain. Skin is composed of an epidermis, a dermis, and a subcutaneous layer, and there are appendages such as hair follicles, sebaceous glands, and nerve cells. In particular, the interaction between the epidermis and the dermis is very important for skin development and the formation of its appendages.

[0004] Currently, most studies related to human skin development and diseases are conducted using animal experiments and in vitro artificial skin models. However, animal experiment models are different from the human body in terms of genetic / biological characteristics, and artificial skin models commonly used for toxicity and drug screening include only a basic epidermis and dermis that do not generate appendages such as sweat glands, hair follicles, and melanocytes present in actual skin. Therefore, there is an increasing need to develop a skin model that can mimic the complexity and functions of in vivo skin tissue and is more similar to actual skin.

[0005] In recent years, human pluripotent stem cells have been used to... A 3D organoid culture method has been developed that can replicate the tissues and functions within body organs. Luganoids are cultured organisms that can reproduce the morphology and function of actual organ tissues or organs. Also called a look-alike or similar organ. In recent years, various human skin cells, nerve cells, and fat cells have been found. Furthermore, a skin organoid culture method has been developed that resembles actual skin, in which appendages and other structures are formed. This can happen. However, conventional skin organoid culture methods (see Non-Patent Literature 1) can form The created organoids exhibited a three-dimensional circular structure different from actual skin, and were used in drug toxicity efficacy experiments. Furthermore, it is difficult to use as a model for skin grafting and atopic dermatitis.

[0006] On the other hand, atopic dermatitis is a condition characterized by dry skin lesions accompanied by erythema, itching, rashes, and keratinized skin. It is a chronic / intractable / inflammatory skin disease that presents with symptoms. It affects 15-30% of children in developed countries worldwide. It affects 2-10% of adults, and currently in South Korea, 10% of the population and 20% of the child population are affected. I suffer from atopic dermatitis. According to statistics from the Health Insurance Review and Assessment Service, in South Korea, Nearly one million patients visit hospitals each year for sexually transmitted diseases, and the estimated number of patients based on prevalence rates. The number is approximately 2 million. Currently, the known cause of the disease is filaggrin (fi This is due to a complex interaction of factors such as laggrin gene mutations, immunological factors, and skin barrier damage. It is known that this can occur. Also, recent research suggests that atopic dermatitis can be a latent condition. One possible cause is the presence of Staphylococcus aureus bacteria on the skin. ) Increase in colonization and decrease in bacterial diversity (dysbiosis) s) has been presented. However, the exact disease mechanism and appropriate treatments are still unclear. It has not yet been done. Furthermore, research on the disease mechanism and drug screening of atopic dermatitis is also being conducted. Currently, research is being conducted using conventional cell lines in two-dimensional culture methods, in vitro artificial skin models, and The research primarily involves the use of animal models.

[0007] Therefore, it has a structure that is more similar to actual skin, and the complexity and function of skin tissue in vivo. The development of improved skin organoid models that can be replicated is necessary. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Latest Research and Development Trends in 3D Skin Models, Chang Sung-jae, BRIC View 2019-T21 [Overview of the project] [Problems that the invention aims to solve]

[0009] Therefore, the present inventors have developed a three-dimensional, layered skin organoid that closely resembles actual human skin. For efficient manufacturing of air-liquid interfaces (A Applying the LI culture method, a 3D air-liquid interface-skin organoid model was created from human pluripotent stem cells. Dell (ALI-Skin Organoid) is manufactured, and Staphylococcus aureus is added to ALI-Skin Organoid. By inoculating with bacteria, the mechanisms and disease factors of atopic skin disease can be elucidated, and medications can be developed. Atopic dermatitis organophysiology allows for the advancement of research into the efficacy of disease treatment agents through physical screening. We developed the Id model.

[0010] Therefore, an object of the present invention is a method for producing a skin organoid from a human pluripotent stem cell (pluripote nt stem cell) including the following steps .

[0011] (a) culturing an organoid derived from a pluripotent stem cell in the presence of a Wnt agonist

[0012] (b) culturing the culture of step (a) in a medium for skin organoid maturation for 40 days or more , and

[0013] (c) cutting the culture of step (b) and culturing it at an air-liquid interface

[0014] Another object of the present invention is a method for producing a skin organoid, including the step of treating the skin organoid produced by the above method with Staphylococcus aureus. The skin organoid is characterized in that it is a skin simulation model of atopic dermatitis . .

[0015] The present invention provides a skin organoid produced by the above method, characterized in that the skin organoid is a skin simulation model of atopic dermatitis .<00,screening method for a therapeutic agent for atopic dermatitis, including the step of treating the skin organoid with a candidate substance for a therapeutic agent for atopic dermatitis . .

[0017] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description . ​​[Means for solving the problem]

[0018] Therefore, in order to achieve the above-mentioned objective of the present invention, the present invention comprises the following steps of human pluripotency From stem cells (pluripotent stem cells) to skin organoids (org This provides a method for manufacturing anoids.

[0019] (a) The step of culturing pluripotent stem cell-derived organoids in the presence of a Wnt agonist,

[0020] (b) A step in which the culture from step (a) is cultured in a skin organoid maturation medium for 40 days or more. , and

[0021] (c) A step in which the culture from step (b) is cut and cultured at the air-liquid interface.

[0022] In one embodiment of the present invention, the Wnt agonist may be CHIR-99021. That's fine, but it's not limited to that.

[0023] In another embodiment of the present invention, the Wnt agonist is used on pluripotent stem cells on day 5-7 of culture. It may be added to, but is not limited to.

[0024] In yet another embodiment of the present invention, the culture medium for maturing skin organoids is glutamac Selected from the group consisting of S, 2-mercaptoethanol, B-27, N2, and normosine. It may, but is not limited to, contain one or more ingredients.

[0025] In yet another embodiment of the present invention, the following steps are included before the performance of step (a) above. This is acceptable, but not limited to it.

[0026] (1) The stage of culturing embryoid bodies of pluripotent stem cells,

[0027] (2) The step of inducing differentiation of the embryoid body into nonneuroectoderm,

[0028] (3) The step of inducing differentiation of the nonneuroectoderm into cranial neural crest-like cells.

[0029] In yet another embodiment of the present invention, step (1) is carried out in the presence of Y-27632. It may be anything, but is not limited to this.

[0030] In yet another embodiment of the present invention, step (2) is SB431542, FGF2, This may also be done in the presence of BMP4, but is not limited to this. .

[0031] In yet another embodiment of the present invention, step (3) is FGF2 and LDN19318 This may, but is not limited to, the presence of 9.

[0032] In yet another embodiment of the present invention, the culture in step (a) is carried out for 5 to 14 days. This is acceptable, but it is not limited to this.

[0033] In yet another embodiment of the present invention, the culture in step (b) is carried out for 40 to 100 days. It may be anything, but is not limited to this.

[0034] In yet another embodiment of the present invention, the culture in step (c) is carried out for 3 to 4 weeks. This is acceptable, but not limited to it.

[0035] In yet another embodiment of the present invention, the method is to (d) prepare the culture from step (c) to 1 The procedure may further include, but is not limited to, a step of culturing under dry conditions for 10 days. It's not that.

[0036] In yet another embodiment of the present invention, step (d) is the formation of the stratum corneum of the skin organoid. It may be for the purpose of ripening, but is not limited to that.

[0037] In yet another embodiment of the present invention, step (c) is the culture of step (b) into four equal parts Cut into uniform sizes and placed on collagen-coated Transwell culture inserts. It is also possible to cultivate the dermis layer with the collagen side and the epidermis layer exposed to the air. However, this is not the only limiting factor.

[0038] In yet another embodiment of the present invention, the air-liquid interface culture in step (c) is performed on skin organs This may be carried out using a nodule maturation medium, but is not limited to that.

[0039] In yet another embodiment of the present invention, the skin organoid is characterized by suppressed cartilage formation. This is acceptable, but it is not limited to this.

[0040] In yet another embodiment of the present invention, the skin organoid has a diameter of 2 to 20 mm. It may be anything, but is not limited to this.

[0041] In yet another embodiment of the present invention, the skin organoid is located below the epidermal layer and the dermis and The subcutaneous fat layer may be located there, but is not limited to it.

[0042] In yet another embodiment of the present invention, the skin organoid is KRT5(Kerati n 5, CK5), KRT10, KRT15, ​​KRT17, Loricrin (Loricri n), filaggrin, SOX2 (SRY-Box Transc One or more selected from the group consisting of ription Factor 2) and Melane-A. It may express, but is not limited to, this.

[0043] In yet another embodiment of the present invention, the pluripotent stem cells are embryonic stem cells, or induced pluripotent stem cells. Stem cells are acceptable, but are not the only option.

[0044] Furthermore, the present invention relates to the step of treating the skin organoid produced by the above method with Staphylococcus aureus. A method for producing a skin organoid, including a layer, wherein the skin organoid is atopic The present invention provides a method characterized by being a skin model that simulates dermatitis.

[0045] Furthermore, the present invention relates to a skin organoid produced by the above method, wherein the skin organoid The do is a skin organoid characterized by being a skin model that simulates atopic dermatitis. provide.

[0046] In one embodiment of the present invention, the skin organoid is selected from the group consisting of the following characteristics It may have one or more characteristics, but is not limited to that.

[0047] Increased cell death in the dermis layer of the skin.

[0048] KRT5, KRT10, and filaggrin are skin epidermal barrier-related markers. rin), loricrin, and claudin4 A decrease in the expression of one or more selected groups,

[0049] Keratinocyte differentiation Decreased expression of the gene KRT or KRTAP,

[0050] The antimicrobial peptide gene β-D Fensin, PGLYRP2 (Peptidoglycan Recognition Protein 2), TLR1 (Tol Like Receptor 1), TLR2, LGALS9 (Galectin 9), L CN2 (Lipocalin 2) and S100A (S100 calcium-binding protein) Increased expression of one or more selected from the group consisting of )

[0051] Inflammatory cytokines derived from epidermal or dermal cells The genes Cxcl (CXC motif chemokine ligand) and Ccl (CC) are genes. Motif chemokine ligand, TSLP (Thymic stromal lymphocyte) opoietin), IL-1 (interleukin-1), TNF (tumor Increased expression of one or more factors selected from the group consisting of necrosis factors,

[0052] Decreased expression of epidermal stem cell marker KRT15,

[0053] Formation of Staphylococcus aureus colonies (colonization),

[0054] Furthermore, the present invention involves treating the skin organoids with candidate substances for atopic dermatitis treatment. This invention provides a screening method for atopic dermatitis treatment agents, including a step-by-step process.

[0055] Furthermore, the present invention relates to the screening application of the aforementioned skin organoids as a treatment for atopic dermatitis. To provide.

[0056] Furthermore, the present invention includes Cutibacterium acnes or its culture solution as an active ingredient. The present invention provides pharmaceutical compositions for the prevention or treatment of atopic dermatitis.

[0057] In one embodiment of the present invention, the composition is used for filaggrin expression. It may increase, but is not limited to.

[0058] Furthermore, the present invention includes Cutibacterium acnes or its culture solution as an active ingredient. The present invention provides a food composition for the prevention or improvement of atopic dermatitis.

[0059] Furthermore, the present invention includes Cutibacterium acnes or its culture solution as an active ingredient. The present invention provides a cosmetic composition for the prevention or improvement of atopic dermatitis.

[0060] Furthermore, the present invention includes Cutibacterium acnes or its culture solution as an active ingredient. A method for preventing or treating atopic dermatitis, comprising the step of administering a pharmaceutical composition to an individual. provide.

[0061] Furthermore, the present invention includes Cutibacterium acnes or its culture solution as an active ingredient. The present invention provides a pharmaceutical composition for use in the prevention or treatment of atopic dermatitis.

[0062] Furthermore, the present invention relates to the atopic dermatitis of Cutibacterium acnes or its culture medium. It provides applications for producing drugs for the prevention or treatment of [the disease]. [Effects of the Invention]

[0063] The inventors have found that by treating the organoid with a Wnt agonist on day 6, the size of the organoid can be reduced. Not only does it increase significantly, but the formation of cartilage, which is an unnecessary component of skin organoids, is suppressed. It was confirmed that it is controlled. The ALI-skin organoid of the present invention is manufactured by conventional culture methods. Compared to actual skin organoids, skin cells, appendages, nerve cells, and fat cells are more prominent in real skin. It was observed that it was formed with a structure similar to that of actual skin, and that hair follicle growth was greatly improved. We confirmed that organoids are formed with a similar structure. In addition, atopic skin As a result of treating Staphylococcus aureus to create an inflammation model, damaged skin barrier and epithelial cells were observed. Increased cytokines derived from cells, decreased expression of skin barrier function-related genes, keratinocyte differentiation-related Decreased expression of linked genes, antimicrobial peptides ) Increased expression of genes, increased expression of cytokine genes derived from epidermal and dermal cells and Atopic dermatitis is characterized by decreased skin stem cell expression and the occurrence of Staphylococcus aureus colonies. The features are well-reproduced, and the skin organoid of this invention can be used as an actual skin model and for atopic dermatitis. It is expected to be useful as a model for sexually transmitted dermatitis.

[0064] Furthermore, the atopic dermatitis model of the present invention is useful as a microbiome therapeutic agent. Cutibacterium acnes, a symbiotic bacterium found in humans. um acnes) rescues the skin barrier from destruction caused by Staphylococcus aureus infection, and Staphylococcus aureus It was confirmed that it provides a protective mechanism against cocci colonies. Therefore, The disruption of the skin barrier and cell damage caused by Tybacterium acnes, which is transmitted by Staphylococcus aureus. It is expected to be useful in treating atopic dermatitis through significant preventive and therapeutic effects. It will be done. [Brief explanation of the drawing]

[0065] [Figure 1] Figure 1 shows the skin organoid formation timeline (63-138 days) and step-by-step photographs using human induced pluripotent stem cell lines developed using a conventional method. Scale bars 500 μm [Figure 2] Figure 2 shows photographs of skin organoids prepared using a conventional method at 140 days, confirming the presence of pigmented hair follicles (upper panel) and albino hair follicles (lower panel). Scale bars 500 μm [Figure 3a-3c] Figures 3a and 3c show fluorescent staining images of skin organoids prepared using conventional methods, stained with differentiation markers. Figure 3a shows skin organoids stained with epidermal progenitor cell markers (TFAP+KRT5+) (left) and dermal progenitor cells (TFAP+PDGFRα+) (right) on day 22. Figure 3b shows skin hair follicle dermal papilla cell expression marker (Sox2) staining images on day 67 (left) and day 83 (right). Figure 3c shows hyaline cartilage chondrocyte differentiation marker (S100α; left) and Coll2A1; right) staining images. Scale bars 50 μm [Figure 4] Figure 4 shows the epidermal layer formation timeline in skin organoids prepared using a conventional method. It also shows the results of immunohistochemical analysis for KRT5 and KRT14 (basement membrane markers), KRT10 (spinous layer marker), loricrin, and filaggrin (stratum corneum markers). Scale bars 50 μm [Figure 5] Figure 5 shows the method for culturing three-dimensional ALI-skin organoids from human pluripotent stem cells according to the present invention by adding Wnt signaling pathway activation (0.5-8 μM Wnt agonist (CHIR-99021)). [Figure 6] Figure 6 shows the quantification of skin organoid diameter (mm) based on images of skin organoids on days 6, 14, 25, 49, and 85. CHIR(-): No CHIR-99021 added; CHIR(+): CHIR-99021 added. Mean±SEM; n=9; *** indicates P<0.0005. [Figure 7] Figure 7 shows photographs of skin organoids derived from induced pluripotent stem cells at days 14, 25, 38, 65, or 85. CHIR(-): No CHIR-99021 was added; CHIR(+): CHIR-99021 was added. In the CHIR(-) skin organoid photographs at days 38, 65, and 85, the red circles indicate cartilage structures. Scale bars 1 mm. [Figure 8] Figure 8 shows the results of analyzing the gene expression levels of the cartilage expression markers COL2A1, ACAN, and SOX9 in skin organoids derived from induced pluripotent stem cells at day 69 via Real-time PCR. CHIR(-): No CHIR-99021 was added; CHIR(+): CHIR-99021 was added. n=3; *** indicates P<0.0005. [Figure 9] Figure 9 shows a method for producing a three-dimensional air-liquid interface-skin organoid model (ALI-skin organoid) from human induced pluripotent stem cells using the air-liquid interface (ALI) culture method. [Figures 10a-10b] Figures 10a and 10b show the results of comparing ALI-cultured skin organoids with skin organoids cultured using a conventional protocol (whole skin organoids). Figure 10a shows a representative photograph comparing ALI-cultured skin organoids and conventional skin organoids at day 138, and Figure 10b shows the results of H&E (hematoxylin & eosin) tissue staining. Scale bars 500 μm [Figure 11]Figure 11 shows the results of staining the subcutaneous fat and sebaceous glands around hair follicles in ALI skin organoids with Oil Red O. X100 (left) and X200 (right). [Figure 12] Figure 12 shows a 200x200 image of hair follicle H&E staining of ALI-skin organoids after a total of 119 days of culture. (Skin organoid culture 68 days + ALI culture 51 days) [Figure 13a] Figure 13a shows ALI-skin organoid photographs at a total of 114 days. Skin organoids were cultured for 85 days, followed by 3 weeks of ALI culture at 100% humidity, and then ALI culture for 0, 2, 4, or 6 days under dry conditions to mature skin keratinocytes. Scale bars 500 μm. [Figure 13b] Figure 13b shows the results of H&E (hematoxylin & eosin) histological staining of ALI-skin organoids after a total of 114 days. Skin organoids were cultured for 85 days, followed by 3 weeks of ALI culture at 100% humidity, and then ALI culture for 0, 2, 4, or 6 days under dry conditions to mature skin keratinocytes. Scale bars 500 μm [Figure 14] Figure 14 shows fluorescent staining images of markers related to the structure of the epidermal layer of ALI-cutaneous organoids. (a) ALI-cutaneous organoid basement membrane (KRT5) and spinous layer (KRT10) marker staining images, (b) ALI-cutaneous organoid stratum corneum (Loricrin) marker staining images, and (c) ALI-cutaneous organoid stratum corneum (Filaggrin) marker staining images. Scale bars 50 μm [Figure 15]Figure 15 shows fluorescent staining images of ALI-skin organoid hair follicle structure-related markers. (a) ALI-skin organoid epidermal cell (E-cadherin) and dermal cell (PDGGR) marker staining images, (b) ALI-skin organoid hair bulge area (KRT15) marker staining images, (c) ALI-skin organoid epidermal layer melanocyte (melanA) marker staining images, (d) ALI-skin organoid hair follicle papilla cell (Sox2) and Merkel cell (Sox2) marker staining images, (e) ALI-skin organoid hair follicle melanocyte (MelanA) marker staining images, and (f) ALI-skin organoid outer root sheat (KRT17) marker staining images. Scale bars 50 μm [Figure 16] Figure 16 shows fluorescent staining images of cutaneous organoids cultured for 85 days, followed by ALI culture at 100% humidity for 3 weeks, and then ALI culture for 0, 2, 4, or 6 days under dry conditions to mature keratinocytes. Image a shows ALI-stained images of cutaneous organoids with basement membrane (KRT5) and stratum corneum (Filaggrin) markers, and image b shows ALI-stained images of cutaneous organoids with stratum corneum (Loricrin) and stratum spinosum (KRT10) markers. Scale bars 50 μm [Figure 17] Figure 17 shows fluorescent staining images of dermal organoids cultured for 85 days, followed by ALI culture at 100% humidity for 3 weeks, and then ALI culture for 0, 2, 4, or 6 days under dry conditions to mature keratinocytes. The images show ALI-stained dermal organoids stained with basement membrane (KRT5; red), dermal collagen 3 (COL3; green; left), and vimentin (VIM; green; right) markers. Scale bars 50 μm [Figure 18]Figure 18 shows the results of staining subcutaneous fat (lipid tox; red; left) and sebaceous glands (lipid tox; red; right) around hair follicles of skin organoids that were cultured for 85 days, ALI cultured at 100% humidity for 3 weeks, and then ALI cultured under dry conditions for 0, 2, 4, or 6 days to mature skin keratinocytes. Scale bars 50 μm [Figure 19] Figure 19 shows photographs modeling atopic dermatitis by inoculating ALI-skin organoids with Staphylococcus aureus. Specifically, the images show (top) H&E stained ALI-skin organoids and (bottom) Staphylococcus aureus (green) and basal epithelial layer (KRT5, red) stained with markers, 24 hours after surface inoculation of ALI-skin organoids with Staphylococcus aureus (ATCC 29213; 0 CFU (PBS only), 1 × 10⁵ CFU, 1 × 10⁶ CFU, or 1 × 10⁷ CFU). [Figure 20] Figure 20 shows a model of atopic dermatitis created by infecting ALI-skin organoids with Staphylococcus aureus. The images show (top) staining with cell proliferation (Ki67; green) and basal epithelial layer (KRT5; red) markers, and (bottom) staining with cell death (TUNEL, red) and basal epithelial layer (KRT5; green) markers, 24 hours after surface inoculation of Staphylococcus aureus into ALI-skin organoids. Scale bars 50 μm [Figure 21] Figure 21 shows a model of atopic dermatitis created by infecting ALI-skin organoids with Staphylococcus aureus. The images show, 24 hours after surface inoculation of Staphylococcus aureus into ALI-skin organoids, (top) stained with markers for the stratum corneum (Filaggrin; green) and basal epithelium (KRT5; red), (middle) stained with markers for the stratum corneum (Loricrin; green) and basal epithelium (KRT14; red), and (bottom) stained with markers for the stratum spinosum (KRT10; green) and basal epithelium (KRT5; red). Scale bars 50 μm [Figure 22]Figure 22 shows a model of atopic dermatitis created by infecting ALI-skin organoids with Staphylococcus aureus. The results were obtained by fluorescent staining 24 hours after surface inoculation of ALI-skin organoids with Staphylococcus aureus, and then observing the expression of cytokines in skin epithelial cells. The image shows stained photographs of Staphylococcus aureus-infected ALI-skin organoid epithelial cell cytokines (TSLP; green) and basal epithelial layer (KRT14; red) markers. Scale bars 50 μm [Figure 23] Figure 23 shows the results of a model of atopic dermatitis created by infecting ALI-skin organoids with Staphylococcus aureus. After 24 hours of surface inoculation of ALI-skin organoids with Staphylococcus aureus, a decrease in epidermal stem cell expression was confirmed by fluorescence staining. (Top) Photographs of Staphylococcus aureus-infected ALI-skin organoids stained with epidermal stem cell (KRT15; green) and basal epithelial layer (KRT5; red) markers, and (Bottom) Photographs of epidermal stem cell marker (p63; green) and basal epithelial layer (KRT5; red) markers. Scale bars 50 μm [Figure 24] Figure 24 shows the results of a model of atopic dermatitis created by infecting ALI-skin organoids with Staphylococcus aureus. The expression of cytokines in skin epithelial cells was confirmed by fluorescence staining 24 hours after surface inoculation of Staphylococcus aureus into the ALI-skin organoids. The image shows stained photographs of the tight junctional protein (tight junctional protein) marker (Claudin 4 (CLDN4); green) and the basal epithelial layer (KRT5; red). Scale bars 50 μm [Figures 25a-25f]Figures 25a to 25f show the results of a model of atopic dermatitis created by infecting ALI-skin organoids with Staphylococcus aureus. The changes in gene expression were compared using RNA sequencing analysis 18 hours after surface inoculation of ALI-skin organoids with Staphylococcus aureus. Significant differences in gene expression were observed between biological replicas of ALI-skin organoids treated with Staphylococcus aureus and those of an untreated control group. Figure 25a shows differentially expressed genes (DEGs; dark blue) represented in a volcano plot. Figure 25b visualizes differentially expressed genes through a heat map. Of the 4,255 differentially expressed genes (DEGs), 2,162 were highly expressed, while 2,093 were less expressed by S. aureus inoculation (adjusted p-value < 0.05). Red indicates the control group, and blue indicates ALI-skin organoids treated with Staphylococcus aureus. Figure 25c shows the results of RNA-sequencing analysis comparing the expression of skin barrier function-related genes between ALI-skin organoids infected with Staphylococcus aureus and an untreated control group. Figure 25d shows the results of RNA-sequencing analysis comparing the expression of genes related to keratinocyte differentiation between ALI-skin organoids treated with Staphylococcus aureus and an untreated control group. Figure 25e shows the results of RNA-sequencing analysis comparing the expression of antimicrobial peptides (AMPs)-related genes between ALI-skin organoids treated with Staphylococcus aureus and an untreated control group. Figure 25f shows the results of RNA-sequencing analysis comparing the expression of inflammatory cytokines-related genes between ALI-skin organoids treated with Staphylococcus aureus and an untreated control group.Figure 26a shows the ALI skin organoid culture method, pretreatment with Cuticulobacterium acnes, and inoculation with Staphylococcus aureus. Figure 26b shows the results of immunohistochemical staining analysis for filaggrin (green) and KRT5 (red) after treatment with Cuticulobacterium acnes. Scale bars 50 μm. [Modes for carrying out the invention]

[0066] This invention relates to human pluripotent stem cells, comprising the following steps. This invention provides a method for producing skin organoids from ell.

[0067] (a) The step of culturing pluripotent stem cell-derived organoids in the presence of a Wnt agonist,

[0068] (b) A step in which the culture from step (a) is cultured in a skin organoid maturation medium for 40 days or more. , and

[0069] (c) A step in which the culture from step (b) is cut and cultured at the air-liquid interface.

[0070] The present invention will be described in detail below.

[0071] In the present invention, the term "organoid" refers to the appearance of the surface area of ​​a tissue or organ or It refers to a three-dimensional assembly of one or more cell types that mimics the actual structure or function. Luganoids can similarly reproduce the physiologically active functions of the human body and construct organ analogs from patient tissues. By doing so, disease modeling based on the patient's genetic information and drug testing through repeated trials are possible. Cleaning and other services can be performed.

[0072] In the present invention, "organoid culture" refers to the process of generating or maintaining organoids. This includes all actions that can be performed, such as cells being isolated from cells or specific tissues. This may involve differentiating into tissues or organ cells having a specific function, and / or organo It may also be something that allows the id to survive, grow, or multiply.

[0073] In this invention, pluripotent stem cells are defined as follows: This term collectively refers to stem cells that can differentiate into almost all types of cells that make up the endoderm, mesoderm, and ectoderm. In the present invention, the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells. This is acceptable, but not limited to it.

[0074] In this invention, the term "embryonic stem cell (ES)" is used. C) refers to the extraction of the inner cell mass from an embryo in the blastula stage, which is just before the fertilized egg implants in the mother's uterus. These cells are cultured in vitro and possess pluripotency, meaning they can differentiate into all types of animal cells. This refers to cells possessing NCY, and preferably, human embryonic stem cells of human origin.

[0075] In this invention, the term "induced pluripotent stem cells" is used. "Stem cells (iPSCs, reverse-differentiated stem cells)" are animal somatic cells that have undergone reverse differentiation technology. This method is used to establish undifferentiated stem cells that have differentiation potential similar to embryonic stem cells. These are stem cells with differentiation capabilities at a similar level to ESCs.

[0076] In this invention, "embryoid body" refers to a body produced in a suspension culture state. It refers to a spherical mass of stem cell-derived cells that can potentially differentiate into endoderm, mesoderm, and ectoderm. Having the ability to do so ensures most differentiation inductions for tissue-specific differentiated cells. It is manufactured as a precursor during the development process.

[0077] In the present invention, the Wnt agonist may be CHIR-99021, however It is not limited to that.

[0078] In the present invention, the Wnt agonist is added to induced pluripotent stem cells on day 5 to 7 of culture. It may be anything, but is not limited to this.

[0079] In the present invention, the culture medium for maturing skin organoids is glutamax, 2-mercapto Contains one or more components selected from the group consisting of ethanol, B-27, N2, and normosine. This may be, but is not limited to. The terms used herein are "Culture medium for maturation of skin organoids" refers to a medium for maturating pluripotent stem cells or organoids derived from pluripotent stem cells. This refers to the culture medium necessary for culturing ids in the form of skin organoids. One embodiment of the present invention In this context, the culture medium for maturing skin organoids is Advanced DMEM / in a 1:1 ratio. In F12 and Neurobasal medium, add 1×GlutaMax™ and 0.5×B-27 Minus Vitamin 1A, 0.5 x N2, Supplement, 0.1 mM 2-Mercaptoethamine Contains 100 μg / ml normosine.

[0080] In the present invention, the culture medium for maturing skin organoids is Advanced DMEM / F A mixed medium of 12 and Neurobasal medium may be used, but is not limited to this. isn't it.

[0081] In this invention, "air-liquid interface culture" ALI culture refers to a culture vessel that is partially open or partially filled with culture medium. Cultivation may be carried out in a culture container, but is not limited to this. Air-liquid interface Culture may also involve exposing the surface of cells or organoids to air.

[0082] In the present invention, the air-liquid interface culture is performed on a collagen-coated plate. It may be done, but is not limited to.

[0083] In the present invention, the following steps may be included before the performance of step (a): This is not the only limitation.

[0084] (1) The stage of culturing embryoid bodies of pluripotent stem cells,

[0085] (2) The step of inducing differentiation of the embryoid body into nonneuroectoderm,

[0086] (3) The step of inducing differentiation of the nonneuroectoderm into cranial neural crest-like cells.

[0087] In the present invention, even if step (1) is performed in the presence of Y-27632 That's fine, but it's not limited to that.

[0088] In the present invention, step (2) is the presence of SB431542, FGF2, and BMP4. It may be carried out below, but is not limited to this.

[0089] In the present invention, step (3) is carried out in the presence of FGF2 and LDN193189. It may be anything, but is not limited to this.

[0090] In the present invention, the culture in step (a) may be carried out for 5 to 14 days. However, it is not limited to this. For example, the culture in step (a) may be 5 to 13 days, 5 to 12th, 5th-11th, 5th-9th, 6th-13th, 6th-12th, 6th-11th, 6th-10th, 6 ~9 days, 7-13 days, 7-12 days, 7-11 days, 7-10 days, 7-9 days, or approximately 8 days It may be done.

[0091] In the present invention, the culture in step (b) may be carried out for 40 to 100 days. However, it is not limited to this. For example, the culture in step (b) above may last 40 to 95 days. 40-90 days, 45-95 days, 45-90 days, 45-80 days, 50-80 days, 40-7 5th, 40-70th, 55-75th, 58-75th, 59-74th, 60-72nd, and This may be carried out for 61 to 71 days.

[0092] In the present invention, step (c) is to cut the culture from step (b) and culture it at the air-liquid interface. It may be in stages. In one embodiment of the present invention, the culture in stage (b) is cut into four equal parts with scissors. Afterward, each cut culture was spread onto a 12-well insert plate and cultured using the ALI method. After culturing in an incubator under 100% humidity conditions for 3-4 weeks, allow 2-6 days for maturation of the lamellar epithelial layer. Under dry conditions (0% humidity conditions) The cultures were performed in an incubator.

[0093] In the present invention, step (c) is to cut the culture from step (b) into four uniform sizes. The dermis layer on top of the collagen-coated Transwell culture insert is collagen On the other hand, the epidermal layer may be cultured in a way that exposes it to air, but is not limited to this. It is not something that can be done.

[0094] Therefore, in the present invention, the method is to (d) prepare the culture from step (c) for 1 to 10 days The procedure may further include, but is not limited to, a step of culturing under intermittent dry conditions. There is none. The aforementioned stage (d) is 1-10 days, 1-9 days, 1-8 days, 1-7 days, 1-6 days, 2 ~10 days, 2-9 days, 2-8 days, 2-7 days, 2-6 days, 3-10 days, 3-9 days, 3-8 days 3-7 days, 3-6 days, 4-10 days, 4-9 days, 4-8 days, 4-7 days, or 4-6 days It may be done, but is not limited to this.

[0095] The aforementioned step (d) may be for the maturation of the stratum corneum of the skin organoid, It is not limited to that.

[0096] In the present invention, the culture in step (c) may be carried out for 3 to 4 weeks, This is not the only limitation.

[0097] In the present invention, the air-liquid interface culture in step (c) is performed using a culture medium for skin organoid maturation. It may be done, but is not limited to this.

[0098] The culture period of this invention depends on the amount and type of tissue, the type of culture medium used, and the size of the desired organoid. The size, quantity, and characteristics may be adjusted accordingly.

[0099] In the present invention, the skin organoid may be one in which cartilage formation is suppressed. However, this is not the only limitation.

[0100] In the present invention, the skin organoid is such that the subcutaneous fat layer is located below the dermis layer. You may do so, but you are not limited to it.

[0101] In the present invention, the skin organoid is KRT5 (Keratin 5), KRT1 0, KRT15, ​​KRT17, Loricrin, Fillaggrin aggrin), SOX2(SRY-Box Transcription Facto A group consisting of r 2), melan-A, collagen 3 (COL3), and vimentin (VMT). It may express one or more selected from, but is not limited to, this. .

[0102] Furthermore, the present invention relates to the step of treating the skin organoids produced by the above method with Staphylococcus aureus. A method for producing a skin organoid, including a layer, wherein the skin organoid is atopic The present invention provides a method characterized by being a skin model that simulates dermatitis.

[0103] The method of the present invention will be described in detail below, and will be applied to the method for producing the skin organoids described above. Parts that overlap with the explanation will be omitted.

[0104] Furthermore, the present invention relates to a skin organoid produced by the above method, wherein the skin organoid The do is a skin organoid characterized by being a skin model that simulates atopic dermatitis. provide.

[0105] In this specification, the skin organoid is described as having a flattened shape rather than a sphere. It has certain characteristics, but is not limited to them.

[0106] In this specification, even if the skin organoid has a diameter of 2 to 20 mm However, it is not limited thereto. In one embodiment of the present invention, the skin of the present invention The organoids have a larger diameter compared to organoids cultured using conventional methods. I confirmed it.

[0107] In this specification, "atopic dermatitis" refers to a condition characterized by severe itching and generalized dry skin. This is a chronic inflammatory skin disease characterized by recurrent eczematous lesions in specific areas, and it exhibits a genetic predisposition and dietary risk factors. Allergies accompanied by allergic diseases such as allergic substances, allergic asthma, and allergic rhinitis. It is characterized by a gyneic (ghee) movement. The etiology of atopic dermatitis is largely related to the skin barrier. The etiologies can be divided into those related to abnormal immune responses and those related to sudden changes in the filaggrin gene. The mutation appears in about 40% of atopic dermatitis patients, resulting in severe symptoms or persisting into adulthood. It has been reported that if it persists, it appears more frequently. The mechanism is that it is congenitally damaged. In humans with a damaged skin barrier, external allergens can more easily pass through the stratum corneum. It can trigger atopic dermatitis by causing sensitization in the body.

[0108] In the present invention, the skin organoid is one or more selected from the group consisting of the following characteristics. It may have the characteristics of, but is not limited to.

[0109] Increased cell death in the dermis layer of the skin.

[0110] KRT5, KRT10, and filaggrin are skin epidermal barrier-related markers. rin), loricrin, and claudin4 A decrease in the expression of one or more selected groups,

[0111] Keratinocyte differentiation Decreased expression of the gene KRT or KRTAP,

[0112] The antimicrobial peptide gene β-D Fensin, PGLYRP2 (Peptidoglycan Recognition Protein 2), TLR1 (Tol Like Receptor 1), TLR2, LGALS9 (Galectin 9), L CN2 (Lipocalin 2) and S100A (S100 calcium-binding protein) Increased expression of one or more selected from the group consisting of )

[0113] Inflammatory cytokines derived from epidermal or dermal cells The genes Cxcl (CXC motif chemokine ligand) and Ccl (CC) are genes. Motif chemokine ligand, TSLP (Thymic stromal lymphocyte) opoietin), IL-1 (interleukin-1), TNF (tumor Increased expression of one or more factors selected from the group consisting of necrosis factors,

[0114] Decreased expression of epidermal stem cell marker KRT15, ​​and

[0115] Formation of Staphylococcus aureus colonies (colonization),

[0116] In this specification, "filaggrin" refers to various substances expressed by keratinocytes during the differentiation stage. It is one of the structural proteins that facilitates differentiation from the basal layer to the stratum corneum of the epidermis. It may be involved and form the main component of natural moisturizing factors (NMF), which are essential for maintaining moisture in skin tissue. It is used as a primary indicator of skin moisture retention and skin barrier function.

[0117] In this specification, "loricrin" refers to the skin barrier expressed by keratinocytes during the differentiation stage. It is a type of protein.

[0118] In this specification, "Thymic stromal lymphopoietin (T SLP is a cytokine with a structure similar to IL-7, and is involved in the production of keratinocytes in the skin. It is expressed in epithelial cells, smooth muscle cells, and lung fibroblasts, among others. TSLP activates dendritic cells. Sexualization is performed to differentiate helper T cells into Th2 cells, and the differentiation of Th2 cells is atopic It is important in inducing symptoms of genital dermatitis.

[0119] In this specification, "Staphylococcus aureus" " is a pathogenic bacterium that increases in atopic dermatitis patients and further worsens atopic symptoms. .

[0120] In this specification, Staphylococcus aureus is defined as 1 × 10¹ to 1 × 10²⁰ CFU, 1 × 10¹ to 1×10¹⁸CFU, 1×10¹~1×10¹⁵CFU, 1×10¹~1×10¹⁰CF U, 1×101~1×108CFU, 1×101~1×107CFU, 1×103~1× 1020CFU, 1×103~1×1018CFU, 1×103~1×1015CFU, 1×10³~1×10¹¹ CFU, 1×10³~1×10⁹ CFU, 1×10³~1×1 07CFU, 1×105~1×1020CFU, 1×105~1×1015CFU, 1× Even when treated at concentrations of 10⁵ to 1 × 10¹⁰ CFU, or 1 × 10⁵ to 1 × 10⁷ CFU That's fine, but it's not limited to that.

[0121] The aforementioned skin organoid may be used in vivo for the treatment of a subject. The present invention relates to the subject This invention includes a method for treating skin diseases. In one embodiment, as disclosed in this invention, the method is to treat skin In subjects who require improvement, treatment, or relief of the symptoms of a skin disease, the improvement, treatment, or relief of the symptoms This includes reduction. The method further makes skin organoids effective in treating the skin disease of the subject. This includes administering it in a certain amount.

[0122] The treatment method for the subject involves administering skin organoids or transferring skin organoids. This may further include planting, in which case the subject may smell as a result of the administration or transplantation. The symptoms of the skin disease are improved, treated, or alleviated. The aforementioned improvement, treatment, or alleviation is natural. The skin disease or symptoms of the skin disease that can be detected using sensory perception or artificial devices A change in meaning is also acceptable.

[0123] Skin diseases that can be treated using the method of the present invention include diseases or conditions that appear on the skin, for example, Unrestricted use for acne, atopic dermatitis, skin swelling, boils, urticaria, psoriasis, and seborrheic dermatitis. , contact dermatitis, skin discoloration symptoms, bacterial dermatitis, fungal dermatitis, eczema, edema, sudden rash Folliculitis, follicular keratosis, warts, actinic keratosis, milia, xerosis, hidradenitis, keloids These include diseases, allergic dermatitis, skin abscesses, and neurodermatitis.

[0124] The skin organoids of the present invention may be included in a pharmaceutical composition at a concentration of 1 pg to 30 g w / v%. However, this is not the only limiting factor.

[0125] In this invention, the term "individual" is not limited to any vertebrate, but specifically refers to humans, maws, etc. Snakes, rats, guinea pigs, rabbits, monkeys, pigs, horses, cows, sheep, antelopes, dogs and It may be applied to cats, and preferably to humans.

[0126] In this invention, "administration" means introducing the pharmaceutical composition of the present invention to a patient by any appropriate method. This means that the administration route of the composition of the present invention is by oral or other means, as long as it can reach the target tissue. It may be administered via various parenteral routes.

[0127] In the present invention, "prevention" means delaying the progression of skin diseases by administering the composition according to the present invention. It refers to all actions, and "treatment" means the administration of the pharmaceutical composition according to the present invention to treat skin diseases. "Improvement" means any action that leads to an improvement or a favorable change in symptoms. Administration of the composition by Mei reduces parameters related to skin diseases, such as the severity of symptoms. It means all actions that cause something to happen.

[0128] In this invention, pharmaceutical composition ) provides suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. It may also be included.

[0129] In this invention, "carrier" is also called a vehicle. This refers to compounds that facilitate the addition of proteins or peptides into cells or tissues. For example, dimethyl sulfoxide (DMSO) enters the cells or tissues of living organisms. It is a commonly used carrier that facilitates the introduction of many organic materials.

[0130] In this invention, "diluent" refers to the bio-diluent of the target protein or peptide. In addition to stabilizing the physical active form, water is used to dissolve proteins or peptides. It is defined as a compound that is diluted. Salts dissolved in a buffer are used as diluents in this field. The buffer solution commonly used is phosphate-buffered saline, which is used to measure the salinity of human body fluids. This is because it is a copy. Buffer salts can control the pH of a solution at low concentrations, therefore buffer diluents are It is rare for compounds to alter their biological activity. The azera acid used here contains These compounds are administered to human patients either by themselves or in combination with other components in therapy. Alternatively, it may be administered as a pharmaceutical composition mixed with appropriate carriers and excipients.

[0131] Furthermore, the pharmaceutical compositions according to the present invention can be prepared by conventional methods as powders, granules, tablets, etc. Topical preparations such as capsules, suspensions, emulsions, syrups, and aerosols, and sterile injection preparations. The composition may be used in the form of a solution, and may contain carriers, excipients and The diluents include lactose, dextrose, sucrose, oligosaccharides, and sorbitol. Mannitol, xylitol, erythritol, maltitol, starch, acacia Ingredients: Alginate, gelatin, calcium phosphate, calcium silicate, cellulose methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydro Xybenzoate, propylhydroxybenzoate, talc, magnesium stearate Examples include t and mineral oil. When formulating, fillers, bulking agents, and binders are typically used. It is prepared using diluents or excipients such as wetting agents, decomposing agents, and surfactants. Oral administration Solid preparations for administration include tablets, pills, powders, granules, capsules, etc. The solid preparation contains the compound with at least one adjunct agent, for example, starch, calcium calcium carbonate, sucrose ) or prepared by mixing lactose, gelatin, etc. Also, In addition to pure floating agents, lubricants such as magnesium stylate talc are also used. (Oral liquid) Formulations include suspensions, liquid preparations, emulsions, and syrups, but the most commonly used are... In addition to simple diluents such as water and liquid paraffin, various excipients such as humectants and sweeteners are also used. Fragrances, preservatives, etc. may be included. Parenteral administration formulations may contain sterile aqueous solutions or non-aqueous solvents. This includes suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propionic acid. Polyethylene glycol (polypropylene glycol), polyethylene glycol, olive Vegetable oils such as oat oil, or injectable esters such as ethyl oleate may be used. The bases for suppositories include witepsol, macrogol, and twin( (tween)61, cocoa butter, lauric acid butter, glycerol gelatin, etc. may also be used.

[0132] The pharmaceutical composition of the present invention may be administered orally or parenterally, preferably parenterally. Oral administration methods include intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, local administration, and transdermal administration. It may be administered as follows.

[0133] The appropriate dosage of the pharmaceutical composition of the present invention depends on the formulation method, administration method, patient's age, weight, and sex. The response varies depending on factors such as the patient's condition, food, administration time, administration route, excretion rate, and response sensitivity. It may be prescribed separately.

[0134] The pharmaceutical composition of the present invention can be easily put into practice by a person with ordinary skill in the art to which the invention belongs. Formulation using pharmaceutically acceptable carriers and / or excipients, depending on the applicable method. Therefore, it may be manufactured in unit dose form or manufactured by being contained within a large-capacity container. i. In this case, the dosage form is in the form of a solution, suspension, or emulsion in an oil or aqueous medium. It may also be in the form of an X-form, powder, granules, tablet, or capsule. The following may further be included: a dispersant or a stabilizer.

[0135] Furthermore, the present invention involves treating the skin organoids with candidate substances for atopic dermatitis treatment. This invention provides a screening method for atopic dermatitis treatment agents, including a step-by-step process.

[0136] Furthermore, the present invention relates to the screening application of the aforementioned skin organoids as a treatment for atopic dermatitis. To provide.

[0137] Furthermore, the present invention includes Cutibacterium acnes or its culture solution as an active ingredient. The present invention provides pharmaceutical compositions for the prevention or treatment of atopic dermatitis.

[0138] In the present invention, the composition increases filaggrin expression. It may be anything, but is not limited to this.

[0139] Furthermore, the present invention includes Cutibacterium acnes or its culture solution as an active ingredient. The present invention provides a food composition for the prevention or improvement of atopic dermatitis.

[0140] Furthermore, the present invention includes Cutibacterium acnes or its culture solution as an active ingredient. The present invention provides a cosmetic composition for the prevention or improvement of atopic dermatitis.

[0141] Furthermore, the present invention includes Cutibacterium acnes or its culture solution as an active ingredient. A method for the prevention or treatment of atopic dermatitis is proposed, which includes the step of administering a pharmaceutical composition to an individual. To provide.

[0142] Furthermore, the present invention includes Cutibacterium acnes or its culture solution as an active ingredient. The present invention provides a pharmaceutical composition for use in the prevention or treatment of atopic dermatitis.

[0143] Furthermore, the present invention relates to the atopic dermatitis of Cutibacterium acnes or its culture medium. It provides applications for producing drugs for the prevention or treatment of [the disease].

[0144] In this invention, "Cutibacterium a Propionibacterium acnes (cnes) is an anaerobic, Gram-positive rod-shaped bacterium. These are the normal bacterial flora found on the surface of human skin and mucous membrane tissue. They are anaerobic microorganisms. Propionibacterium acnes is It grows inside the hair follicle.

[0145] In this invention, the term "culture" refers to the growth of microorganisms under appropriately artificially controlled environmental conditions. This refers to all actions taken to achieve a certain result, and in this invention, it is a concept that includes "fermentation."

[0146] The bacterial cells of "Cutibacterium acnes" mentioned above are the live bacteria themselves obtained from the culture medium. This includes not only any processing methods for lactic acid bacteria known to those skilled in the art, for example, This includes, but is not limited to, crushed bacterial cells, dried products, frozen products, etc. The term "culture medium" includes "fermentation liquid," and refers to the culture medium itself that is cultured in a liquid culture medium. , the culture solution is filtered or centrifuged to remove the bacterial strain, and the resulting filtrate (centrifugation supernatant) This includes any processed products derived from the culture medium itself.

[0147] The content of Cutibacterium acnes or its culture solution in the composition of the present invention is It can be adjusted as appropriate according to the patient's symptoms, the progression of symptoms, and the patient's condition, for example, all sets Based on the weight of the finished product, this ranges from 0.0001% to 99.9% by weight, or from 0.001% to 50% by weight. It may be, but is not limited to, %. The content ratio is obtained by removing the solvent in the dry This value is based on the amount of dryness.

[0148] In the present invention, the description of the pharmaceutical composition is as described above.

[0149] The present invention uses Cutibacterium acnes or its culture solution as a food additive. In that case, add the Cutibacterium acnes or its culture solution as is, or It may also be used with other foods or food ingredients, and may be used as appropriate in the usual manner. This is also acceptable. The amount of active ingredients mixed should be appropriate depending on the purpose of use (prevention, health, or therapeutic treatment). It may be determined. Generally, during the manufacture of food or beverages, the Cutibacterium of the present invention Acnes or its culture solution may be used in an amount of 15% by weight or less, or 10% by weight or less, relative to the raw materials. It may be added. However, if the purpose is health and hygiene or health regulation In the case of long-term intake, the amount may be less than or equal to the range mentioned above, and there are no safety concerns. Therefore, the active ingredient may be used in amounts greater than the aforementioned range.

[0150] There are no particular restrictions on the types of food to which the substance can be added. Examples of foods to which the substance can be added include meat, soy - Sage, bread, chocolate, candy, snacks, sweets, pizza, ramen Other noodles, gums, dairy products including ice cream, various soups, drinking water, tea These include energy drinks, alcoholic beverages, and vitamin complexes, and have health benefits in the usual sense. Includes all food items.

[0151] The health beverage composition according to the present invention, like ordinary beverages, contains various flavorings or natural carbohydrates. These may be included as additional ingredients. The natural carbohydrates mentioned above are glucose and fructose. Monosaccharides such as maltose and sucrose, disaccharides such as dextrose Polysaccharides such as nitrates and cyclodextrin, as well as xylitol, sorbitol and These are sugar alcohols such as erythritol. Sweeteners include thaumatin and stevia extract. Natural sweeteners such as fermented fruit extracts, as well as synthetic sweeteners such as saccharin and aspartame, are used. The proportion of natural carbohydrates is generally about 0.0 per 100 mL of the composition of the present invention. It is 1 to 0.20 g, or approximately 0.04 to 0.10 g.

[0152] In addition to the above, the composition of the present invention contains various nutrients, vitamins, electrolytes, flavorings, colorings, Pectin acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters Agents, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, etc. It may also contain natural fruit juice, fruit juice It may contain fruit pulp for the manufacture of beverages and vegetable drinks. These ingredients may also be used independently. These can be used in combination. The proportion of such additives is not very important, but It is generally selected from the range of 0.01 to 0.20 parts by weight per 100 parts by weight of the bright composition. It is.

[0153] The dosage form of the cosmetic composition according to the present invention is a skin lotion, a skin softener, a skin toner , an astringent, a lotion, a milk lotion, a moisture lotion, a nourishing lotion, a massage cream, a nourishing cream, a mist, a moisture cream, a hand cream, a hand lotion, a foundation, an essence, a nourishing essence, a pack , a soap, a cleansing foam, a cleansing lotion, a cleansing cream, a cleansing oil, a cleansing balm, a body lotion or a body cleanser. It may be in the form.

[0154] The cosmetic composition of the present invention may further contain a composition selected from the group consisting of a water-soluble vitamin, an oil-soluble vitamin, a high-molecular peptide, a high-molecular polysaccharide, and a sphingolipid. It may be.

[0155] Any water-soluble vitamin may be used as long as it can be formulated in cosmetics. For example , vitamin B1, vitamin B2, vitamin B6, pyridoxine, pyridoxine hydrochloride, vita min B12, pantothenic acid, nicotinic acid, nicotinamide, folic acid, vitamin C, vita min H, etc. may be mentioned, and their salts (thiamine hydrochloride, sodium ascorbate, etc.) and derivatives (sodium ascorbate-2-phosphate, magnesium ascorbate-2-phosphate sodium salt, etc.) are also included in the water-soluble vitamins that can be used in the present invention. Water-soluble vitamins are obtained by ordinary methods such as the microbial conversion method, the purification method from microbial cultures, the enzymatic method or the chemical synthesis method. It is obtained.

[0156] Any oil-soluble vitamin that can be incorporated into cosmetics is acceptable, for example. Vitamin A, carotene, vitamin D2, vitamin D3, vitamin E (d1-alpha- Examples include copherol, d-alpha-tocopherol, and d-alpha-tocopherol. These are derived from (ascorbyl palmitate, ascorbyl stearate, dipal Ascorbyl mitinate, dl-alpha-tocopherol acetate, dl-alpha-nicotinate Tocopherol vitamin E, DL-pantothenyl alcohol, D-pantothenyl alcohol Oil-soluble vitamins used in this invention also include (such as pantothenyl ethyl ether). Oil-soluble vitamins are obtained by microbial conversion, purification from microbial cultures, enzymes, or chemical processes. It can be obtained by conventional methods such as synthesis.

[0157] Any high molecular weight peptide that can be incorporated into cosmetics is acceptable, for example. Collagen, hydrolyzed collagen, gelatin, elastin, hydrolyzed elastin, keratin Examples include tin. High molecular weight peptides are purified from microbial culture media by enzymatic methods or It can be obtained by purification using conventional methods such as chemical synthesis, or usually from pigs or cattle. It may also be used after being refined from natural materials such as the dermis of silkworms or silk fibers of silkworms.

[0158] Any high molecular weight polysaccharide that can be incorporated into cosmetics is acceptable, but for example, Droxyethylcellulose, xanthan gum, sodium hyaluronate, chondroitin Examples include sulfuric acid or its salts (such as sodium salts). For example, chondroitin sulfate. Alternatively, the salts or other substances can usually be refined from mammals or fish.

[0159] Any sphingolipid that can be incorporated into cosmetics is acceptable, for example. Examples include ceramides, phytosphingosine, and sphingoglycolipids. The quality is usually refined by conventional methods from mammals, fish, shellfish, yeast, or plants. It can be obtained by chemical synthesis or by other means.

[0160] The cosmetic composition of the present invention contains the above essential components, along with other components that are typically incorporated into cosmetics as needed. Other ingredients may be added.

[0161] Other ingredients that may be added include oils and fats, moisturizers, emollients, and surfactants. Properties, organic and inorganic pigments, organic powders, UV absorbers, preservatives, fungicides, antioxidants, plants Extracts, pH adjusters, alcohol, colorants, fragrances, blood circulation promoters, cooling agents, antiperspirants, purified water, etc. Some examples include:

[0162] The oil components include ester-based oils and fats, hydrocarbon-based oils and fats, silicone-based oils and fats, and fluorinated oils and fats. Examples include animal fats and vegetable oils.

[0163] Examples of ester-based oils include glyceryl tri-2-ethylhexanoate and 2-ethylhexanoate. Cetyl, isopropyl myristate, butyl myristate, isopropyl palmitate, Ethyl stearate, octyl palmitate, isocetyl isostearate, stearin Butyl phosphate, ethyl linoleate, isopropyl linoleate, ethyl oleate, myristic acid Isocetyl acid, isostearyl myristate, isostearyl palmitate, myristic acid Octyldodecyl acid, isocetyl isostearate, diethyl sebacate, diadipate Isopropyl, Isoalkyl Neopentanoate, Glyceryl Tri(caprylate / caprate) , Trimethylolpropane Tri(2-ethylhexanoate), Trimethylol Propane Triisostearate, Pentaerythritol Tetra(2-ethylhexanoate), Cetyl Caprylate, Decyl Laurate, Hexyl Laurate, Decyl Myristate, Myristyl Myristate , Cetyl Myristate, Stearyl Stearate, Decyl Oleate, Cetyl Ricinoleate , Isostearyl Laurate, Isotridecyl Myristate, Isocetyl Palmitate, Octyl Stearate , Isocetyl Stearate, Isodecyl Oleate, Octyldodecyl Oleate, Octyldodecyl Linoleate, Isopropyl Isostearate, , Cetostearyl 2-ethylhexanoate, Stearyl 2-ethylhexanoate, Hexyl Isostearate, Ethylene Glycol Dioctanoate, Ethylene Glycol Dioleate, , Propylene Glycol Dicaprate, Propylene Glycol Di(caprylate / caprate), , Propylene Glycol Dicaprylate, Neopentyl Glycol Dicaprate, Neopentyl Glycol Dioctanoate, Glyceryl Tricaprylate, Glyceryl Triundecanoate, , Glyceryl Triisopalmitate, Glyceryl Triisostearate, Octyldodecyl Neopentanoate, Isostearyl Octanoate, Octyl Isononanoate, , Hexyldecyl Neodecanoate, Octyldodecyl Neodecanoate, Isocetyl Isostearate, Isostearyl Isostearate, , Octyldodecyl Isostearate, Polyglyceryl Oleate Esters, Polyglyceryl Isostearate Esters, Tricetyl Citrate, Triisalkyl Citrate, , Triisooctyl Citrate, Lauryl Lactate, Myristyl Lactate, Cetyl Lactate , , Isocetyl Isostearate, Isostearyl Isostearate, , Isostearyl Isostearate, Octyldodecyl Isostearate, Polyglyceryl Oleate Esters, Polyglyceryl Isostearate Esters, Tricetyl Citrate, Triisalkyl Citrate, , Triisooctyl Citrate, Lauryl Lactate, Myristyl Lactate, Cetyl Lactate , Triisopropyl Citrate, Triisooctyl Citrate, Lauryl Lactate, Myristyl Lactate, Cetyl Lactate Tyl, octyldecyl lactate, triethyl citrate, acetyltriethyl citrate, citrate Acetyl tributyl acid, trioctyl citrate, diisostearyl malate, hydroxy 2-ethylhexyl stearate, di-2-ethylhexyl succinate, diisopropyl adipate Chil, diisopropyl sebacate, dioctyl sebacate, cholesteryl stearate, Cholesteryl isostearate, cholesteryl hydroxystearate, cholesteryl oleate Steryl, dihydrocholesteryl oleate, phytosteryl isostearate, ole Phytosteryl stearate, 12-stearoyl hydroxystearate isocetyl, 12-s Stearyl thearoylhydroxystearate, 12-stearoylhydroxystearyl Examples include ester compounds such as isostearyl phosphate.

[0164] Examples of hydrocarbon oils include squalene, liquid paraffin, α-olefin oligomer, and Soparaffin, ceresin, paraffin, liquid isoparaffin, polybutene, microcrystalline Examples include hydrochloric acid wax and hydrocarbon oils such as petrolatum.

[0165] Examples of silicone-based oils include polymethyl silicone, methylphenyl silicone, and methyl Cyclopolysiloxane, octamethylpolysiloxane, decamethylpolysiloxane, dodec Methylcyclosiloxane, dimethylsiloxane / methylcetyloxysiloxane copolymer Body, dimethylsiloxane / methyl stearoxane copolymer, alkyl-modified silico Examples include corn oil and amino-modified silicone oil.

[0166] Examples of fluorinated oils include perfluoropolyethers.

[0167] Examples of animal or vegetable oils include avocado oil, almond oil, olive oil, sesame oil, and rice bran oil. Safflower oil, soybean oil, corn oil, rapeseed oil, apricot kernel oil, palm kernel oil, palm oil, hi Masu oil, sunflower oil, grape seed oil, cottonseed oil, coconut oil, kukui nut oil, wheat germ oil, rice Wheat germ oil, shea butter, evening primrose oil, macadamia nut oil, meadowfoam oil, egg yolk oil, beef tallow Horse oil, mink oil, orange roughy oil, jojoba oil, candelilla wax, carnauba wax Examples include animal or vegetable oils such as camphor oil, liquid lanolin, and hydrogenated castor oil.

[0168] Examples of humectants include water-soluble low molecular weight humectants, lipid-soluble molecular weight humectants, water-soluble polymers, and lipid-soluble polymers. These are some examples.

[0169] Examples of water-soluble low molecular weight humectants include serine, glutamine, sorbitol, mannitol, and pyropropyl alcohol. Sodium lydone carboxylate, glycerin, propylene glycol, 1,3-butylene Glycol, ethylene glycol, polyethylene glycol B (degree of polymerization n=2 or higher), Repropylene glycol (degree of polymerization n=2 or higher), polyglycerin B (degree of polymerization n=2 or higher) Examples include lactic acid and lactate salts.

[0170] Examples of lipid-soluble low-molecular-weight humectants include cholesterol and cholesterol esters. ru.

[0171] Examples of water-soluble polymers include carboxyvinyl polymers, polyaspartates, and tragacan. T, xanthan gum, methylcellulose, hydroxymethylcellulose, hydroxyethyl Cellulose, hydroxypropylcellulose, carboxymethylcellulose, water-soluble cellulose Examples include tin, chitosan, and dextrin.

[0172] Lipid-soluble polymers include polyvinylpyrrolidone eicosene copolymer and polyvinylpyrrolidone. Dong-hexadecene copolymer, nitrocellulose, dextrin fatty acid ester, polymer Examples include silicone.

[0173] Emollients include long-chain acyl glutamate cholesteryl esters and hydroxys Cholesteryl therate, 12-hydroxystearic acid, stearic acid, rosinic acid, Examples include noline fatty acid cholesteryl esters.

[0174] Examples of surfactants include nonionic surfactants, anionic surfactants, and cationic surfactants. Examples include fertilizers and amphoteric surfactants.

[0175] Nonionic surfactants include self-emulsifying glyceryl monostearate and propylene glycol. Licor fatty acid esters, glycerol fatty acid esters, polyglycerol fatty acid esters, Sorbitan fatty acid ester, POE (polyoxyethylene) sorbitan fatty acid ester, POE sorbitol fatty acid ester, POE glycerin fatty acid ester, POE alkyl POE fatty acid ester, POE hydrogenated castor oil, POE castor oil, POE-POP (Polyoxyethylene / Polyoxypropylene) copolymer, POE / POP alkyl A Tel, polyether-modified silicone, lauric acid alkanolamide, alkylamine oxy Examples include sids and hydrogenated soybean phospholipids.

[0176] Examples of anionic surfactants include fatty acid soaps, α-acyl sulfonates, and alkyl sulfons. Alkylallyl sulfonates, alkylnaphthalene sulfonates, alkyl sulfates Salt, POE alkyl ether sulfate, alkylamide sulfate, alkyl phosphate, POE Alkyl phosphates, alkylamide phosphates, alkylylalkyl taurine salts, N-A silamino acid salts, POE alkyl ether carboxylates, alkyl sulfosuccinates, A Sodium hydroxysulfoacetate, acylated hydrolyzed collagen peptide salt, perfluorine Examples include lukyl phosphate esters.

[0177] Examples of cationic surfactants include alkyltrimethylammonium chloride and stearyl chloride. Trimethylammonium, stearyltrimethylammonium bromide, cetostearyl chloride Trimethylammonium, distearyldimethylammonium chloride, stearyldimethylammonium chloride Tylbenzylammonium, behenyltrimethylammonium bromide, benzalconyl chloride Mu, diethylaminoethylamide stearate, dimethylaminopropyl stearate Examples include mido, lanolin derivatives, and quaternary ammonium salts.

[0178] Examples of amphoteric surfactants include carboxybetaine type, amidebetaine type, and sulfobetaine type. hydroxysulfobetaine type, amidesulfobetaine type, phosphobetaine type, amino acid Examples include amphoteric surfactants such as ruvonate type, imidazoline derivative type, and amidoamine type. It is possible.

[0179] Organic and inorganic pigments include silicic acid, anhydrous silicic acid, magnesium silicate, talc, and cerisa. Thread, mica, kaolin, red iron oxide, clay, bentonite, titanium-coated mica, oxysalts Bismuth oxide, zirconium oxide, magnesium oxide, zinc oxide, titanium oxide, aluminum oxide nium, calcium sulfate, barium sulfate, magnesium sulfate, calcium carbonate, magnesium carbonate Nesium, iron oxide, ultramarine, chromium oxide, chromium hydroxide, calamine, and complexes thereof, etc. Inorganic pigments, polyamides, polyesters, polypropylenes, polystyrenes, polyurethanes Vinyl resin, urea resin, phenolic resin, fluororesin, silicon resin, acrylic resin, Lamin resin, epoxy resin, polycarbonate resin, divinylbenzene-styrene copolymer Body, silk powder, cellulose, CI pigment yellow, CI pigment orange Examples include various organic pigments and composite pigments of these inorganic and organic pigments.

[0180] Examples of organic powders include metal soaps such as calcium stearate and sodium zinc cetirate. , alkyl metal phosphates such as zinc laurate and calcium laurate, N-lau N-lauroyl-β-alanine calcium, N-lauroyl-β-alanine zinc, N-lauroyl Acyl amino acid polyvalent metal salts such as calcium lysine, N-lauroyl taurine calcium M, N-palmitoyl-taurine calcium and other amide sulfonic acid polyvalent metal salts, N-I Psilon-lauroyl-L-lysine, N-epsilon-palmitoyllysine, N-α-P Lithoyl ornithine, N-α-lauroyl arginine, N-α-hydrogenated beef tallow fatty acid acylu N-acyl basic amino acids such as ruginine, N-lauroylglycylglycine, etc. Alpha-amino acids such as acyl polypeptides, alpha-aminocaprylic acid, and alpha-aminolauric acid. Fatty acid, polyethylene, polypropylene, nylon, polymethyl methacrylate, polystyrene Examples include ethylene, divinylbenzene-styrene copolymer, and tetrafluoroethylene.

[0181] Examples of UV absorbers include para-aminobenzoic acid, ethyl para-aminobenzoate, and para-aminobenzoic acid. Amyl benzoate, octyl para-aminobenzoate, ethylene glycol salicylate, salicylate Phenyl salicylate, octyl salicylate, benzyl salicylate, butylphenyl salicylate, Homomentyl lysate, benzyl cinnamate, 2-ethoxyethyl paramethoxycinnamate Octyl paramethoxycinnamate, mono-2-ethylhexane diparamethoxycinnamate Lyceryl, Isopropyl paramethoxycinnamate, Diisopropyl diisopropyl cinnamate Acid ester mixture, urocanic acid, ethyl urocanate, hydroxymethoxybenzoate Non, hydroxymethoxybenzophenone sulfonic acid and its salts, dihydroxymethoxy Benzophenone, dihydroxymethoxybenzophenone disulfonate sodium, dihydro Roxybenzophenone, tetrahydroxybenzophenone, 4-tert-butyl-4' -Methoxydibenzoylmethane, 2,4,6-trianilino-p-(carbo-2'-ethylmethane) 1,3,5-Triazine, 2-(2-hydroxy-5-methyl) Examples include benzotriazole (Tylphenyl).

[0182] As disinfectants, hinokitiol, triclosan, and trichlorohydroxydiphenyl A Tel, chlorhexidine gluconate, phenoxyethanol, resorcinol, isopropyl Methylphenol, azulene, salicylic acid, zinc pyrithione, benzalkonium chloride Examples include photosensitizer 301, mononitroguaiacol sodium, and undecylenic acid. ru.

[0183] Antioxidants include butylhydroxyanisole, propyl gallate, and erythorbic acid. These are some examples.

[0184] pH adjusters include citric acid, sodium citrate, malic acid, sodium malate, Fumaric acid, sodium fumarate, succinic acid, sodium succinate, sodium hydroxide, li Examples include sodium monohydrogen phosphate.

[0185] Examples of alcohols include higher alcohols such as cetyl alcohol.

[0186] Furthermore, the ingredients that may be added are not limited to those mentioned above, and also the aforementioned Any of the ingredients can be blended within a range that does not impair the purpose and effects of the present invention, however, the total weight Alternatively, it may be incorporated at a weight percentage of 0.01-5% or 0.01-3%.

[0187] When the dosage form of the present invention is a lotion, paste, cream, or gel, the carrier component is Animal fibers, plant fibers, waxes, paraffin, starch, tracant, cellulose derivatives polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide It can also be used in the following ways.

[0188] If the dosage form of the present invention is a powder or spray, the carrier component may be lactose, talc, Silica, aluminum hydroxide, calcium silicate, or polyamide powder It may also be used, and especially in the case of sprays, chlorofluorohydrocarbons may be added. The propellant may include ropane / butane or dimethyl ether.

[0189] When the dosage form of the present invention is a solution or emulsion, the carrier component may be a solvent, solvating agent, or emulsion. A compounding agent is used, for example, water, ethanol, isopropanol, ethyl carbonate, etc. Tyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-Butyl glycol oil, glycerol aliphatic ester, polyethylene glyco There are fatty acid esters of ru or sorbitan.

[0190] When the dosage form of the present invention is a suspension, the carrier component may be water, ethanol, or propylene glycol. Liquid diluents such as kohl, ethoxylated isostearyl alcohol, polyoxyethylene Suspensioning agents such as sorbitol esters and polyoxyethylene sorbitan esters, and microcrystals. Cellulose, aluminum methhydroxyl, bentonite, agar or tracant It can be used in any way.

[0191] When the dosage form of the present invention contains a surfactant-containing cleanser, an aliphatic alcohol is used as the carrier component. Sulfates, aliphatic alcohol ether sulfates, sulfosuccinate monoesters, isethion Acids, imidazolinium derivatives, methyl taurate, sarcosinate, fatty acid amide ether Sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fat Acid diethanolamide, vegetable oil, linolenic acid derivative, or ethoxylated glycerol fat Acid esters may also be used.

[0192] The terminology used in this invention is intended to be as widely used as possible, taking into consideration the functions of this invention. I have selected commonly used terms, but this does not reflect the intent or judgment of engineers working in this field. For example, it may change due to the emergence of new technologies. Also, in certain cases, the applicant may choose at their discretion. Some terms are used, and in this case, their meanings will be described in detail in the description section of the relevant invention. Therefore, the terms used in this invention are not merely names of terms, but rather the meanings that those terms possess. This should be defined based on the overall content of the present invention.

[0193] Terms such as "first," "second," etc. may be used to describe various components, but the said configuration The elements should not be limited by the terms used herein. The terms refer to one component as another. Used solely for the purpose of distinguishing from constituent elements. For example, without departing from the scope of the rights of the present invention. However, the first component may be named the second component, and similarly the second component may also be named the second component. It may be named as one component. The terms and / or refer to multiple related items. Includes either a combination of items or multiple related listed items.

[0194] In the entirety of the specification of this invention, if a part "includes" a certain component, this means in particular Unless otherwise stated, it does not exclude other components, but rather includes other components. It also means that it is acceptable. The term "about" as used throughout the specification of this invention is "actual". When the meaning of "qualitatively" is mentioned, and the specific manufacturing and material tolerances are presented, A numerical value or a value close to it is used to aid in understanding the present invention, and is accurate and absolute. To prevent unscrupulous infringers from unfairly exploiting disclosed information that mentions specific figures. It will be used.

[0195] Throughout the specification of this invention, the expression in the form of Markouche contains "these combinations The term is selected from a group of components described in the expression in the Marcoush form. It means a mixture or combination of one or more elements, selected from the group consisting of the aforementioned elements. It means including one or more of the character "る". [Examples]

[0196] The following are preferred embodiments to aid in understanding the present invention. However, the following embodiments are This is provided to make the present invention easier to understand, and the present invention is demonstrated by the following examples. The content is not limited.

[0197] Examples

[0198] Example 1. Fabrication of 3D skin organoids and characteristics of organoids fabricated by conventional methods gender analysis

[0199] Example 1-1. Culture of human induced pluripotent stem cells

[0200] Induced pluripotent stem cell lines (iPSCs; CMC3s) derived from healthy subjects are Y-27632(1 V (V) was cultured in Essential 8 (Gibco) culture medium supplemented with 0 μM without supporting cells. Culture on a culture dish coated with itronectin (ThermoFisher). The culture medium was changed daily, and the ReLeSR™ (Stem Cell) culture was performed in a cycle of approximately 5 days. The culture was subcultured using (technology).

[0201] Examples 1-2. Method for differentiating 3D skin organoids using human induced pluripotent stem cell lines.

[0202] A document disclosing a conventional skin organoid differentiation protocol using induced pluripotent stem cell lines ( Lee, J. et al. Hair-bearing human skin gene rated entirely from pluripotent stem cel Based on ls.Nature 582, 399-404 (2020), skin organoids The cells were cultured. Induced pluripotent stem cell colonies were obtained using ReLeSR™ (Stem Cell T). After separating into small cell groups using ecology, Accutase (Ther The cells were isolated into single cells using moFisher. Embryoid bodies y) For formation, 1000 single cells are formed using U-bottom low-attachment Dispensed into each well of an ent 96-well plate (Corning). Embryoid body The culture medium is Essential 8 (Gibc) supplemented with Y-27632 (20 μM). (o) was used. The embryoid bodies were cultured for 2 days until they grew to a size of 300-500 μm. On day 2 of differentiation, the formed embryoid body becomes the non-neural ectoderm. rm) For differentiation induction, 2% Matrigel (Corning), 10 μM SB431542 (Tocris), 4ng / ml FGF2 (PeproTec h) The culture was transferred to a culture medium containing 15 ng / ml of BMP4 PeproTech. Day 6 From cranial neural crest-like cells To induce the growth, the culture medium contains 200 ng / ml of FGF2 and 50 μg / ml of LDN193. 189 (Tocris) was added to the culture medium. Also, on day 14, organoids were placed in 3 ml of skin. 6-well plate containing skin organoid maturation medium (low-attachment Transfer to a 6-well plate (Corning) and use an orbital shaker. Skin organoids were cultured using (65rpm; Thermo Fisher) until the day of analysis. The culture medium was changed every 3 days. Skin organoid maturation medium was mixed with Adva in a 1:1 ratio. nced DMEM / F12 (Gibco) and Neurobasal (Gibco) medium Contains 1X GlutaMax™ (Gibco) and 0.5X B-27 Minus Vitamin B. 1A (Gibco), 0.5X N2 (Gibco), Supplement, 0.1mM 2-Me Lucaptoethanol (Gibco) and 100 μg / ml Normocin (Invi Includes vogen. Skin organoids analyzed by EVOS XL Core bright-field imaging. The image was taken at 40x magnification using a system microscope (Thermo Fisher).

[0203] As a result, as shown in Figure 1, hair follicles were observed on days 60-70. The layered epithelium was It was located in the inner layer of the luganoid, and the dermis and subcutaneous layer were surrounded by it.

[0204] Furthermore, as shown in Figure 2, the skin organoids grew to a diameter of 5 mm or more for the first time in 140 days. These contained several pigments (upper panel of Figure 2) or albino hair follicles (lower panel of Figure 2). Organoids produced using the skin organoid differentiation method (Lee et al. 2020) are round and compacted. It had a collective shape and exhibited an inside-out form.

[0205] Examples 1-3. Fluorescent immunohistochemistry staining

[0206] Skin organoids differentiated using a conventional culture method (Lee et al. 2020) were freeze-cut and then fluorescently stained. We confirmed the expression of skin markers and their hierarchical structure through color. First, the formed skin organoids After fixing with 4% PFA, the material underwent a dehydration process with 30% sucrose solution. Afterward, freeze in a gelatin / sucrose solution and use a Cryostat instrument to determine the exact temperature. Slides were prepared with a thickness of 10-12 μm. Frozen sections were treated with 5% normal goat serum and 0.5% uric acid. Serum albumin, 0.25% fish gelatin, and 0.3% Triton X-1 in PBS The primary antibody was blocked for 1 hour at room temperature with a blocking buffer composed of 00. The primary antibody was blocked overnight at 4°C. The cells were diluted in a buffer and cultured. Next, the sections were washed three times with PBS and then sterilized with a barrier buffer. The cells were cultured with antibody at room temperature for 1 hour. The sections were washed three times with PBS, and the nuclei were 4'6'-dia. Staining was performed using midino-2-phenylindole (DAPI). The section was washed once with PBS and then mounted using a fluorescent mounting medium (DAKO). Conventional method The differentiation markers of cultured skin organoids were identified through fluorescence staining analysis, and the formation and The structure was confirmed by frozen section slide staining. The primary antibodies used are as follows: :Anti-KRT5(1:500;abcam;ab52635), Anti-KRT 10(1:100;Santa Cruz Biotechnology;sc-238 77), Anti-KRT17 (1:100; Santa Cruz Biotechn. ology;sc-393091),Anti-Filaggrin(1:100;Sa nta Cruz Biotechnology;sc-66192), Anti-Vi Mentin (1:100; Santa Cruz Biotechnol) ogy;sc-6260),Anti-COL3A Collagen 3 (1:100;San ta Cruz Biotechnology;sc-27124),Anti-Lor icrin(1:500;abcam;ab85679),Anti-Staphylo coccus aureus(1:100;Santa Cruz Biotechno logy;sc-58038),Anti-PDGFRα(1:250;Cell Si gnaling Technology;3164S),Anti-MelanA(1: 250;abcam;ab51061),Anti-TSLP(1:250;abcam ;ab47943), Anti-Lipid Tox(1:200;Invitroge n;H34477), Anti-Claudin4(1:200;Invitrogen ;32-9400), Anti-KRT15(1:200;Invitrogen MA 5-11344),Anti-p63(1:250;abcam;ab735),Ant i-TFAP(1:50;Santa Cruz Biotechnology;sc- 12726),Anti-COL2A1(1:50;Millipore;ab261) ,Anti-Fibronectin(1:100;abcam;ab6328),An ti-S100α(1:50;Santa Cruz Biotechnology;s c-53438),Anti-E-cadherin(1:250;abcam;ab1 416).

[0207] As shown in Figure 3a, in skin organoids differentiated by the conventional method, epidermal progenitor cells (T) FAP+KRT5+;epidermal progenitor cells) and true Dermal progenitor cells (TFAP+PDGFRα+) The formation and structure of ls) were confirmed.

[0208] As shown in Figure 3b, Sox2 was observed in skin organoid hair follicles cultured for 67 and 87 days. +Expression of dermal papilla cells was confirmed. 67 In skin organoids differentiated on day 1, hyaline cartilage Differentiation was observed in )

[0209] Furthermore, as shown in Figure 3c, S100α+ chondrocytes and Coll2A1 produce cartilage. This was shown.

[0210] Next, the skin organoids cultured using conventional methods were stratified. ) Formation and structural timeline of the epidermal layer The investigation was conducted using the LIDE staining method.

[0211] As shown in Figure 4, skin organoids matured at days 33, 46, 67, 87 and 140. The expression of epidermal proteins was confirmed. The KRT5+ and KRT14+ basal layers were cultured for 33 days. It has been observed to be expressed in nourished skin organoids, and KRT10 is expressed in stratum spinosum dermal cells. Spinous keratinocytes are first observed in the surface epithelium at approximately 46 days. It was discovered. Loricrin and filaggrin were released. The cornified epidermal layer These appeared 67 days after differentiation.

[0212] In other words, conventional methods for producing skin organoids exhibit the following differentiation and expression characteristics. 1) The fabricated organoids have a round aggregate shape and are inside-out (ins It has an ide-out morphology. The layered epithelium is present in the inner layer of the organoid. The dermis and subcutaneous layer surround the outside. 2) The innermost core of the 3D skin organoid A mature keratinized epidermal layer was observed. 3) Differentiation markers for the epidermis and dermis were identified, and hair Frozen section slides showing the formation and structure of skin appendages such as encapsulating cells and mammary gland cells, and hyaline cartilage. This was confirmed through staining. 4) Final maturation of keratinization of the epithelial layer began around day 70.

[0213] Therefore, the inventors have found a way to efficiently manufacture three-dimensionally layered skin organoids and to implement Conventional 3D skin organoid culture for the formation of skin organoids similar to human skin The cultivation method has been modified. This invention relates to the air-liquid interface. Applying the ALI culture method to create 3D air-liquid interface-skin organoids from human pluripotent stem cells. We manufactured a model (ALI-skin organoid). The ALI culture method is widely used in skin tissue culture. A method used to promote the lamination of the epidermis and expose the epidermal layer to air. It closely resembles the process of differentiation of living skin.

[0214] Example 2. Production of ALI skin organoids by activation of the Wnt signaling pathway.

[0215] The inventors of this invention have found that Wnt signaling pathway activation (0.5-8 μM Wnt agonist (C) HIR-99021)) Experiments revealed that the Wnt signaling pathway affects skin organoids. The impact was confirmed.

[0216] First, the skin organoids were prepared based on the methods of Examples 1 and 2, but with some modifications. 6 From day [number], cranial neural crest-like cells To induce ) the culture medium, add 200 ng / ml of FGF2 and 50 μg / ml of LDN1 93189 (Tocris) was added. Also, to activate the Wnt signaling pathway... A 0.5-8 μM Wnt agonist (CHIR-99021) was added to the culture medium on day 6. (See Figure 5). On day 14 of culture, the organoids were placed in 3 ml of skin organoid maturation medium. 6-well plate (low-attachment 6-well plate) Move to Corning, Orbital shaker (65rpm; Thermo Skin organoids were cultured in a Fisher culture medium until the day of analysis. The culture medium was changed every three days. The skin organoids to be analyzed were examined using an EVOS XL Core bright-field microscope (T The image was taken with a Hermo Fisher at 40x magnification.

[0217] As shown in Figures 6 and 7, on day 6 of differentiation, 0.5-8 μM Wnt agoni was added to the culture medium. As a result of adding CHIR-99021, which is st, the skin oil produced by the conventional method was The size of organoids was significantly increased compared to ganoids.

[0218] Furthermore, as shown in Figure 7, CHIR-99021 was not treated using the conventional culture method. In the (CHIR(-)) group, cartilage differentiation occurred in skin organoids at days 38, 65, and 85. (The red circle indicates cartilage.) However, by treating with CHIR-99021, Skin organoids with activated Wnt become larger in size and develop spheres without cartilage formation. Upon confirming that it differentiates into cysts, we found that Wnt activation is unnecessary in skin organoids. This demonstrates that it significantly inhibits the formation of cartilage, a vital component of the cartilage.

[0219] Example 3. Cartilage-related genetics of ALI skin organoids induced by activation of the Wnt signaling pathway. Confirmation of sub-marker expression

[0220] One method for evaluating gene expression is lysis buffer. Cells are lysed using () to extract genes, and gene expression is determined using RT-PCR. Quantification was performed. To evaluate gene expression, total RNA was collected using the RNeasy kit (Qiage). (n, catalog number 74106), and the cDNA was reverse transcr iptase III (Thermo Fisher Scientific, catalog) Prepared according to the manufacturer's guidelines using part number 4368814. Real-time PCR is SRBR Green Master Mix (Thermo Fishe The detection was performed using r Scientific (catalog number A25776), and St ep One Plus Real-time PCR System (Applied Bi (osystems) was used. Gene expression was controlled by GAPDH (glyceral pathway). The expression of de-3-phosphate dehydrogenase was normalized. The degree of gene expression is specific to COL2A, ACAN, and SOX9 mRNA. The primers were evaluated using a primer. The specific sequences of the primers are shown in Table 1 below.

[0221] [Table 1]

[0222] As shown in Figure 8, skin organoids treated with CHIR-99021 (CHIR(+)) ) showed a higher cartilage expression marker compared to the group not treated with CHIR-99021 (CHIR(-)). - The expression levels of the COL2A, ACAN, and SOX9 genes are significantly reduced. I confirmed that.

[0223] Example 4. Human induced pluripotent stem cells derived from the air-liquid interface (Air-liquid interface) Preparation and characterization of skin organoids using the rface (ALI) culture method.

[0224] Example 4-1. Air-liquid interface (ALI) culture Creation of skin organoids using nourishing methods

[0225] After culturing the skin organoids from Example 2 for approximately 40-100 days, the organoids were divided into four equal parts. One size Dumount #3 forceps (Fine Science To ols) and Spring Scissors (Fine Science Tools) Transwell cultures were cut using a tool and coated with collagen (Corning). Transwell culture insert (pore size 0.4μ) m, 12-well plate; Corning) On the dermis, the collagen side is on the dermis side, and the air side is on the epidermis side. The ALI skin organoids were cultured spread out so as to be exposed to the environment. At the air-liquid interface, for another 3-4 weeks, 100 After culturing in a % humidity incubator, dry conditions are used for 2-6 days to allow the lamellar epithelial layer to mature. condition;0% humidity condition incubat The cultures were performed using (or). The method for producing ALI-skin organoids is shown in Figure 9.

[0226] Example 4-2. Air-liquid interface (ALI) Characterization of skin organoids using culture methods: Confirmation of histological structure

[0227] To confirm the histological structure of skin organoids, H&E staining and Oil Red O2 staining were performed. Taining was performed. The H&E staining method was as follows: Organoids were stained with 4% paraform After fixing with marine, the material was dehydrated with alcohol and then cleared with xylene. Afterward, it was embedded in paraffin. Frozen sections were made to a thickness of 12 μm to prepare slides, and Hem After staining with atoxylin and Eosin, the images were scanned under an optical microscope (Olympus Ix70). I confirmed it.

[0228] The method for staining with Oil Red O is as follows: Organoids are treated with 4% parahol. After fixation with Marine, freeze sections were made to a thickness of 12 μm and slides were prepared. The solution is Oil O Red staining solution (oil o red 0.5g(Sigma Aldrich)in 100ml isopropa Stained with (nol), then stained with Hematoxylin, and then examined under an optical microscope (Olympus I Confirmed on x70.

[0229] As shown in Figure 10a, the ALI-skin organoid of the present invention is cultured by conventional methods. Compared to other skin organoids, it not only showed a morphology that was more similar to actual skin, but also had hair. We observed a significant improvement in the growth of the sac.

[0230] Furthermore, as shown in Figure 10b, the ALI-skin organoid of the present invention is histologically continuous. It develops into an epithelial structure, and the surface has a normal basket weave pattern. It forms a mature lamellar epithelial layer having a weave pattern, but conventional Whole skin organoids are layers of epithelium. However, the layers are located inside the organoid, and the dermis and subcutaneous layers are surrounded on the outside, It exhibited an inside-out morphology different from that of skin tissue. Therefore, Furthermore, the ALI-skin organoid of the present invention can be used as a skin model representing actual skin tissue. We confirmed that it was suitable.

[0231] Furthermore, as shown in Figure 11, ALI-skin organoids produced by the ALI culture method of the present invention It was confirmed that the subcutaneous fat layer differentiated below the dermis layer, similar to actual skin. Furthermore, it was observed that sebaceous glands, which develop around hair follicles, also differentiate.

[0232] Furthermore, as shown in Figure 12, ALI-skin organoids produced by the ALI culture method of the present invention It was found that a well-developed human hair follicle structure is formed. Hair follicle bulge stem cells (hair (Follicle bulge stem cells) Also clearly shows sebaceous glands, hair bulbs, and hair shafts. It was observed that this definitely manifests as hair follicles that resemble human skin structure. Furthermore, it shows hair follicles with significantly improved morphology compared to conventional organoids. To instigate.

[0233] Furthermore, as shown in Figure 13a, for the maturation of the skin keratinocytes of the present invention, on day 0, day 2, and day 4, Alternatively, ALI-skin organoids cultured under dry conditions for 6 days exhibit a shape similar to skin tissue. He showed a certain attitude.

[0234] Furthermore, as shown in Figure 13b, the H&E staining results were obtained for 4 and 6 days under the final drying conditions. In the ALI-skin organoids, mature keratinocytes differentiate (red stained areas). This was observed.

[0235] Example 4-3. Air-liquid interface (ALI) culture Characterization of skin organoids using nourishing methods: Fluorescent immunohistochemistry

[0236] Using the fluorescence immunohistochemistry staining methods of Examples 1-3, the ALI-skin organoids of the present invention were stained. We confirmed the expression of various skin-related factors.

[0237] As shown in Figure 14, the ALI-skin organoid of the present invention has mature epidermis and mature skin KRT10, a marker for the spinous stratum corneum, is expressed, and loricr, a marker for the stratum corneum of the epidermis, is expressed. Differentiation between in and filaggrin was also observed.

[0238] Furthermore, as shown in Figure 15, the ALI-skin organoid of the present invention has elongated hair follicles in the dermis. Papillary and melanocyte expression was observed, and KRT15+ hair follicle bulge stem cells were also found to be present. Confirmed. Also, ALI-skin organoids are hair shaft outer sheath It was observed that (s of hair shafts) was expressed. This is the A of the present invention. LI-Skin organoids exhibit improved hair follicle growth and expression reminiscent of human skin structure. This suggests that...

[0239] Furthermore, as shown in Figure 16, the ALI-skin organoid of the present invention has horns of mature epidermis The differentiation of the laminar markers loricrin and filaggrin was observed under dry conditions for 4 days or We observed that it began to appear in ALI-skin organoids cultured on day 6.

[0240] Furthermore, as shown in Figure 17, ALI cultured under dry conditions for 6 days in the final stage of the present invention - In skin organoids, collagen 3 (COL3) and vimentin are dermal layer markers. We confirmed that (VMT) had differentiated.

[0241] Furthermore, as shown in Figure 18, in the final 6-day stage of the present invention, ALI- cultured under dry conditions In skin organoids, the expression of subcutaneous tissue and sebaceous gland adipocytes can be confirmed with Lipidtox. Ta.

[0242] Example 5. Modeling of atopic dermatitis caused by Staphylococcus aureus infection.

[0243] Example 5-1. Modeling of atopic dermatitis caused by Staphylococcus aureus infection: Histological structure Confirmation

[0244] Staphylococcus aureus, a common cause of skin infections, is frequently found on the skin of atopic dermatitis patients. However, it is not found in the skin of healthy people. To model atopic dermatitis, yellow When inoculating with Staphylococcus, confirm whether it penetrates the epidermal layer of skin organoids, ALI-Skin organoid Colonization of Staphylococcus epidermidis using Luganoids We investigated this matter.

[0245] Based on Example 4, ALI skin organoids cultured for 3-4 weeks were subjected to Staphylococcus aureus (S) Infection was carried out using the .aureus strain ATCC 29213. The suspension was divided into 100 × g After centrifuging for 10 minutes, the absorbance of the suspension was calculated at 600 nm to obtain 1 × 10¹⁰ CFU / Phosphate-buffered saline at concentrations of ml, 1 × 10⁹ CFU / ml, and 1 × 10⁸ CFU / ml. The suspension was then resuspended. Next, 1 μl of the bacterial suspension was placed on an ALI skin organoid at 37°C and 5% C. Inoculation was performed under O2 conditions for 24 hours. Subsequently, the yellow blot was obtained by the immunofluorescence staining method of Examples 1-3. Plasmodium falciparum, basal epithelial layer marker KRT5, cell proliferation marker Ki67, stratum corneum marker f Ilaggrin and loricrin, basal epithelial layer marker KRT14, spinous layer marker KRT10 Furthermore, KRT15 and p63, which are markers for skin epidermal stem cells, were identified. In addition, cell death was observed. This was confirmed through TUNEL analysis.

[0246] As shown in Figure 19, the epidermis and stratum corneum of ALI-skin organoids inoculated with Staphylococcus aureus Immunostaining analysis confirmed the detection of Staphylococcus aureus in both the skin and the skin. Staphylococcus aureus is significantly present from the epidermis layer along the hair follicles to the dermis layer of ALI-skin organoids. Once infected, Staphylococcus aureus causes colonization. I confirmed it.

[0247] Furthermore, as shown in Figure 20, true ALI-skin organoids inoculated with Staphylococcus aureus Increased cell death levels were observed in the cortex, and a significant decrease in KRT5 expression levels was observed. Structural damage to the epidermal and dermal layers of the inoculated ALI-cutaneous organoid was observed. It was done.

[0248] Furthermore, as shown in Figure 21, the table of ALI-skin organoids inoculated with Staphylococcus aureus When the expression levels of filaggrin and loricrin in the stratum corneum are significantly reduced, the skin barrier Damage was observed.

[0249] Furthermore, as shown in Figure 22, ALI-skin organoids inoculated with Staphylococcus aureus TSLP expression in totopic dermatitis keratinocytes is significantly increased, and basal epithelial layer markers are When we confirmed that a certain KRT14 level significantly decreased, the symptoms of atopic dermatitis improved. It was confirmed that it would be triggered.

[0250] Furthermore, as shown in Figure 23, ALI-skin organoids inoculated with Staphylococcus aureus... The expression levels of the skin epidermal stem cell markers KRT15 and p63 were significantly reduced. I confirmed it.

[0251] Furthermore, as shown in Figure 24, ALI-skin organoids inoculated with Staphylococcus aureus... Claudine 4 (CLDN4) and KRT5, which are skin barrier assembly protein markers, were significant. We confirmed that it decreased to [a certain level].

[0252] Example 5-2. Modeling of atopic dermatitis caused by Staphylococcus aureus infection: RNA-se Analysis of changes in gene expression using Quencing

[0253] ALI-skin treated with 1 × 10⁶ CFU of Staphylococcus aureus by the method of Example 5-1 Skin organoids (n=3) and untreated ALI skin organoids (control group; n=3) To compare and evaluate gene expression between these two periods, RNA sequencing analysis was used. First, genes were extracted using the gene extraction method of Example 3. Gene quality check, Reverse transcription and sequencing encing) is Illu of Macrogen Inc. (Seoul, Korea) The sequencing was performed using the mina sequencing platform and protocol. The expressed gene is defined as having a >2x change in expression and an adjusted P-value < 0.05. Next, functional notes were recorded using Gene Ontology and the KEGG database. Analysis and gene set enrichment analysis were performed. Of the 46,427 genes identified, 1 At least one zero fragment per kilobase of 00 million mapped read transcripts 26,458 genes with discrepancies were excluded, and the genes expressed differentially were selected. Ally expressed genes (DEGs) must be processed for analysis. It left behind 19,969 genes that were not preserved.

[0254] As shown in Figures 25a and 25b, Staphylococcus aureus is applied to the surface of ALI-skin organoids. Changes in gene expression were compared 18 hours after inoculation using RNA-sequencing analysis. The study analyzed ALI-skin organoids treated for Staphylococcus aureus and untreated control groups. The biological replicas between the two showed clear differences in the expression of numerous genes, and 4,255 Of the differentially expressed genes (DEGs), 2,162 genes were highly expressed, and 2, 093 genes were expressed at a low level after inoculation with Staphylococcus aureus.

[0255] As shown in Figure 25c, in ALI-skin organoids treated with Staphylococcus aureus, Compared to the light group, FLG (filaggrin), LCE (late keratinized film), Loricrin ( We discovered a significant decrease in the expression of genes related to skin barrier function, such as loricrin. did.

[0256] Furthermore, as shown in Figure 25d, the RNA-sequencing analysis revealed that Staphylococcus aureus In ALI-treated skin organoids, KRT and KRTAP levels were higher compared to the control group. Which keratinocyte differentiation We confirmed that the expression of genes related to ) was remarkably low.

[0257] Furthermore, the inventors have identified the mechanisms for bacterial recognition, antimicrobial peptide (AMP) production, and inflammatory signaling. We clarified the host cell's response to bacterial infection, including [specific bacteria]. As shown in Figure 25e, RNA sequencing analysis revealed that Staphylococcus aureus was treated with ALI-skin. Skin organoids showed higher levels of Defb (Defensin Beta) and Pglyrp2 compared to the control group. Peptidoglycan-recognizing proteins 2), TLR1 and TLR2 (Tall-like receptors 1 and 2) ), Lgals9 (Galectin 9), Lcn2 (Lipocalin 2) and S100A (S10 Major antimicrobial peptides such as calcium-binding proteins (Antimicrobial We confirmed an increase in the expression of genes related to peptides (AMPs).

[0258] Furthermore, as shown in Figure 25f, CXCL (CXC motif chemokine ligand), CCL (CC motif chemokine ligand), TSLP, IL-1 (interleukin- 1) Inflammatory cytokines such as TNF (tumor necrosis factor) The expression of genes related to inflammatory cytokines has increased significantly. I confirmed that it would be added.

[0259] Example 6. Cutibacterial treatment of ALI skin organoids infected with Staphylococcus aureus Confirmation of the therapeutic effect of Cutibacterium acnes

[0260] The results shown in Example 5 showed that ALI skin organoids infected with Staphylococcus aureus were We confirmed that it accurately mimicked the characteristics of totopic dermatitis. Therefore, in Example 6, Health The collaborative culture of symbiotic microorganisms found in the skin of certain individuals is used to improve the expression of atopic dermatitis. We investigated whether the type could be prevented or reduced. Cutibacterium acnes develops from the skin of healthy people. It is one of the most common symbiotic microorganisms observed. Microbiome therapy using [unclear] is effective in preventing atopic dermatitis and improving skin barrier function. We investigated the therapeutic effects of this treatment.

[0261] ALI skin organoids cultured for 3-4 weeks based on Example 4 were cultured under dry conditions for 4 days. Afterward, based on Example 5, Cutibacterium acnes was administered at 1 × 10⁹ CFU / ml. The bacterial suspension was then suspended in phosphate-buffered saline at the specified concentration. Next, the bacterial suspension was placed on the ALI skin organoid. 1 μl was inoculated at 37°C under 5% CO2 conditions for 48 hours. After pretreatment of Acnes, yellow buds were used according to Example 5. C. urticaria was suspended in phosphate-buffered saline at a concentration of 1 × 10⁹ CFU / ml. Staphylococcus aureus suspension on pre-treated ALI skin organoids with Cterium acnes. 1 μl was inoculated at 37°C under 5% CO2 conditions for 18 hours, and then cultured under dry conditions. Untreated group (0 (Control group), Staphylococcus aureus (SA) 1 × 10⁶ alone, Cutibacterium acnes (CA) 1 × 10⁶ CFU + SA 1 × 10⁶ CFU, or CA 1 × 10⁷ CFU + SA Organoids were inoculated with 1 × 10⁶ CFU. ALI skin organoid culture method and The method of inoculating with Tibacterium acnes is shown in Figure 26a.

[0262] After vaccination, ALI cutaneous organs infected with Cutibacterium acnes by Staphylococcus aureus To confirm whether Noid has an effect of protecting the skin barrier, the skin barrier function is... Immunostaining was performed on filaggrin, an important protein. The immunofluorescence staining method of Examples 1-3 revealed the epithelial layer marker KRT5 (CK5) and We analyzed and identified filaggrin, an important protein in the skin barrier. Ta.

[0263] As shown in Figure 26b, ALI skin organs treated with only 1 × 10⁶ Staphylococcus aureus In the id, filaggrin expression was significantly reduced. On the other hand, 1 × 10⁶ CFU cutiva ALI skin organoids pre-treated with Cterium acnes showed only Staphylococcus aureus. The expression level of filaggrin was significantly increased compared to the treated samples. These results suggest that... - Tibacterium acnes rescues the skin barrier from damage caused by Staphylococcus aureus infection, This demonstrates the provision of a protective mechanism against Staphylococcus aureus colonies. Therefore, Cutibacterium acnes is one of the symbiotic microorganisms found on the skin of healthy people. S has a significant preventive and therapeutic effect against Staphylococcus aureus-mediated disruption of the skin barrier and cell damage. By demonstrating this, it is expected to be used as a treatment for atopic dermatitis. .

[0264] In summary, the skin organs generated by the activation of the Wnt signaling pathway of the present invention Compared to skin organoids produced by conventional methods, the size of the organoids is different. This increased significantly and suppressed the formation of cartilage, which is an unnecessary component in skin organoids. Furthermore, the ALI-skin organoid of the present invention is a skin organoid produced by conventional culture methods. Compared to ganoids, the skin cells, appendages, nerve cells, and fat cells are more similar to those of actual skin. We observed that the structure formed significantly improved hair follicle growth. Furthermore, the present invention relates to atopic dermatitis The organoid model of skin dermatitis shows increased cytokines derived from damaged skin barriers and epithelial cells. Atopic dermatitis can manifest as decreased epidermal stem cell expression and the formation of colonies of Staphylococcus aureus. When the characteristics of dermatitis were accurately reproduced, it became a screening model for atopic dermatitis treatment agents. It is expected to be useful in this way.

[0265] Furthermore, using the atopic dermatitis model of the present invention, the human symbiotic bacterium Cutiva Cutibacterium acnes is infected with Staphylococcus aureus. It rescues the skin barrier from destruction and protects the protective mechanism against Staphylococcus aureus colonies. By providing it, we confirmed that it can be usefully used as a microbiome therapeutic agent. Based on the above results, Cutibacterium acnes can be used for the treatment of atopic dermatitis. It is expected to be usable.

[0266] The above description of the present invention is illustrative and based on common knowledge of the art to which the present invention pertains. Those who possess the technical concept or essential features of the present invention may, without changing them, modify them in other specific forms. It can be understood that it can be easily transformed into this state. Therefore, the implementation described above Examples must be understood to be illustrative in every respect and not limiting. do not have. [Industrial applicability]

[0267] The inventors have found that by treating the organoid with a Wnt agonist on day 6, the size of the organoid can be reduced. Not only does it increase significantly, but the formation of cartilage, which is an unnecessary component of skin organoids, is suppressed. It was confirmed that it is controlled. The ALI-skin organoid of the present invention is manufactured by conventional culture methods. Compared to actual skin organoids, skin cells, appendages, nerve cells, and fat cells are more prominent in real skin. It was observed that it was formed with a structure similar to that of actual skin, and that hair follicle growth was greatly improved. We confirmed that organoids are formed with a similar structure. In addition, atopic skin As a result of treating Staphylococcus aureus to create a model of fire, damaged skin barrier and epithelium were observed. Increased cell-derived cytokines, decreased expression of skin barrier function-related genes, keratinocyte differentiation-related Decreased gene expression, antimicrobial peptides Increased gene expression, increased cytokine gene expression derived from epidermal and dermal cells, and epidermal stem cells Atopic dermatitis is characterized by decreased cyst expression and the occurrence of Staphylococcus aureus colonies. When copied, the skin organoid of the present invention was used as an actual skin model and atopic skin It can be useful as a flame model and has potential for industrial applications.

[0268] On the other hand, the atopic dermatitis model of the present invention is useful as a microbiome therapeutic agent. Cutibacterium acnes, a bacterium that coexists with humans. M acnes) rescues the skin barrier from destruction caused by Staphylococcus aureus infection, Staphylococcus aureus It was confirmed that it provides a protective mechanism against fungal colonies. Therefore, cutie Bacterium acnes is significant in disrupting the skin barrier and causing cell damage mediated by Staphylococcus aureus. It can be usefully used in the treatment of atopic dermatitis through its preventive and therapeutic effects, and is industrially usable. It has a sex.

Claims

1. Use for manufacturing pharmaceutical compositions for the prevention or treatment of atopic dermatitis, comprising Cutibacterium acnes or its culture medium as an active ingredient.

2. The use according to claim 1, wherein the pharmaceutical composition increases filaggrin expression.

3. Use for manufacturing a food composition for the prevention or improvement of atopic dermatitis, comprising Cuticulobacterium acnes or its culture solution as an active ingredient.

4. Use for manufacturing cosmetic compositions for the prevention or improvement of atopic dermatitis, comprising Cuticulobacterium acnes or its culture solution as an active ingredient.

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