Novel method for preparing skin organoid

Abstract

The present invention relates to a method for preparing a skin organoid. In addition, the present invention relates to: a skin organoid prepared by the preparation method; a preparation for preventing or treating skin diseases, the preparation comprising the skin organoid; a method for evaluating the efficacy of a substance for preventing or treating skin diseases using the skin organoid; and a method for screening such a substance. The skin organoid prepared according to the present invention has a high degree of similarity with human skin tissue, may include hair follicles, and can regenerate damaged skin tissue when implanted into a living body. Therefore, the method for preparing a skin organoid according to the present invention can be used to prepare a skin organoid having a similar morphology and function to living skin tissue, and the prepared skin organoid can be used as a regenerative therapeutic agent for skin diseases, or can be advantageously used in a method for evaluating the efficacy of or screening a therapeutic agent for skin diseases.

Description

A novel method for manufacturing skin organoids

[0001] The present invention relates to a novel method for producing skin organoids. Furthermore, the present invention relates to skin organoids produced by the method, organoid preparations capable of regenerating damaged skin using the skin organoids, and methods for evaluating and screening the efficacy of skin disease treatment substances using the skin organoids.

[0002] Skin diseases caused by skin damage or aging have been treated with methods such as chemical peels, laser irradiation, or medications, but these methods have been associated with drawbacks such as pain, scarring, and side effects. Recently, skin organoids have emerged as a novel strategy for treating skin diseases that can overcome these drawbacks, and numerous studies are currently underway. Organoids are collections of cells differentiated from specific stem cells. They possess structural and functional properties that closely resemble actual tissues in the body, making them ideal for regenerating damaged tissue.

[0003] In addition, organoids can mimic the structure and function of specific tissues, so they can be used to evaluate or screen the efficacy of therapeutics and the like, replacing cell and animal testing, and are therefore highly anticipated in the industry.

[0004] However, because the skin is composed of a multilayer structure, there are many limitations in manufacturing it into organoids compared to other tissues.

[0005] Accordingly, the inventors of the present invention have conducted various studies to develop skin organoids with a multilayer structure that can mimic the structural and functional aspects of skin. As a result, a method for producing skin organoids using a concentration gradient of specific culture components was developed. Skin organoids produced by this method are structurally and functionally similar to actual skin tissue and can include hair follicles. Skin organoids produced in this manner can regenerate damaged skin tissue when transplanted in vivo, and their usefulness in evaluating or screening therapeutic agents for skin diseases has been experimentally demonstrated, leading to the completion of the present invention.

[0006] Each description and embodiment disclosed in this invention can also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this invention fall within the scope of this invention. Furthermore, the scope of this invention is not limited by the specific descriptions described below.

[0007] Additionally, terms not specifically defined herein should be understood to have the meanings commonly used in the technical field to which the present invention pertains. Furthermore, unless specifically defined in context, the singular includes the plural, and vice versa.

[0008]

[0009] One aspect of the present invention provides a method for producing a skin organoid, comprising the following steps:

[0010] a) A step of culturing pluripotent stem cells in the first medium and differentiating them into ectoderm; and

[0011] b) A step of culturing the product of step a) in a second medium to differentiate it into cranial neural crest stem cells.

[0012] Alternatively, the manufacturing method may also be represented as comprising the following steps:

[0013] a) A step of culturing pluripotent stem cells in a first medium to differentiate them into surface ectoderm; and

[0014] b) A step of culturing the product of step a) in a second medium to differentiate it into cranial neural crest cells.

[0015] As used herein, the term "organoid" refers to a mass of cells with a three-dimensional structure. Organoids are defined as miniature, simplified versions of organs, manufactured through artificial culture processes rather than being collected, acquired, or harvested from animals or other sources.

[0016] Step a) above is a step of culturing pluripotent stem cells in a first medium. Here, surface ectoderm can be induced to differentiate.

[0017] The term "stem cell" as used herein refers to a cell that has pluripotency, which allows differentiation into cells derived from endoderm, mesoderm, and ectoderm, or multipotency, which allows differentiation into cells closely related to tissues or functions. The ectoderm can be differentiated into epidermis, glandular tissue, neuroepithelium, or the nervous system, and in the skin organoid of the present invention, can be differentiated into the epidermal layer.

[0018] As used herein, the term "differentiation" refers to the process by which cells become specialized to acquire a form and function appropriate for their respective roles during cell division and proliferation. For example, this may be the process by which pluripotent stem cells transform into ectoderm, or the process by which ectoderm transforms into the epidermal layer.

[0019] The term "pluripotent stem cell" as used herein refers to a cell that has the ability to differentiate into all cells constituting an organism and the ability to self-renew, and can be used interchangeably with the terms "pluripotent stem cell" or "pluripotent stem cell". The pluripotent stem cell may be, for example, an embryonic stem cell, an embryonic germ cell, or an induced pluripotent stem cell, but is not limited thereto. Preferably, the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell. The term "induced pluripotent stem cell" may be used interchangeably with the terms "induced pluripotent stem cell" or "artificial pluripotent stem cell".

[0020] In the present invention, step b) is a step of culturing the material obtained in step a) in a second medium. Here, cranial neural crest stem cells can be induced to differentiate.

[0021] As used herein, the term "cranial neural crest cell" refers to a cell that forms the dermis layer after differentiating into mesenchymal cells and neuro-glial cells.

[0022] As used herein, the term "dermis" refers to the thick layer of cells beneath the epidermis, comprising approximately 90% of the skin. The dermis is known to play a role in maintaining skin elasticity and radiance, supplying nutrients to the epidermis, and maintaining and protecting other skin tissues.

[0023] As used herein, the term "subcutaneous fat layer" refers to the tissue located beneath the skin and may be used interchangeably with the term "subcutaneous tissue layer." The adipocytes in the subcutaneous fat layer are known to produce subcutaneous fat, which plays a role in storing nutrients, regulating body temperature and moisture, maintaining skin elasticity, and cushioning against external impact.

[0024] The first and second media may contain BMP (Bone morphogenic protein). The BMP may be, for example, BMP2 protein, BMP3 protein, BMP4 protein, BMP6 protein, BMP7 protein, BMP8 protein, BMP9 protein, BMP10 protein, BMP11 protein, or BMP15 protein, but is not limited thereto.

[0025] Organoids have the characteristic that the manufacturing results significantly vary depending on the culture conditions. For example, culture conditions such as culture date, culture temperature, culture medium, and culture substrate are important factors that determine the characteristics of organoids. Among them, culture medium conditions play the greatest role in growing organoids with characteristics similar to the desired tissue or organ by regulating various signaling pathways in stem cells. Since organoids cultured under different medium conditions exhibit very different characteristics in terms of similarity to actual tissue or viability, the present invention has established optimal culture medium conditions that can obtain a sufficient number of organoids that are very similar in shape and function to actual tissue and can be utilized for screening therapeutic substances or therapeutic agents.

[0026] Specifically, in the method for producing skin organoids according to the present invention, the concentration of BMP included in the second medium is lower than the concentration of BMP included in the first medium. For example, the concentration of BMP in the first medium may be 10 ng / mL or more, 15 ng / mL or more, 20 ng / mL or more, or 25 ng / mL or more, and the concentration of BMP in the second medium may be 10 ng / mL or less, 5 ng / mL or less, 2.5 ng / mL or less, or 1 ng / mL or less.

[0027] In the production of skin organoids, when the BMP concentration is reduced in step b) compared to step a) to form a downward concentration gradient, various advantages are provided in the induction of cranial neural crest cells, melanocytes, and hair follicles, compared to when the BMP concentrations in steps a) and b) are the same or similar. As a result, the skin organoids produced according to the production method of the present invention can structurally and functionally mimic actual skin, and for example, can mimic a multilayer structure or include hair follicles.

[0028] The term "melanocyte" as used herein refers to a cell that exists in the skin epidermis, dermis, or mucosal epithelium, and is derived from cranial neural crest cells, and is known to be involved in determining the color of skin or hair.

[0029] The term "hair follicle" as used herein refers to a sac that surrounds a hair and is formed across the epidermis and dermis, and is known to play a role in protecting the hair root, which is a hair root within the skin, and supplying nutrients to the hair root to help the hair grow.

[0030] Additionally, the first and second media may further comprise a transforming growth factor β (TGFβ) inhibitor. The TGFβ inhibitor may be, but is not limited to, A83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-364947, and SJN-2511, for example.

[0031] Additionally, the skin organoid may comprise or be composed of a multilayer structure including an epidermal layer, a dermal layer, and a subcutaneous tissue layer. Additionally, the skin organoid may comprise a hair follicle.

[0032] The manufacturing method according to the present invention may additionally include a step c) of seeding pluripotent stem cells in a third medium prior to step a).

[0033] The manufacturing method according to the present invention may further include a step d) of inducing caudalization of the material obtained in step b) using a fourth medium. The fourth medium may include a BMP inhibitor and / or a mitogenic growth factor. The BMP inhibitor may be, for example, Dorsomorphin, LDN-193189, Noggin, DMH1, CTGF (Connective tissue growth factor), Follistatin, Sclerostin, DAN, Chordin, Gremlin, Cerberus 1, or Inhibin, but is not limited thereto. The above-mentioned mitogenic growth factor is a protein that activates a signaling pathway related to MAPK (Mitogen-activated protein kinase), and may be, for example, HGF (Hepatocyte Growth Factor), IGF (insulin-like growth factor), EGF (Epidermal growth factor), FGF (Fibroblast growth factor), KGF (Keratinocyte growth factor), PDGF (Platelet-derived growth factor), VEGF (Vascular endothelial growth factor), NRG (Neuregulin), and BDNF (Brain-derived neurotrophic factor), but is not limited thereto.

[0034] Optionally, the manufacturing method according to the present invention may further include a step e) of inducing self-assembly of the epidermis of the product obtained in step d) using a fifth medium.

[0035] Optionally, the manufacturing method according to the present invention may further include a step f) of maturing the product obtained in step e) using a sixth medium to produce a skin organoid.

[0036] The first to sixth media may include Matrigel, antibiotics, antioxidants, fetal bovine serum (FBS), GlutaMAX, N2, 2-mercaptoethanol (β-Mercaptoethanol), and / or B27, which are commonly included in cell or organoid culture media in the art. The antibiotics may be, for example, penicillin, streptomycin, gentamicin, primocine, or normocin, but are not limited thereto. The antioxidant may be, but is not limited to, nicotinamide, 1-thioglycerol, valproic acid, or N-acetyl-L-cysteine, for example. The basal medium that may be used herein may be a medium for animal cells commonly used in the art, and may be, but is not limited to, RPMI1640, F-10, F-12, DMEM (Dulbecco's Modified Eagle's Medium), A-DMEM, advanced DMEM / F12, MEM (Minimal essential Medium), BME (Basal Medium Eagle), αMEM (α Minimal essential Medium), GMEM (Glasgow's Minimal essential Medium), IMEM (Iscove's Modified Dulbecco's Medium), McCoy's 5A, Neurobasal medium, or a mixture thereof.

[0037]

[0038] Another aspect of the present invention provides a skin organoid manufactured by the above manufacturing method.

[0039] The above skin organoids can be used for the prevention or treatment of skin diseases. Furthermore, the skin organoids can be used for methods for evaluating or screening the efficacy of agents for the prevention or treatment of skin diseases.

[0040] In the skin organoid according to the present invention, each term has the same meaning as described in the method for producing the skin organoid, unless specifically mentioned.

[0041]

[0042] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating a skin disease, comprising a skin organoid manufactured by the above manufacturing method.

[0043] In a pharmaceutical composition for preventing or treating a skin disease comprising a skin organoid according to the present invention, each term has the same meaning as described in the method for producing the skin organoid, unless otherwise specified.

[0044] The skin organoids of the present invention possess a histological structure and function very similar to human skin. Therefore, when transplanted onto skin, they can regenerate damaged skin, making them suitable for use as preventative or therapeutic agents for skin diseases or conditions.

[0045] The pharmaceutical composition according to the present invention may be a cell therapy agent or a regenerative therapy agent.

[0046] As used herein, the term "cell therapy" refers to a pharmaceutical product manufactured using living autologous, allogenic, or xenogenic cells for the purpose of prevention, treatment, or diagnosis. The cell therapy product may be used to regenerate damaged or defective cells or tissues, and may be used to restore the function and morphology of damaged or defective cells or tissues.

[0047] The pharmaceutical composition according to the present invention can be administered to a subject in need of prevention or treatment of a skin disease by including an effective amount of a skin organoid.

[0048] The above skin diseases may be caused by skin damage or aging, and may include, but are not limited to, atopic dermatitis, psoriasis, seborrheic dermatitis, urticaria, photosensitivity dermatitis, acne, skin boils, pimples, skin wrinkles, loss of skin elasticity, skin aging, wound healing, skin pigmentation disorders, skin barrier damage, skin cancer, or hair loss.

[0049] As used herein, the term "treatment" refers to any action that improves or completely cures the symptoms of a skin disease by transplanting or administering a skin organoid according to the present invention. Furthermore, the term "prevention" refers to any action that suppresses or delays the symptoms of a skin disease by transplanting or administering a skin organoid according to the present invention.

[0050] The effective amount may be a "therapeutically effective amount" or a "prophylactically effective amount." As used herein, the term "therapeutically effective amount" refers to any amount that, when a drug or therapeutic agent is used alone or in combination with another therapeutic agent, can exhibit a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or a prevention of damage or disability caused by the disease. As used herein, the term "prophylactically effective amount" refers to any amount that inhibits the occurrence or recurrence of a skin disease in a subject. The level of the effective amount may be determined based on factors such as the severity of the subject's condition, age, sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0051] The term "administration" as used herein refers to physically introducing a composition into a subject using any of various methods and delivery systems known to those skilled in the art. Routes of administration for the pharmaceutical compositions of the present invention include, but are not limited to, submucosal, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as injection or infusion. In addition, the administration can be non-surgical using a device such as a catheter, or surgically, such as injection or implantation after incision of the diseased site. The number of administrations for the compositions of the present invention can be performed, for example, once, multiple times, and over one or more extended periods of time.

[0052] The pharmaceutical composition of the present invention may vary depending on the age, sex, and weight of the subject, and specifically, depending on the symptoms of the subject, the pharmaceutical composition may be administered once or several times a day, or at intervals of several days to several months. In addition, the dosage may be 5x10 skin organoids included in the pharmaceutical composition of the present invention. 5 dog / ㎠ to 5x10 6 It can be adjusted to be administered per ㎠, but is not limited thereto, and may be increased or decreased depending on the route of administration, severity of disease, gender, weight, age, etc.

[0053] The pharmaceutical composition of the present invention may further include suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. Examples of carriers, excipients, and diluents that may be included in the composition include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0054] The term "subject" includes a human or any non-human animal, which non-human animal may be a vertebrate, such as a primate, dog, cow, horse, pig, rodent, such as a mouse, rat, guinea pig, etc. In this specification, the "subject" is used interchangeably with "individual" and "patient."

[0055] The pharmaceutical composition of the present invention may be administered in combination with another therapeutic agent. In this case, the pharmaceutical composition of the present invention and the other therapeutic agent may be administered simultaneously, sequentially, or separately. The other therapeutic agent may be, but is not limited to, a drug such as a compound or protein that has the effect of preventing, treating, or improving skin diseases.

[0056] Furthermore, the pharmaceutical composition of the present invention may be formulated to be administered simultaneously, sequentially, or separately with other therapeutic agents. For example, the skin organoids and the other therapeutic agents may be administered simultaneously in a single formulation, or they may be administered simultaneously, sequentially, or separately as separate formulations. To enable simultaneous, sequential, or separate administration, the skin organoids and the other therapeutic agents included in the pharmaceutical composition of the present invention may be formulated separately in separate containers, or they may be formulated together in the same container. Furthermore, the skin organoids and the other therapeutic agents included in the pharmaceutical composition of the present invention may be the same or different from each other in terms of their pharmaceutically effective doses, administration times, administration intervals, administration routes, treatment periods, etc.

[0057]

[0058] Another aspect of the present invention provides a method for preventing or treating a skin disease, comprising the step of transplanting a skin organoid prepared according to the above method into a subject.

[0059] In the method for preventing or treating skin diseases according to the present invention, each term has the same meaning as described above unless specifically stated otherwise.

[0060] In the method for preventing or treating a skin disease according to the present invention, the skin organoid can be administered to a subject simultaneously, sequentially, or separately with another therapeutic agent.

[0061] The above "simultaneous" administration refers to administering the skin organoid and another therapeutic agent simultaneously as a single formulation. It also refers to administering the skin organoid and another therapeutic agent simultaneously as separate formulations, in which case the routes of administration of the skin organoid and the other therapeutic agent may be different. The above "sequential" administration refers to administering the skin organoid and another therapeutic agent relatively consecutively, allowing for the shortest possible time between administrations. The above "separate" administration refers to administering the skin organoid and another therapeutic agent at set intervals. The method of administering the skin organoid and another therapeutic agent can be appropriately selected by a physician or expert in the art, taking into account the therapeutic efficacy, side effects, etc. of the subject.

[0062]

[0063] Another aspect of the present invention provides a method for evaluating the efficacy of a substance or cosmetic for preventing or treating a skin disease and a screening method, the method comprising a step of treating a skin organoid produced by the above production method with a substance or cosmetic for preventing or treating a skin disease.

[0064] In the method for evaluating the efficacy of a substance or cosmetic for preventing or treating skin diseases and the screening method according to the present invention, each term has the same meaning as described above unless specifically stated otherwise.

[0065] The term 'cosmetics' used in the present invention is not limited to products that are intended to improve skin conditions, and may be, for example, preparations developed for the purposes of moisturizing the skin, enhancing skin elasticity, and improving skin wrinkles.

[0066] As used herein, the term "candidate substance" refers to a substance expected to treat, prevent, or improve a skin disease. Specifically, any substance expected to directly or indirectly improve or alleviate a skin disease may be used without limitation, and includes all potential substances, such as compounds, genes, or proteins.

[0067] The above candidate substance can be processed using methods known in the art. Specifically, the candidate substance can be processed by co-culturing the skin organoid with the candidate substance, or by transplanting or administering the candidate substance into a living body containing the skin organoid. However, this is not limited to these methods, and those skilled in the art will be able to use methods suitable for the purposes of the present invention.

[0068] The efficacy evaluation method and screening method of the present invention may include step a) treating the organoid of the present invention with a candidate substance, and step b) comparing the skin organoid treated with the candidate substance with a control group.

[0069] As used herein, the term "control" refers to skin organoids that have not been treated with a candidate substance, or skin organoids that have not been treated with a substance intended to prevent or treat a skin disease. This may also include a positive control group. The control group may be used to compare results with skin organoids treated with the candidate substance.

[0070] The skin organoid manufactured according to the present invention has a high tissue similarity to human skin tissue, can contain hair follicles, and can regenerate damaged skin tissue, especially when transplanted into a living body.

[0071] Therefore, the method for producing a skin organoid according to the present invention can be utilized to produce a skin organoid similar in shape and function to a living tissue, and the produced skin organoid can be used as a regenerative treatment material for skin diseases, or can be usefully utilized as a method for evaluating the efficacy of a skin disease treatment material or screening a skin disease treatment material.

[0072] Figure 1 is an image of a skin organoid produced using induced pluripotent stem cells (scale bar: 500 μm). D1, D2, D4, D6, and D8 represent 1 day, 2 days, 4 days, 6 days, and 8 days, respectively, from the start of culture.

[0073] Figure 2 is an image of a skin organoid produced using embryonic stem cells (scale bar 500 μm). D1, D2, D4, D6, D8, and D40 represent 1 day, 2 days, 4 days, 6 days, 8 days, and 40 days from the start of culture, respectively.

[0074] Figure 3 is an image of skin organoids manufactured using induced pluripotent stem cells (n=3).

[0075] Figure 4 is an image of a skin organoid manufactured using embryonic stem cells (n=3).

[0076] Figures 5a and 5b are images showing the results of histochemical staining of DAPI and AP2α protein, an ectoderm expression marker, after harvesting skin organoids 4 days after starting culture using induced pluripotent stem cells to confirm the similarity of skin organoids to skin tissue.

[0077] Figure 5b is an image showing the results of histochemical staining of DAPI, E-cadherin (ECAD), an ectoderm expression marker, and SOX10 protein, a cranial neural crest cell expression marker, after harvesting skin organoids 4 days after starting culture using induced pluripotent stem cells to confirm the similarity of skin organoids to skin tissue.

[0078] Figure 6a is an image showing the results of histochemical staining of DAPI and AP2α protein, an ectoderm expression marker, after harvesting skin organoids 4 days after initiating culture using embryonic stem cells to confirm the similarity of skin organoids to skin tissue.

[0079] Figure 6b is an image showing the results of histochemical staining of DAPI, E-cadherin, an ectoderm expression marker, and SOX10 protein, a cranial neural crest cell expression marker, after harvesting skin organoids 4 days after starting culture using embryonic stem cells to confirm the similarity of skin organoids to skin tissue.

[0080] Figure 7a is an image showing the results of histochemical staining of DAPI, an ectoderm expression marker, AP2α protein, after harvesting skin organoids 8 days after starting culture using induced pluripotent stem cells to confirm the similarity of skin organoids to skin tissue.

[0081] Figure 7b is an image showing the results of histochemical staining of DAPI, E-cadherin, an ectoderm expression marker, and SOX10 protein, a cranial neural crest cell expression marker, after harvesting skin organoids 8 days after starting culture using induced pluripotent stem cells to confirm the similarity of skin organoids to skin tissue.

[0082] Figure 8a is an image showing the results of histochemical staining of DAPI, an ectoderm expression marker, AP2α protein, after harvesting skin organoids 8 days after initiating culture using embryonic stem cells to confirm the similarity of skin organoids to skin tissue.

[0083] Figure 8b is an image showing the results of histochemical staining of DAPI, E-cadherin, an ectoderm expression marker, and SOX10 protein, a cranial neural crest cell expression marker, after harvesting skin organoids 8 days after starting culture using embryonic stem cells to confirm the similarity of skin organoids to skin tissue.

[0084] Hereinafter, the present invention will be described in more detail through examples. These examples are intended to more specifically explain the present invention, and the scope of the present invention is not limited by these examples.

[0085]

[0086] Manufacturing example

[0087] Embryoid bodies (EBs) were formed using induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). Specifically, hPSCs, such as iPSCs and ESCs, were dissociated into single cells using a cell dissociation solution, and the number of cells was counted and 3x10 per well of a 96-well low attachment plate. 3 Dog or 5x10 3Embryoid bodies (EBs) were formed by dividing the cells. EBs, which are clusters of hPSCs, are commonly used to effectively induce differentiation. Initially, they consist of mostly undifferentiated hPSCs. However, depending on the culture conditions, the undifferentiated cells gradually differentiate into surface ectoderm (SE) or cranial neural crest (CNC) cells.

[0088] The present invention uses a first medium to induce differentiation of surface ectoderm from pluripotent stem cells or embryonic stem cells, such as induced pluripotent stem cells and embryonic stem cells, and uses a second medium to induce differentiation of cranial neural crest (CNC) cells from pluripotent stem cells or embryonic stem cells, such as induced pluripotent stem cells and embryonic stem cells, and is characterized in that the BMP included in the first medium is at a higher concentration than the BMP included in the second medium.

[0089] In this manufacturing example, the content of BMP included in the first medium and the content of BMP included in the second medium were set differently. The manufacturing example described in Table 1 below used induced pluripotent stem cells, and the manufacturing example described in Table 2 below used embryonic stem cells. Manufacturing examples 1 and 5 and manufacturing examples 6 and 10 have no difference in the BMP concentration between the first and second medium. Manufacturing examples 3, 4 and 11 and manufacturing examples 8, 9 and 12 have a BMP concentration gradient that goes down from the first medium to the second medium.

[0090] BMP concentration (ng / mL) included in the first medium BMP concentration (ng / mL) included in the second medium Manufacturing example 12.52.5 Manufacturing example 2250 Manufacturing example 3251 Manufacturing example 4251.5 Manufacturing example 52525 Manufacturing example 11101

[0091] BMP concentration (ng / mL) included in the first medium BMP concentration (ng / mL) included in the second medium Manufacturing example 62.52.5 Manufacturing example 7250 Manufacturing example 8251 Manufacturing example 9251.5 Manufacturing example 102525 Manufacturing example 12101

[0092] The first medium was DMEM / F12 as the basic medium, to which BMP4 was added as a BMP, and SB431542 was added as a TGFβ inhibitor. The day on which embryoid bodies (EBs) were cultured in the first medium was designated as D0, and differentiation into surface ectoderm was confirmed after 2 days of culture. Then, on the second day (D2) of culture, the medium was replaced with the second medium, and after 2 days of culture, differentiation of undifferentiated cells into cranial neural crest (CNC) cells was confirmed.

[0093] Afterwards, the medium was treated with LDN193189 as a BMP inhibitor and cultured for an additional 2 days. Then, on the 6th day (D6) of culture, the medium was replaced with a mixed medium of Advanced DMEM / F12 medium and Neurobasal medium supplemented with GlutaMAX supplement, B-27 supplement, 2-Mercaptoethanol, and Normocin, which are commonly used in organoid culture, to promote maturation of skin organoids. After the medium exchange on the 8th day (D8), skin organoids were harvested and photographed on the 2nd day (D10), 22nd day (D30), and 42nd day (D50), respectively.

[0094]

[0095] Experimental Example 1

[0096] Figure 1 is a photograph taken with a phase contrast microscope at D1, D2, D4, D6, and D8 when the same iPSC#1 cells were used according to the conditions of Table 1. In addition, Figure 2 is a photograph taken with a phase contrast microscope at D1, D2, D4, D6, D8, and D40 when the same hESC#1 cells were used according to the conditions of Table 2.

[0097] Figure 3 shows the results of three independent repeated experiments, which were performed using a phase contrast microscope at D10, D30, and D50 when iPSC#2 cells were used according to the conditions of Manufacturing Examples 1 and 11, respectively (n=3). In addition, Figure 4 shows the results of three independent repeated experiments, which were performed using a phase contrast microscope at D10, D30, and D50 when ESC#2 cells were used according to the conditions of Manufacturing Examples 6 and 12, respectively (n=3).

[0098] As can be seen in Figures 1 and 3, in Manufacturing Examples 3, 4, and 12, which are conditions for descending the BMP concentration gradient, it was observed that the formation of cyst structures, which are morphologically important in the early stage of skin differentiation, was well induced, indicating that the differentiation of skin organoids was induced uniformly. On the other hand, in the remaining Manufacturing Examples 1, 2, and 5, it was observed that cyst structures were not formed, indicating that the early stage of skin differentiation was not well induced. In the Manufacturing Examples, which are conditions for differentiation without a concentration gradient, the rate of failure in the early stage of skin organoid differentiation was high, and even if the skin organoids were differentiated, they were observed to be formed in a morphologically irregular, uneven, or shrunken form.

[0099] As can be seen in FIGS. 2 and 4, in Manufacturing Examples 6, 7, 8, 9, and 12, the formation of morphologically important cyst structures in the early stage of skin differentiation was well induced, but in Manufacturing Example 10, the cyst structure was not formed, so the early stage of skin differentiation was not well induced. In addition, in Manufacturing Examples 6 and 7, although cyst formation was induced in the early stage of skin differentiation, there was a problem in that the skin layer induction was not performed properly during the skin maturation process, or the size and shape of the skin organoids were formed unevenly. On the other hand, in Manufacturing Examples 8, 9, and 12, which were under the condition of decreasing the BMP concentration gradient, the skin layer induction was performed normally during the maturation process, and it was observed that organoids with uniform size and shape were formed.

[0100] Through the above experiments, it was demonstrated that differentiation into surface ectoderm cells, which play an important role in the differentiation of the epidermal layer of the skin, and cranial neural crest (CNC) cells, which play an important role in the differentiation of the dermal layer, can be efficiently induced through a gradient of BMP concentrations in the first and second media in the early stages of differentiation of skin organoids, and that uniform and mature skin organoids can be produced.

[0101]

[0102] Experimental Example 2

[0103] In this experimental example, immunohistochemical analysis was performed to confirm whether the previously manufactured human skin organoids were histologically similar to actual human skin.

[0104] Specifically, immunofluorescence staining was performed on skin organoids harvested on day 4 (D4) and day 8 (D8) from the start of culture using antibodies against DAPI, which stains cell nuclei; E-cadherin (ECAD) protein as a surface ectoderm expression marker; and SOX10 and AP2α proteins as cranial neural crest cell expression markers. The results are shown in Figs. 5 to 8.

[0105] Figures 5a to 6b are photographs of skin organoids (Preparation Example 1, Preparation Example 3, Preparation Example 6, and Preparation Example 8) harvested and stained on the fourth day (D4) from the start of culture, and Figures 7a to 8b are photographs of skin organoids (Preparation Example 1, Preparation Example 3, Preparation Example 6, and Preparation Example 8) harvested and stained on the eighth day (D8) from the start of culture.

[0106] As a result, it was confirmed that surface ectoderm was induced better in Manufacturing Example 3, which used a higher concentration of BMP in the first medium through a BMP concentration gradient, than in Manufacturing Example 1, which used induced pluripotent stem cells without a BMP concentration gradient. In addition, it was confirmed that cranial neural crest cells were induced better in Manufacturing Example 3, which formed a BMP concentration gradient, than in Manufacturing Example 1, which used induced pluripotent stem cells without a BMP concentration gradient.

[0107] Similar to the results of the above-mentioned induced pluripotent stem cells, it was confirmed that ectoderm was better induced in Production Example 8, which used a higher concentration of BMP in the first medium through a BMP concentration gradient, than in Production Example 6, which used embryonic stem cells without a BMP concentration gradient. In addition, it was confirmed that cranial neural crest cells were better induced in Production Example 8, which formed a BMP concentration gradient, than in Production Example 6, which used the BMP concentration gradient without a BMP concentration gradient.

[0108] The above results demonstrate that when the BMP concentration in the second medium is lower than that in the first medium, i.e., when a downward BMP concentration gradient is formed, surface ectoderm and cranial neural crest cells are significantly better differentiated, and thus the multilayer structure of the produced skin organoids is better formed. This suggests that the method of the present invention is efficient in producing skin organoids and can enhance skin tissue similarity.

Claims

1. a) A step of culturing pluripotent stem cells or embryoid bodies (EBs) in a first medium containing BMP to differentiate them into surface ectoderm; and b) a step of culturing the product of step a) in a second medium containing BMP to differentiate it into cranial neural crest cells; a method for producing a skin organoid, comprising: A method for producing skin organoids, wherein the BMP concentration of the second medium is lower than the BMP concentration of the first medium.

2. In paragraph 1, A method for producing a skin organoid, wherein the above pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

3. In paragraph 1, A method for producing a skin organoid, wherein the above BMP is selected from the group consisting of BMP2, BMP3, BMP4, BMP6, BMP7, BMP8, BMP9, BMP10, BMP11 and BMP15.

4. In paragraph 1, A method for producing a skin organoid, wherein the first medium and the second medium additionally contain a TGFβ (Transforming growth factor β) inhibitor.

5. In paragraph 4, A method for producing a skin organoid, wherein the TGFβ inhibitor is selected from the group consisting of A83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-364947, and SJN-2511.

6. A skin organoid manufactured according to the manufacturing method of paragraph 1, wherein the skin organoid comprises a multilayer structure including an epidermal layer, a dermal layer, and a subcutaneous tissue layer.

7. In paragraph 6, A skin organoid, wherein the skin organoid comprises a hair follicle.

8. In paragraph 6, The above skin organoid is a skin organoid for preventing or treating skin diseases.

9. In paragraph 8, A skin organoid, wherein the skin disease is at least one selected from the group consisting of atopic dermatitis, psoriasis, seborrheic dermatitis, urticaria, photosensitivity dermatitis, acne, skin boils, pimples, skin wrinkles, deterioration of skin elasticity, skin aging, wound healing, skin pigmentation disease, skin barrier damage, skin cancer, and hair loss.

10. In paragraph 6, The above skin organoid is a skin organoid used for evaluating the efficacy of cosmetics or skin disease treatment or prevention preparations.

11. In paragraph 6, The above skin organoid is a skin organoid used for screening cosmetics or skin disease treatment or prevention agents.

12. A pharmaceutical composition for treating or preventing skin disease comprising a skin organoid according to Article 6.

13. In paragraph 12, A pharmaceutical composition, wherein the skin disease is at least one selected from the group consisting of atopic dermatitis, psoriasis, seborrheic dermatitis, urticaria, photosensitivity dermatitis, acne, skin boils, pimples, skin wrinkles, deterioration of skin elasticity, skin aging, wound relief, skin pigmentation disease, skin barrier damage, skin cancer, and hair loss.

14. A method for evaluating the efficacy of a cosmetic or skin disease treatment or prevention agent using the skin organoid of Article 6.

15. A method for screening cosmetics or skin disease treatment or prevention agents using the skin organoid of Article 6.

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