Method for producing human pluripotent stem cell-derived skin epithelial organoid and uses thereof

Abstract

The present invention relates to a method for preparing a human pluripotent stem cell-derived skin epithelial organoid and uses thereof. As the skin epithelial organoid according to the present invention is produced under specific conditions, stable organoid culture is possible even under passaging, freezing, and thawing conditions. In addition, as it is differentiated into a form that mimics skin epithelial tissue and hair follicle structures and can be used as a 3D in vitro skin model, there are advantages in that the organoid can be used not only for studying molecular genetic and cell biological interaction mechanisms, but also as a skin disease model for screening a therapeutic agent for skin disease or a drug for skin regeneration, and enables evaluation of fine dust or skin toxicity.

Description

Method for preparing human pluripotent stem cell-derived skin epithelial organoids and uses thereof

[0001] The present invention relates to a method for producing skin epithelial organoids derived from human pluripotent stem cells and the use thereof.

[0002] Pluripotent stem cells (PSCs) are cells capable of differentiating into all types of cells or tissues that make up an organism. They are a collective term for cells that possess pluripotency, capable of self-renewal indefinitely and differentiating into cells with specific functions depending on the environment. In particular, because it is possible to produce induced pluripotent stem cells (iPSCs), which are patient-specific pluripotent stem cells, through reprogramming technology, high utility is expected in the regenerative medicine industry and a wide range of fields.

[0003] Recently, research on techniques for producing pluripotent skin tissue stem cells, progenitor cells, or differentiated skin tissue-associated cells using pluripotent stem cells has been actively underway. Existing skin models have primarily been validated using 2D culture techniques utilizing differentiated cells such as keratinocytes or dermal fibroblasts. This has limitations in mimicking phenotypes based on cell-to-cell interactions and in vivo conditions.

[0004] Previous attempts to generate skin organoids from induced pluripotent stem cells (iPSCs) involved methods such as co-culturing after differentiation into keratinocytes and dermal fibroblasts, and transplanting embryoid bodies into immunodeficient mice for maturation. More recently, a method for reproducing skin tissue through self-organization in an ex vivo environment has been reported. However, these methods faced limitations, including inconsistencies in differentiation patterns, long differentiation times, and the inability to perform passages. To date, no technology for manufacturing pluripotent stem cell-derived organoids that mimic human skin epithelial tissue has been reported.

[0005] Accordingly, the inventors, after diligent efforts, were able to produce an organoid capable of mimicking intercellular interactions and phenotypes according to in vivo conditions by manufacturing the organoid under specific culture conditions, and completed the present invention by confirming that the aforementioned problems associated with attempts to generate skin organoids from existing induced pluripotent stem cells were effectively resolved.

[0006] One objective of the present invention is a) a step of differentiating pluripotent stem cells into ectoderm by treating them with a differentiation medium containing a BMP activator, an FGF family, a TGF-β inhibitor, and an extracellular matrix; b) a step of differentiating the ectoderm into cranial neural crest-like cells by treating them with a differentiation medium containing a BMP inhibitor and an FGF family; c) a step of inducing differentiation into skin epithelial organoids by adding a culture medium to the cranial neural crest-like cells; and d) a step of maturing the skin epithelial organoids by treating them with a first maturation medium containing a ROCK inhibitor, a TGF-β inhibitor, a Wnt activator, and a ligand for a tyrosine kinase receptor. and e) a step of culturing the matured skin epithelial organoid by subculturing it at least 5 times in a second maturation medium containing a TGF-β inhibitor, a Wnt activator, and a ligand for a tyrosine kinase receptor; the present invention provides a method for producing a skin epithelial organoid comprising: a step of culturing the matured skin epithelial organoid by subculturing it at least 5 times in a second maturation medium containing a TGF-β inhibitor, a Wnt activator, and a ligand for a tyrosine kinase receptor.

[0007] Another objective of the present invention is to provide a method for producing a hair follicle organoid comprising the step of mixing and culturing the above-mentioned skin epithelial organoid and dermal cells.

[0008] Another objective of the present invention is to provide a method for producing skin epithelial tissue comprising: i) a step of differentiating pluripotent stem cells into ectoderm by treating them with a differentiation medium containing a BMP activator, an FGF family, a TGF-β inhibitor, and an extracellular matrix; ii) a step of differentiating the ectoderm into cranial neural crest-like cells by treating them with a differentiation medium containing a BMP inhibitor and an FGF family; iii) a step of inducing differentiation into skin epithelial organoids by adding a culture medium to the cranial neural crest-like cells; iv) a step of maturing the skin epithelial organoids by treating them with a first maturation medium containing a ROCK inhibitor, a TGF-β inhibitor, a Wnt activator, and a ligand for a tyrosine kinase receptor; and v) a step of reorganizing the skin epithelial organoids through an air-liquid interface culture on top of a transwell.

[0009] Another objective of the present invention is to provide an in vitro skin mimicry model obtained by the above method.

[0010] Another objective of the present invention is to provide a screening method for a skin disease treatment / skin regeneration drug comprising the step of treating the above-described in vitro skin mimic model with a test substance.

[0011] Another objective of the present invention is to provide a method for evaluating fine dust / skin toxicity comprising the step of treating the fine dust / sample in the above-described in vitro skin mimic model.

[0012] In the following, redundant details will be omitted to prevent clutter. In other words, the content of the invention is not limited solely to the following, and should be interpreted in accordance with the overall context of the invention.

[0013] The terms used in this invention are used merely to describe specific embodiments and are not intended to limit the invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this application, terms such as "comprising" or "having" are intended to indicate the presence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0014] Where in this specification, when a quantity, concentration, or other value or parameter is given as an enumeration of a range, a preferred range, a preferred upper limit, and a preferred lower limit, it should be understood that any pair of any upper range limit or preferred value and any lower range limit or preferred value are specifically disclosed, regardless of whether the range is disclosed separately.

[0015] Where a range of numerical values ​​is mentioned in this specification, unless otherwise stated, the range is not intended to be limited to the specific value mentioned when defining the range. Furthermore, unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the embodiments pertain. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this application.

[0016] In one preferred embodiment, the present invention comprises: a) a step of differentiating pluripotent stem cells into ectoderm by treating them with a differentiation medium containing a BMP activator, an FGF family, a TGF-β inhibitor, and an extracellular matrix; b) a step of differentiating the ectoderm into cranial neural crest-like cells by treating them with a differentiation medium containing a BMP inhibitor and an FGF family; c) a step of inducing differentiation into skin epithelial organoids by adding a culture medium to the cranial neural crest-like cells; and d) a step of maturing the skin epithelial organoids by treating them with a first maturation medium containing a ROCK inhibitor, a TGF-β inhibitor, a Wnt activator, and a ligand for a tyrosine kinase receptor. and e) a step of culturing the matured skin epithelial organoid by subculturing it at least 5 times in a second maturation medium containing a TGF-β inhibitor, a Wnt activator, and a ligand for a tyrosine kinase receptor; the present invention provides a method for producing a skin epithelial organoid, comprising: a step of culturing the matured skin epithelial organoid by subculturing it at least 5 times in a second maturation medium containing a TGF-β inhibitor, a Wnt activator, and a ligand for a tyrosine kinase receptor.

[0017] Each step of the method for manufacturing a skin epithelial organoid according to the present invention will be described in more detail below.

[0018] a. Differentiation into ectoderm

[0019] This step is a process of differentiating pluripotent stem cells into ectoderm by treating them with a differentiation medium containing a BMP activator, FGF family and TGF-β inhibitor, and the extracellular matrix.

[0020] In a preferred embodiment, the method according to the present invention can induce the formation of 3D spheroids of pluripotent stem cells before the start of differentiation into the ectoderm, preferably 1, 2, 3, or 4 days prior or earlier. At this time, cell viability can be enhanced by adding a ROCK inhibitor, preferably thiazobibin or Y-27632, to the pre-differentiation medium and culturing. Preferably, cell viability can be enhanced by adding Y-27632 and culturing.

[0021] In a preferred embodiment, the method according to the present invention can be performed at the time of differentiation (day 0 of differentiation).

[0022] The term "pluripotent stem cell (PSC, PS cell)" as used in the present invention includes any cell capable of differentiating into almost any cell, namely a cell derived from any of the three germ layers (germ epithelium), including the endoderm (internal stomach wall, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, genitourinary system), and ectoderm (epithelial tissue and nervous system). A pluripotent stem cell may be an embryonic stem cell or an induced pluripotent stem cell.

[0023] The above "embryonic stem cell (ESC, ES cell)" refers to a pluripotent cell derived from the inner cell mass of a blastocyst, which is an early-stage embryo. For the purposes of the present invention, embryonic germ cells are also included.

[0024] The above "induced pluripotent stem cell (iPSC, iPS cell)" refers to a type of pluripotent stem cell that is artificially induced from normally pluripotent cells, such as adult somatic cells, by inducing "forced" expression of a specific gene.

[0025] The “extracellular matrix” mentioned above is a three-dimensional matrix, a type of basement membrane matrix used for cell culture, and is primarily used to provide a 3D culture environment. In some embodiments, the pluripotent stem cells are embedded in the ECM. The culture medium of the present invention can diffuse into the three-dimensional ECM.

[0026] Details regarding ECM disclosed in Polymer Hydrogels to Guide Organotypic and Organoid Cultures (Adv Funct Mater, 2020, Valentina Magno et al.) and Engineering the Extracellular Matrix for Organoid Culture (Int J Stem Cells. 2022 Feb 28;15(1):60-69) are incorporated by reference into the present invention.

[0027] The extracellular matrix includes, but is not limited to, fibrin, laminin, collagen, and / or alginate, for example. Examples of extracellular matrix-producing cells are chondrocytes that produce collagen and proteoglycans, fibroblasts that produce type IV collagen, laminin, interstitial procollagen, and fibronectin, and colonic myofibroblasts that produce collagen (types I, III, and V), chondroitin sulfate proteoglycans, hyaluronic acid, fibronectin, and tenasin-C. These are "naturally occurring ECMs." Naturally occurring ECMs may be commercially available. Examples of commercially available extracellular matrix include extracellular matrix proteins (Invitrogen) and basement membrane preparations from Angelbreath-Holm-Swarm (EHS) mouse sarcoma cells (e.g., Cultrex (trademark) basement membrane extract (Trevigen, Inc.), Type I collagen (Invitrogen), Vitrogel (trademark) (TheWell Bioscience Inc.), Regenix (trademark) (Cellartgen Inc.), Geltrex (ThermoFisher), or Matrigel (trademark) (BD Biosciences)).

[0028] As a preferred example of the present invention, the culture medium may further include an extracellular matrix (ECM), and in this case, it is preferable to use an extracellular matrix concentration of 0.5 to 5%.

[0029] The basic medium for use in the present invention will generally comprise a nutrient solution containing standard cell culture components, e.g., amino acids, vitamins, lipid supplements, inorganic salts, carbon energy sources, and buffers. In some embodiments, the culture medium is further supplemented with one or more standard cell culture components selected from, e.g., amino acids, vitamins, liquid supplements, inorganic salts, carbon energy sources, and buffers.

[0030] A person skilled in the art will understand from ordinary general knowledge that the type of culture medium that may be used as a base medium among the differentiation and / or maturation media of the present invention is a potentially suitable cell differentiation medium. Potentially suitable cell differentiation media may be commercially available and include, but are not limited to, Dulbecco's Modified Dulbecco's Media (DMEM), Medium of Minimum Essentials (MEM), Knockout-DMEM (KO-DMEM), Glasgow Medium of Minimum Essentials (GMEM), Base Eagle Medium (BME), DMEM / Hams F12, Advanced DMEM / Hams F12, Iscove's Modified Dulbecco's Media and Medium of Minimum Essentials (MEM), Hams F-10, Hams F-12, Medium 199, RPMI 1640 medium, and KnockOut Serum replacement XenoFree medium.

[0031] For example, the above basic medium or culture medium may be DMEM / F12.

[0032] As a preferred application example, the differentiation medium or culture medium comprises at least one of L-ascorbic acid-2-phosphate magnesium, sodium selenium, and insulin as additional components.

[0033] As a most preferred application example, the differentiation medium may be DMEM / F12. Additionally, as an additional culture component, it may include one or more selected from the group consisting of L-ascorbic acid-2-phosphate magnesium, sodium selenium, insulin, sodium hydrocarbon (NaHCO3), transferrin, and NODAL.

[0034] In the step of differentiating pluripotent stem cells according to the present method into ectoderm by treating them with a differentiation medium containing a BMP activator, FGF family, TGF-β inhibitor, and extracellular matrix, differentiation into ectoderm is induced by treating and culturing the BMP activator, FGF family, and TGF-β inhibitor together with the extracellular matrix under any of the aforementioned basic media or differentiation medium conditions.

[0035] The above BMP activator is involved in bone and cartilage development, tooth and limb development, etc. It is selected from BMP-2, BMP-4, BMP-7, BMP-9, small molecules that activate the BMP pathway, proteins that activate the BMP pathway, Noggin, dorsomorphine, LDN189, DMH-1, ventromorphin, and combinations thereof. Preferably, the above BMP activator may be BMP-4. BMP-4 is a protein encoded by the BMP4 gene and is a protein belonging to the TGF-β superfamily. In the present invention, BMP-4 was applied to induce differentiation from the ectoderm to the non-neuroectoderm. More preferably, it may be treated at a concentration of 1 to 10 ng / mL, 1 to 8 ng / mL, or 2.5 to 6 ng / mL, and most preferably at a concentration of 5 ng / mL.

[0036] The above-mentioned FGF (fibroblast growth factor) family is a powerful factor that regulates cell proliferation and differentiation, playing a particularly important role in the normal development of stem cells, tissue maintenance, wound healing, and angiogenesis. The above-mentioned FGF family may be one or more selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, and FGF10. Preferably, it may be FGF2 and FGF10. More preferably, it may be treated at a concentration of 1 to 10 ng / mL, 2 to 8 ng / mL, or 3 to 6 ng / mL, and most preferably at a concentration of 4 ng / mL.

[0037] The above TGF-β inhibitor is any substance that inhibits the function of the TGF-β receptor, for example, a protein, peptide, or small molecule, and as a preferred example, may be any one selected from the group consisting of A83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-36494, and SJN-2511. Preferably, it may be SB-431542. More preferably, it may be treated at a concentration of 1 to 50 μM, 2 to 25 μM, or 5 to 15 μM, and most preferably at a concentration of 10 μM.

[0038] b. Differentiation into cranial neural crest-like cells

[0039] This step is the process of differentiating the ectoderm into cranial neural crest-like cells by treating it with a differentiation medium containing BMP inhibitors and the FGF family.

[0040] In a preferred embodiment, the present method may be performed at least 1, 2, 3, 4 days or thereafter (preferably on the 3rd day of differentiation) from the time of non-neuroectodermal differentiation.

[0041] The differentiation medium used in the method of the present invention comprises the basic medium mentioned above in a. The basic medium is any basic medium suitable for animal or human cells to which the limitations provided herein apply. Basic media for animal or human cell culture typically contain a number of components necessary to support the maintenance of cultured cells. A combination of suitable components can be easily formulated by a person skilled in the art with consideration of the following.

[0042] The basic medium for use in the present invention will generally comprise a nutrient solution containing standard cell culture components, e.g., amino acids, vitamins, lipid supplements, inorganic salts, carbon energy sources, and buffers. In some embodiments, the culture medium is further supplemented with one or more standard cell culture components selected from, e.g., amino acids, vitamins, liquid supplements, inorganic salts, carbon energy sources, and buffers.

[0043] A person skilled in the art will understand from ordinary general knowledge that the type of culture medium that may be used as a base medium among the differentiation media of the present invention is a potentially suitable cell differentiation medium. Potentially suitable cell differentiation media are commercially available and include, but are not limited to, Dulbecco's Modified Dulbecco's Media (DMEM), Medium of Minimum Essentials (MEM), Knockout-DMEM (KO-DMEM), Glasgow Medium of Minimum Essentials (GMEM), Base Eagle Medium (BME), DMEM / Hams F12, Advanced DMEM / Hams F12, Iscove's Modified Dulbecco's Media and Medium of Minimum Essentials (MEM), Hams F-10, Hams F-12, Medium 199, RPMI 1640 medium, and KnockOut Serum Replacement XenoFree medium.

[0044] For example, the above basic medium or culture medium may be DMEM / F12.

[0045] As a preferred application example, the differentiation medium or culture medium comprises at least one of L-ascorbic acid-2-phosphate magnesium, sodium selenium, and insulin as additional components.

[0046] As a most preferred application example, the differentiation medium may be DMEM / F12. Additionally, as an additional culture component, it may include one or more selected from the group consisting of L-ascorbic acid-2-phosphate magnesium, sodium selenium, insulin, sodium hydrocarbon (NaHCO3), transferrin, and NODAL.

[0047] The step of differentiating the ectoderm into cranial neural crest-like cells by treating it with a differentiation medium containing BMP inhibitors and the FGF family induces differentiation into cranial neural crest-like cells by co-treating and culturing with BMP inhibitors and the FGF family under any of the aforementioned basic media or differentiation medium conditions.

[0048] The above-mentioned BMP inhibitor is used to inhibit stem cells from excessively differentiating into cartilage tissue by blocking specific receptors and signaling pathways of bone morphogenetic proteins (BMPs). Additionally, BMP inhibitors can be administered to cells to regulate BMP-induced cell growth and differentiation, and specifically, can be used to regulate tissue differentiation of adult stem cells. As an example, it may be any one selected from the group consisting of Noggin, Dorsomorphin, DMH-1, DMH-2, K02288, LDN-193189, LDN-212854, LDN-214117, and ML347. Preferably, it may be LDN-193189. More preferably, it may be administered at a concentration of 0.01 to 10 μM, 0.1 to 5 μM, or 0.5 to 3 μM, and most preferably at a concentration of 1 μM.

[0049] The above-mentioned FGF (fibroblast growth factor) family is a powerful factor that regulates cell proliferation and differentiation, playing a particularly important role in the normal development of stem cells, tissue maintenance, wound healing, and angiogenesis. The above-mentioned FGF family may be one or more selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, and FGF10. Preferably, it may be FGF2 and FGF10. More preferably, it may be treated at a concentration of 1 to 500 ng / mL, 50 to 350 ng / mL, or 100 to 300 ng / mL, and most preferably at a concentration of 250 ng / mL.

[0050] In the present invention, differentiation into cranial neural crest cells (CNC-like cells) could be induced by treating the non-neuroectoderm with the above-mentioned BMP inhibitor and FGF-2 together.

[0051] c. Preparation of skin epithelial organoids

[0052] This step is a process of inducing differentiation of skin epithelial organoids by adding culture medium to cranial neural crest-like cells.

[0053] In a preferred embodiment, the present method may be performed 2, 3, 4, 5, and 6 days after the point of differentiation of cranial neural crest cells (preferably on the 6th day of differentiation).

[0054] More preferably, the action of BMP inhibitors and FGF-2 is minimized by adding culture medium to cranial neural crest-like cells and culturing them. More specifically, the aforementioned culture medium is commercially available and includes, but is not limited to, Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Knockout-DMEM (KO-DMEM), Glasgow Minimum Essential Medium (GMEM), Basic Eagle Medium (BME), DMEM / HAMS F12, Advanced DMEM / HAMS F12, Iscove's Modified Dulbecco's Media and Minimum Essential Medium (MEM), HAMS F-10, HAMS F-12, Medium 199, RPMI 1640 medium, and KnockOut Serum replacement XenoFree medium.

[0055] For example, the culture medium may preferably be DMEM / F12. As a preferred application, the culture medium comprises at least one of L-ascorbic acid-2-phosphate magnesium, sodium selenium, and insulin as additional components. As a most preferred application, the differentiation medium may be DMEM / F12. Additionally, as additional culture components, it comprises one or more selected from the group consisting of L-ascorbic acid-2-phosphate magnesium, sodium selenium, insulin, sodium hydrocarbon (NaHCO3), transferrin, and NODAL.

[0056] According to one embodiment, on at least 7, 8, 9, and / or 10 days of differentiation, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the differentiation medium of step b. is discarded and new culture medium is added to minimize the action of BMP inhibitors and the FGF family. This process continues for at least 10, 11, 12 days of differentiation or longer.

[0057] In the present invention, the term “organoid” refers to a cell aggregate formed by aggregating and recombining cells isolated from stem cells or organ-derived cells through culture, and may include an organoid or cell cluster formed from a suspension cell culture.

[0058] d. Maturation

[0059] This step is a process of maturing skin epithelial organoids by treating them with a first maturation medium containing a ROCK inhibitor, a TGF-β inhibitor, a Wnt activator, and a tyrosine kinase receptor ligand.

[0060] In a preferred embodiment, the present method may be performed 0 days after the time of preparation of the skin epithelial organoid (at least 10, 11, and / or 12 days from the time of the start of differentiation).

[0061] The above maturation may be performed under a maturation medium. Potentially suitable maturation media used in the method of the present invention may be commercially available and include, but are not limited to, Dulbecco's Modified Dulbecco's Media (DMEM), Medium of Minimum Essentials (MEM), Knockout-DMEM (KO-DMEM), Glasgow Medium of Minimum Essentials (GMEM), Basic Eagle Media (BME), DMEM / Hams F12, Advanced DMEM / Hams F12, Iscove's Modified Dulbecco's Media and Medium of Minimum Essentials (MEM), Hams F-10, Hams F-12, Media 199, RPMI 1640, and KnockOut Serum replacement XenoFree medium.

[0062] For example, the maturation medium may be DMEM / F12, Advanced DMEM / F12, and / or RPMI 1640. If necessary, Advanced DMEM / F12 or Advanced RPMI, which is optimized for serum-free culture and already contains insulin, is used.

[0063] In addition to the essential composition described above, the maturation medium of the present invention may further include one or more additional components selected from the group consisting of Glutamax, HEPES, Primocin, Prostaglandin E2 (PGE2), N-acetylcysteine ​​(NAC), B27, and Nicotinamide.

[0064] The above B27 may be replaced with a generic formulation containing one or more of the ingredients selected from the following list: biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinyl acetate, sodium selenite, tri-iodothyronine (T3), DL-alpha-tocopherol (vitamin E), albumin, insulin, and transferrin.

[0065] In addition, the maturation medium of the present invention may include antibiotics such as P / S (Penicillin-Streptomycin) and / or Primocin, in addition to the essential composition described above.

[0066] As a more preferred application example, in the process of maturing a skin epithelial organoid by treating it with a first maturation medium containing a ROCK inhibitor, a TGF-β inhibitor, a Wnt activator, and a ligand for a tyrosine kinase receptor, said first maturation medium contains a ROCK inhibitor, a TGF-β inhibitor, a Wnt activator, and a ligand for a tyrosine kinase receptor, thereby promoting the maturation of the skin organoid.

[0067] The ROCK inhibitor may preferably be thiazobibin or Y-27632.

[0068] The TGF-β inhibitor is any substance that inhibits the function of the TGF-β receptor, e.g., a protein, peptide, or small molecule, and as a preferred example, may be any one selected from the group consisting of A83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-36494, and SJN-2511. Most preferably, it may be A83-01. More preferably, it may be treated at a concentration of 0.01 to 10 μM, 0.1 to 5 μM, or 0.5 to 3 μM, and most preferably at a concentration of 1 μM.

[0069] The above Wnt activator may be any one selected from the group consisting of WNT-3A, R-spondin 1, R-spondin 2, R-spondin 3, and R-spondin 4. Most preferably, it may be WNT-3A. More preferably, it may be treated at a concentration of 1 to 500 ng / mL, 25 to 250 ng / mL, or 50 to 150 ng / mL, and most preferably at a concentration of 100 ng / mL.

[0070] The ligand of the tyrosine kinase receptor may be any one selected from the group consisting of NRG1 (Neuregulin β1), HRG1 (Heregulin β1), epidermal growth factor (EGF), transforming growth factor-α (TGF-α), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF), and keratinocyte growth factor (KGF). Preferably, treatment may be performed at a concentration of 1 to 300 ng / mL, 250 to 250 ng / mL, or 50 to 150 ng / mL, and most preferably at a concentration of 50 to 100 ng / mL.

[0071] Most preferably, it may be epidermal growth factor (EGF) and keratinocyte growth factor (KGF). More preferably, epidermal growth factor (EGF) may be treated at a concentration of 1 to 300 ng / ml, 10 to 150 ng / ml, or 25 to 100 ng / ml, and most preferably at a concentration of 50 ng / ml.

[0072] More preferably, keratinocyte growth factor (KGF) can be treated at a concentration of 1 to 300 ng / ml, 250 to 250 ng / ml, or 50 to 150 ng / ml, and most preferably at a concentration of 100 ng / ml.

[0073] As a most desirable example, the first maturation medium above contains Y-27632, A83-01, EGF, WNT-3A, and KGF.

[0074] As a most preferred application example, the specific medium composition of the first maturation medium comprises Y-27632, WNT-3A, EGF, KGF, and A83-01, and includes one or more selected from the group consisting of antibiotics, B27 serum-free supplements, Glutamex, HEPES, NAC (N-acetylcysteine), and Forskolin under Advanced DMEM / F-12.

[0075] If necessary, additional serum components such as FBS may be included.

[0076] As a more preferred example of the present invention, the epithelial organoid may be mixed with Matrigel, preferably GFR (growth factor reduced) Matrigel, in a ratio of 1:0.5 to 1:5 and performed in a culture environment in a transwell that mimics in vivo skin interactions. The skin epithelial organoid and GFR Matrigel may be mixed in a ratio of 1:5, 1:4, 1:3, 1:2, 1:1, or 1:0.5, but are not limited thereto. In one embodiment of the present invention, the epithelial progenitor cells and GFR Matrigel were mixed in a ratio of approximately 1:1.

[0077] In particular, the above-mentioned GFR (growth factor reduced) Matrigel is a growth factor-limited Matrigel used to precisely control cell growth, differentiation, and proliferation.

[0078] As a preferred example, the maturation may be performed in a maturation medium containing a ROCK inhibitor starting from the 12th day of differentiation. More preferably, it may be performed in a maturation medium containing FBS and Y-27632. As an even more preferred example, 5% FBS may be applied, and Y-27632 may be applied at a concentration of 1 to 50 μM, 5 to 30 μM, most preferably 10 μM.

[0079] e. Organoid formation and enhancement of growth rate

[0080] This step involves culturing matured skin epithelial organoids by subculturing them at least five times in a second maturation medium containing a TGF-β inhibitor, a Wnt activator, and a ligand for a tyrosine kinase receptor. Through this process, organoid formation can be efficiently induced.

[0081] Specifically, the medium may contain A83-01, EGF, WNT-3A, and KGF. More preferably, this step may be applied, for example, after 1, 2, or 3 days of maturation, and the culture medium is a medium excluded from ROCK inhibitors. Preferably, it is a medium excluded from FBS and Y-27632.

[0082] At this time, skin epithelial organoids can be cultured via the “Air-Liquid Interface” method in the second maturation medium excluding FBS and Y-27632. For a specific explanation regarding this, refer to the description below.

[0083] As a more preferred example of the present invention, the epithelial organoid may be mixed with GFR (growth factor reduced) Matrigel in a ratio of 1:6 to 1:9 and performed in a culture environment in a Transwell that mimics in vivo skin interactions. The skin epithelial organoid and GFR Matrigel may be mixed in ratios of 1:6, 1:7, 1:8, or 1:9, but are not limited thereto. In one embodiment of the present invention, the epithelial progenitor cells and GFR Matrigel were mixed in a ratio of approximately 1:9.

[0084] In particular, the above-mentioned GFR (growth factor reduced) Matrigel is a growth factor-limited Matrigel used to precisely control cell growth, differentiation, and proliferation.

[0085] The culture medium used in the method of the present invention comprises a basic medium. The basic medium is any basic medium suitable for animal or human cells to which the limitations provided herein apply. Basic media for animal or human cell culture typically contain a number of components necessary to support the maintenance of the cultured cells. A combination of suitable components can be easily formulated by a person skilled in the art with consideration of the following.

[0086] The basic medium for use in the present invention will generally comprise a nutrient solution containing standard cell culture components, e.g., amino acids, vitamins, lipid supplements, inorganic salts, carbon energy sources, and buffers. In some embodiments, the culture medium is further supplemented with one or more standard cell culture components selected from, e.g., amino acids, vitamins, liquid supplements, inorganic salts, carbon energy sources, and buffers.

[0087] A person skilled in the art will understand from ordinary general knowledge that the type of culture medium that may be used as a basic medium among the culture media of the present invention is a potentially suitable cell culture medium. Potentially suitable cell culture media may be commercially available and include, but are not limited to, Dulbecco's Modified Dulbecco's Media (DMEM), Medium of Minimum Essentials (MEM), Knockout-DMEM (KO-DMEM), Glasgow Medium of Minimum Essentials (GMEM), Basic Eagle Medium (BME), DMEM / Hams F12, Advanced DMEM / Hams F12, Iscove's Modified Dulbecco's Media and Medium of Minimum Essentials (MEM), Hams F-10, Hams F-12, Medium 199, RPMI 1640 medium, and KnockOut Serum replacement XenoFree medium.

[0088] In addition to the essential composition described above, the culture medium of the present invention may further include one or more additional components selected from the group consisting of Glutamax, HEPES, Primocin, Prostaglandin E2 (PGE2), N-acetylcysteine ​​(NAC), B27, and Nicotinamide.

[0089] The above B27 may be replaced with a generic formulation containing one or more of the ingredients selected from the following list: biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinyl acetate, sodium selenite, tri-iodothyronine (T3), DL-alpha-tocopherol (vitamin E), albumin, insulin, and transferrin.

[0090] In addition, the culture medium of the present invention may include antibiotics such as P / S (Penicillin-Streptomycin) and / or Primocin, in addition to the essential composition described above.

[0091] The concentration of each of these additional components can be appropriately controlled within the general range typically used in culture media.

[0092] In a preferred embodiment, the present invention may be demonstrated by performing a matured organoid in the second maturation medium described above for at least 7 days, preferably 10, 15, 20 days or more, and most preferably 12 to 17 days. In a more preferred embodiment, the present invention may be demonstrated by performing a dilation culture at least 5 times.

[0093] Here, subculture may be, for example, at least 5, 6, 7, 8 or more times. The subculture may preferably be performed at intervals of 5 to 10 days, most preferably 7 days.

[0094] In the present invention, the formation of stable skin epithelial organoids was induced through the corresponding passage culture.

[0095] In one embodiment of the present invention, it was confirmed that the organoid according to the present invention showed increased expression of epithelial cell markers similar to human-derived epithelial cells (KRT5, KRT14, p63, SOX9, INVOLUCRIN), keratinized epithelial cell-related genes (LORICRIN, FILAGGRIN), and hair follicle-derived epithelial cell-related genes (KRT10, KRT11) through five or more passages, and the ease of mass production could be effectively increased by verifying the passage, freezing, and thawing of pluripotent stem cell-derived epithelial organoids.

[0096] Accordingly, the present invention was able to produce an in vitro epithelial model capable of stable organoid culture.

[0097] The above TGF-β inhibitor is any substance that inhibits the function of the TGF-β receptor, for example, a protein, peptide, or small molecule, and as a preferred example, may be any one selected from the group consisting of A83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-36494, and SJN-2511. Most preferably, it may be A83-01. More preferably, it may be treated at a concentration of 0.01 to 10 μM, 0.1 to 5 μM, or 0.5 to 3 μM, and most preferably at a concentration of 1 μM.

[0098] The above Wnt activator may be any one selected from the group consisting of WNT-3A, R-spondin 1, R-spondin 2, R-spondin 3, and R-spondin 4. Most preferably, it may be WNT-3A. More preferably, it may be treated at a concentration of 1 to 500 ng / mL, 25 to 250 ng / mL, or 50 to 150 ng / mL, and most preferably at a concentration of 50 to 100 ng / mL.

[0099] The ligand of the tyrosine kinase receptor may be any one selected from the group consisting of NRG1 (Neuregulin β1), HRG1 (Heregulin β1), epidermal growth factor (EGF), transforming growth factor-α (TGF-α), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF), and keratinocyte growth factor (KGF). Preferably, treatment may be performed at a concentration of 1 to 300 ng / mL, 250 to 250 ng / mL, or 50 to 150 ng / mL, and most preferably at a concentration of 100 ng / mL.

[0100] Most preferably, it may be epidermal growth factor (EGF) and keratinocyte growth factor (KGF). More preferably, epidermal growth factor (EGF) may be treated at a concentration of 1 to 300 ng / ml, 10 to 150 ng / ml, or 25 to 100 ng / ml, and most preferably at a concentration of 50 ng / ml.

[0101] More preferably, keratinocyte growth factor (KGF) can be treated at a concentration of 1 to 300 ng / ml, 250 to 250 ng / ml, or 50 to 150 ng / ml, and most preferably at a concentration of 100 ng / ml.

[0102] As a most preferred example, the second maturation medium above comprises A83-01, EGF, WNT-3A, and KGF. As a most preferred application example, the specific medium composition of the second maturation medium above comprises WNT-3A, EGF, KGF, and A83-01, and comprises one or more selected from the group consisting of antibiotics, B27 serum-free supplements, Glutamex, HEPES, NAC (N-acetylcysteine), and Forskolin under Advanced DMEM / F-12.

[0103] In addition, epithelial organoids can be mixed with Matrigel, preferably GFR (growth factor reduced) Matrigel, and performed in a culture environment in Transwell that mimics in vivo skin interactions.

[0104] In addition, cells can be frozen or thawed under freezing conditions as needed for use.

[0105] According to one embodiment of the present invention, the organoid cell growth rate could be specifically increased by culturing the organoid under the above-described culture medium conditions. In addition, under the above-described culture conditions, the organoid can maintain high expression of KRT10, KRT1, INVOLUCRIN, LORICRIN, and FILAGGRIN, which are genes related to epithelial cell differentiation and keratinized epithelial cells.

[0106] That is, by establishing optimal culture conditions for epithelial organoids as in the method described above, the characteristics of the cells can be maintained even under organ passage culture conditions and / or freezing and thawing conditions.

[0107] In particular, existing skin epithelial organoid manufacturing technology is a keratinocyte-derived culture technology that has had limitations such as low productivity, difficult quality control, and unspecialized effects due to restrictions on the supply of source cells. However, by producing skin epithelial organoids based on a strategy utilizing pluripotent stem cells that is differentiated from existing technology, the present invention enables high productivity, easier quality control, and the maintenance of specialized characteristics as skin epithelial tissue.

[0108] Meanwhile, in another preferred embodiment, the present invention provides a method for producing a hair follicle organoid comprising the step of mixing and culturing a skin epithelial organoid produced by the above-described method and dermal cells.

[0109] As a preferred example, the present method may mix and culture the skin epithelial organoid and dermal cells in a ratio of 0.5 to 2:0.5 to 2, most preferably in an equal ratio.

[0110] According to one embodiment of the present invention, differentiation into a hair follicle structure in a test tube was confirmed by the above-described method.

[0111] In addition, as another preferred embodiment, the present invention comprises: i) a step of differentiating pluripotent stem cells into ectoderm by treating them with a differentiation medium containing a BMP activator, an FGF family, a TGF-β inhibitor, and an extracellular matrix; ii) a step of differentiating the ectoderm into cranial neural crest-like cells by treating them with a differentiation medium containing a BMP inhibitor and an FGF family; iii) a step of inducing differentiation into skin epithelial organoids by adding a culture medium to the cranial neural crest-like cells; iv) a step of maturing the skin epithelial organoids by treating them with a first maturation medium containing a ROCK inhibitor; and v) a step of reorganizing the skin epithelial organoids through an air-liquid interface culture on top of a transwell.

[0112] It is preferable to perform step v) above by including at least 5 subcultures at weekly intervals. Here, the aforementioned second maturation medium may be used for subculture. Specifically, this is a process of culturing by subculturing at least 5 times in a second maturation medium containing a TGF-β inhibitor, a Wnt activator, and a ligand for a tyrosine kinase receptor. Organoid formation can be efficiently induced through this process. The same provisions described in e. above may apply to this.

[0113] In addition, specific details regarding subculture have been explained above, so further explanation is omitted.

[0114] In addition, steps i) to iv) above are identical or similar to a) to d) in the method for manufacturing the skin epithelial organoid, so further explanation regarding this is omitted.

[0115] In the present invention, the “Air-Liquid Interface culture method” may involve culturing in a partially open culture vessel or a culture vessel partially filled with a medium, but is not limited thereto. For example, it may involve exposing the surface of a cell or organoid to air. Although referred to as “air” for convenience, the present invention is not limited to a mixture of gases and compositions found in the surrounding environment. Specifically, the present invention considers and includes a mixture of gases having a composition different from that of the surrounding environment, for example, a mixture concentrated for a specific component or a mixture from which a specific component has been depleted or removed.

[0116] When culturing cells at an air-liquid interface, cells can be cultured on a porous substrate such that the cells come into contact with air on the upper side of the porous substrate and with cell culture medium on the bottom side. For example, a sufficient volume of medium may be added to the bottom of a culture vessel containing a porous substrate (e.g., a filter insert) such that the medium comes into contact with the bottom surface of the cells present on the porous substrate but does not encapsulate or immerse the cells. A suitable porous substrate may be formed from any material that does not adversely affect cell growth and differentiation. An exemplary porous substrate is made of a polymer such as polyethylene terephthalate (PET), polyester, or polycarbonate. A suitable porous substrate may or may not be coated. Examples of commercially available extracellular matrix may be coated with extracellular matrix proteins (Invitrogen) and basement membrane preparations from Angelbreath-Holm-Swarm (EHS) mouse sarcoma cells (e.g., Cultrex (Trademark), basement membrane extract (Trevigen, Inc.), Type I collagen (Invitrogen), Vitrogel (Trademark) (TheWell Bioscience Inc.), or Matrigel (Trademark) (BD Biosciences)). Preferably, it may be coated with Matrigel. In one embodiment of the present invention, the porosity of the substrate must be sufficient to maintain cell viability and promote cell differentiation. Suitable substrates have a porosity of about 0.3 to about 3.0 µm, about 0.3 to about 2.0 µm, about 0.3 to about 1.0 µm, about 0.3 to about 0.8 µm, or about 0.3 to about 0.6 μm, about 0.3 to about 0.5 μm, about 0.5 to about 3.0 μm, about 0.6 to about 3.0 μm, about 0.8 to about 3.0 μm, about 1.0 to about 3.0 μm, about 2.0 to about 3.0 μm, preferably about 0.It includes a filter insert having a pore size of 4 μm and a pore density of about 50 million to about 120 million pores / cm², about 60 million to about 110 million pores / cm², about 70 million to about 100 million pores / cm², preferably about 80 million to about 100 million pores / cm², about 90 million to about 100 million pores / cm², and more preferably about 100 million pores / cm².

[0117] It may be advantageous to replace or regenerate the culture medium daily or every other day. Cells grown on top of porous substrates are generally not single cells; rather, they exist in the form of sheets or as aggregate clusters. Cells cultured at the air-liquid interface can experience much higher oxygen tension compared to cells immersed in the medium.

[0118] According to one embodiment of the present invention, when a skin epithelial organoid is reorganized through the above method, it was confirmed that it structurally mimics skin epithelial tissue.

[0119] The preparation of skin epithelial tissue according to the present invention is not limited thereto, but may proceed for 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days, 2 weeks, 3 weeks or longer.

[0120] Meanwhile, in another preferred embodiment, the present invention provides an in vitro skin mimic model obtained by the above-described method.

[0121] Existing skin models have primarily been validated using 2D culture techniques utilizing differentiated cells such as keratinocytes or dermal fibroblasts. This has had limitations in mimicking phenotypes based on cell-to-cell interactions and in vivo conditions. Additionally, previous attempts to generate skin organoids from induced pluripotent stem cells have had limitations, such as inconsistencies in differentiation patterns, long durations, and the inability to perform subculture.

[0122] However, the present invention was able to consistently differentiate skin epithelial organoids, skin epithelial tissues using the same, and skin hair follicle organoids by the above-described method, and accordingly, was able to specifically mimic the interactions of perfect skin epithelial tissues.

[0123] It is known that the interaction between hair follicle stem cells and epithelial stem cells plays a crucial role in maintaining skin tissue homeostasis and is well known as a major target cell for skin functions associated with wound healing, regeneration, and hair growth. As described above, the present invention can effectively mimic skin epithelial tissue and hair follicle structures from skin epithelial organoids, thereby enhancing functionality as an in vitro model.

[0124] Accordingly, the present invention provides a screening method for a skin disease treatment, comprising the step of treating the above-described in vitro skin mimic model with a test substance as another preferred embodiment.

[0125] The above skin disease may be any one selected from skin diseases caused by fine dust, urticaria, folliculitis, keratosis pilaris, milia, eczema, vitiligo, athlete's foot, rash, burns, blisters, dry skin, hirsutism, scalp dermatitis, dermatitis, hair loss, skin cancer, psoriasis, seborrheic dermatitis, contact dermatitis, and hyperpigmentation. Most preferably, it may be any one selected from skin diseases caused by fine dust, dermatitis, skin cancer, and contact dermatitis.

[0126] In addition, the skin disease caused by the fine dust mentioned above may be due to a decrease in the expression of the P63 gene or an increase in the expression of any one gene selected from the group consisting of KRT1, IVL, FLG, and LOR.

[0127] In this case, the term “treatment” refers to a concept that includes the suppression, elimination, alleviation, relief, improvement, and / or prevention of a disease, or symptoms or conditions resulting from a disease.

[0128] In this case, the “test substance” is a substance expected to improve or treat the aforementioned skin disease.

[0129] The skin-mimicking model according to the present invention was able to consistently differentiate skin epithelial tissue and skin follicle organoids. Accordingly, by specifically mimicking the interactions of perfect skin epithelial tissue, it is capable of studying molecular genetic and cellular biological interaction mechanisms as well as mimicking various skin diseases, and thus has the characteristic of being widely applicable to future disease modeling, new drug and therapeutic development.

[0130] In particular, the skin epithelial organoid model according to the present invention can reproduce skin tissue responses to external harmful factors (e.g., fine dust) at the morphological, molecular, and transcriptomic levels, making it applicable in various research and development fields such as toxicity assessment of environmental hazardous substances, functional analysis, and verification of drug candidates.

[0131] In one embodiment of the present invention, single-cell derived organoids were cultured and then treated with (ultra)fine dust at various concentrations and times to observe structural changes, oxidative stress (ROS), DNA damage (p-H2A.X), apoptosis (cleaved Caspase-3 and Annexin V / PI), cell cycle arrest (CENPF, AURKB), and the activation of immune responses (OAS1, OAS2, OAS3). Additionally, a decrease in basal stem cell markers (△Np63, KRT15) and an increase in differentiation and keratinization-related markers (KRT5, KRT14, KRT1, IVL, FLG, LOR, SPRR) were confirmed, confirming that epidermal homeostasis is disrupted by exogenous stimuli.

[0132] In addition, as another preferred embodiment, the present invention provides a screening method for a skin regeneration drug comprising the step of treating an in vitro skin mimic model obtained by the above-described method with a test substance.

[0133] In this case, the “test substance” is a substance expected to exhibit or influence the skin regeneration described above.

[0134] The skin-mimicking model according to the present invention was able to consistently differentiate skin epithelial tissue and skin follicle organoids, and accordingly, by specifically mimicking the interactions of perfect skin epithelial tissue, it can be utilized as a source for cell therapy and has the characteristic of being applicable to research on therapeutic agents in the fields of skin diseases and regenerative therapy.

[0135] In addition, as another preferred embodiment, the present invention provides a method for evaluating fine dust comprising the step of treating fine dust on an in vitro skin mimic model obtained by the above-described method.

[0136] In addition, as another preferred embodiment, the present invention provides a method for evaluating skin toxicity comprising the step of treating a sample on an in vitro skin mimic model obtained by the above-described method.

[0137] In this case, the “sample” may include natural extracts, compounds, lactic acid bacteria, cosmetic compositions, external skin preparation compositions, and pharmaceutical compositions for application to the skin.

[0138] Existing skin models have been primarily validated using 2D culture techniques with differentiated cells such as keratinocytes or dermal fibroblasts. This has limitations in mimicking intercellular interactions and phenotypes based on in vivo conditions.

[0139] In particular, an approach utilizing 3D organoid models is required to evaluate the effects of interactions within skin tissue regarding microenvironmental changes that may have beneficial or harmful effects on the skin. In one embodiment of the present invention, the ease of mass production is effectively increased by verifying the passage, freezing, and thawing of stem cell-derived epithelial organoids, and the potential for use as a platform to evaluate the effects of environmental stress factors such as (ultra)fine dust has been verified through objective experiments.

[0140] Furthermore, due to these characteristics, and in line with the recent trend of increasing demand for safety assessment technologies that can replace animal testing as interest in animal welfare grows, the in vitro skin mimic model according to the present invention can be utilized for evaluating the toxicity of skin-related drug efficacy tests.

[0141] Meanwhile, the skin epithelial organoid model according to the present invention can be transformed through genetic modification.

[0142] The above genetic modification may be performed at a specific point in time, for example, after 3 to 5 days have elapsed since passage, for the reconstruction of the organoid and stable gene expression.

[0143] The above genetic modification can be performed through a viral vector. For example, any one of a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, or a herpes simplex virus vector may be used, and preferably, a retrovirus vector, among them a lentivirus vector may be used.

[0144] In addition, the genetic modification may be achieved using non-viral delivery technologies, such as liposome-based carriers, or through electroporation or nucleofection.

[0145] In one embodiment of the present invention, a skin epithelial organoid having gene regulatory functions was established by stably introducing a CRISPR Interference (KRAB-dCas9-RFP(Red Fluorescent Protein) Mediated) expression system into a skin epithelial organoid via a lentivirus-mediated method. Specifically, the organoid was infected with a lentivirus vector containing polybrene, washed, reattached to an extracellular matrix-based scaffold (Matrigel, etc.), and cultured in a first maturation medium to reconstitute it into an organoid form. Subsequently, the RFP-positive organoid was isolated and cultured. The gene expression of the skin epithelial organoid transformed in this manner was stably maintained even after cryopreservation and five or more passages.

[0146] Therefore, by additionally introducing gRNA for a specific gene into the skin epithelial organoid model according to the present invention, it can be usefully utilized for selective gene expression inhibition and analysis of the resulting molecular mechanism.

[0147] As the skin epithelial organoid according to the present invention is manufactured under specific conditions, stable organoid culture is possible even under subculture, freezing, and thawing conditions. In addition, since it differentiates into a form that mimics skin epithelial tissue and hair follicle structure and can be used as a 3D in vitro skin model, it has the advantage of enabling research on molecular genetic and cellular biological interaction mechanisms, as well as screening for skin disease treatments or skin regeneration drugs as a skin disease model, and evaluating fine dust or skin toxicity.

[0148] Figure 1 shows a schematic diagram of the fabrication of a human skin epithelial organoid and representative photographs of the organoid at each differentiation stage.

[0149] Figure 2 shows the results of confirming changes in epithelial cell marker expression in human skin epithelial organoids (A: Changes in expression of epithelial cell markers by differentiation stage, B: Comparison of expression with human-derived epithelial cells; IPS: induced pluripotent stem cell, D-12: DMEM / F-12, P.: passage number, MAT: mature, EpiO: epithelial organoid, h.FB: Human Fibroblast, h.KER: human-derived epithelial cell, CRL6-Epio: induced pluripotent stem cell-derived epithelial organoid of Example 1).

[0150] Figure 3 shows the results of analyzing the optimal culture conditions for human skin epithelial organoids (A: Conditions by medium, B: Representative image of organoids according to culture conditions, C: Cell proliferation curve according to culture conditions, D: Comparison of expression of epithelial cell markers (immuno-staining), E: Comparison of expression of epithelial cell markers (Real-time qPCR)).

[0151] Figure 4 shows the results of verifying the phenotype of human skin epithelial organoids (A: Representative image by passage date, B: Cell proliferation curve according to passage, C: RNA-sequencing (heatmap), D: PCA analysis, E: GSEA plot, F: Verification of epithelial cell marker expression).

[0152] Figure 5 shows the results confirming the reorganization of human skin epithelial organoids and the induction of differentiation into skin follicle appendages (A: H&E staining of reconstructed skin epithelial tissue, B: immuno-staining of reconstructed skin epithelial tissue, C: H&E staining of differentiation-induced 3D follicle tissue, D: immuno-staining of differentiation-induced 3D follicle tissue)

[0153] Figure 6 shows the results of evaluating the effects of (ultra)fine dust based on a human skin epithelial organoid model (A: experimental schematic, B: representative photographs of organoids according to fine dust treatment concentrations, C: comparison of cell growth and expression of epithelial cell-specific markers according to (ultra)fine dust exposure).

[0154] Figure 7 shows the results of evaluating structural changes, oxidative stress, cell growth rate, and damage caused by (ultra)fine dust using human pluripotent stem cell-derived skin epithelial organoids (A: Experimental schematic, B: Representative image of organoids according to fine dust treatment time, C: Measurement of ROS production, D: Change in cell proliferation rate, E: Comparison of KI67 and p-H2A.X expression, F: Analysis of gene expression related to cell stress, apoptosis, cell cycle arrest, and aging).

[0155] Figure 8 shows the results of evaluating the apoptotic response in skin epithelial organoids treated with (ultra)fine dust (A: immunofluorescence staining using cleaved Caspase-3, B: flow cytometry based on Annexin V / PI staining).

[0156] Figure 9 shows the results of the analysis of transcriptome expression in skin epithelial organoids treated with (ultra)fine dust (A: hierarchical clustering analysis, BC: gene ontology analysis for each cluster; DF: qRT-PCR analysis).

[0157] Figure 10 shows the results of changes in the expression of basal stem cell markers (△Np63 and KRT15) in skin epithelial organoids treated with (ultra)fine dust (A: immunofluorescence staining; B: qRT-PCR analysis).

[0158] Figure 11 shows the results of changes in the expression of differentiation markers (KRT5, KRT14, KRT1) in skin epithelial organoids treated with (ultra)fine dust (A: immunofluorescence staining; B: qRT-PCR analysis).

[0159] Figure 12 shows the results of analyzing changes in the expression of keratinization-related markers (IVL, FLG, LOR, SPRR families) in skin epithelial organoids treated with (ultra)fine dust (A: GSEA analysis, B: immunofluorescence staining, C: qRT-PCR analysis).

[0160] Figure 13 shows the results of analyzing a transformed skin epithelial organoid produced through lentivirus-mediated genetic modification.

[0161] The present invention will be described in more detail below through examples. These examples are solely for illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0162] Example 1. Preparation of skin epithelial organoids from human pluripotent stem cells

[0163] Differentiation into non-neural ectoderm epithelial progenitor cells was induced for a total of 12 days by regulating the BMP, FGF, and TGF-β signaling pathways of human pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells. Air-liquid interface culture was used as the culture method.

[0164] Specifically, two days prior to the start of differentiation, human embryonic stem cells (H9) or human induced pluripotent stem cells (normal-iPS) cultured in E8 media (DMEM / F12, L-ascorbic acid-2-phosphate magnesium (64 mg / L), sodium selenium (14 µg / L), FGF2 (100 µg / L), insulin (19.4 mg / L), NaHCO3 (543 mg / L) and transferrin (10.7 mg / L), TGF-β1 (2 µg / L)) were isolated as single cells and seeded onto a 96-well u-bottom plate at a density of 3,500 cells per well to induce the formation of 3D spheroids. At this time, Y-27632 was added to the medium to enhance cell viability.

[0165] On the day of differentiation, differentiation into a non-neural ectoderm was induced by replacing the differentiation medium, E6 media (DMEM / F12, L-ascorbic acid-2-phosphate magnesium (64 mg / L), sodium selenium (14 μg / L), insulin (19.4 mg / L), NaHCO3 (543 mg / L), transferrin (10.7 mg / L), NODAL (100 μg / L)), with a medium supplemented with 2% (w / v) GFR (growth factor reduced) Matrigel (Corning), 5 ng / ml BMP-4 (Peprotech), 10 μM SB431542 (Stemgent), and 4 ng / ml Fgf-2 (Peprotech).

[0166] Subsequently, on the third day of differentiation, 25 μl of differentiation medium containing 1 μM of LDN-193189 (Stemgent) and 250 ng / mL of Fgf-2 (Peprotech) was added to each well. Accordingly, differentiation into cranial neural crest (CNC-like cell) was induced.

[0167] In addition, 75 µl of differentiation medium (E6 media) was added on the 6th day of differentiation, and on the 8th and 10th days of differentiation, half of the medium was discarded and half of the new differentiation medium was added.

[0168] On day 12 of differentiation, 8 differentiation-induced spheroids were collected and dissociated into single cells by treatment with trypLE express (Gibco) at 37°C for 15 minutes. 2 x 10 dissociated cells 5 Cells were resuspended in 40 µl of DMEM / F-12 basal medium and mixed with GFR matrigel in a 1:1 ratio.

[0169] Mixed cells were seeded into 6.5 mm Transwells with 0.4 µm pores and cultured at 37°C for 20 minutes to solidify. Maturation medium (Advanced DMEM / F-12 (Gibco), 1x Penicillin / Streptomycin (Gibco)

[0170] 1x B27 serum-free supplement (Gibco), 1x Glutamax (Gibco), 1x HEPES (Gibco), 1 mM NAC (N-acetylcysteine) (Sigma), 10 ng / ㎖ Forskolin (Sigma), 100 ng / ㎖ WNT-3A (R&D), 50 ng / ㎖ EGF, 100 ng / ㎖ KGF (peprotech), 1 μM A83-01) were added to the top and bottom layers of the transwell along with 10 μM Y-27632 (R&D) and 5% FBS.

[0171] After 2 days, cells were resuspended in basal medium and mixed with GFR matrigel in a 1:9 ratio. This mixture was implanted into Transwells and coagulated, then maintained in culture medium to induce organoid formation. At this time, a maturation medium excluding FBS and Y-27632 was used as the culture medium.

[0172] Subsequently, as shown in Figure 1, in order to form a stable organoid, a skin epithelial organoid, which is an in vitro epithelial model derived from human pluripotent stem cells capable of stable organoid culture, was prepared by subculturing at least 5 times in a transwell at weekly intervals.

[0173] Experimental Example 1. Evaluation of changes in gene expression in epithelial tissue of skin epithelial organoids

[0174] Samples were taken at each organoid maturation stage (when subcultured) and mRNA expression levels were quantified via qRT-PCR.

[0175] As shown in Figure 2, the induced pluripotent stem cell-derived epithelial organoid (Example 1; CRL6-Epio) obtained by subculturing five or more times showed that the expression of representative gene markers of basal epithelial cells, KRT5, KRT14, p63, and SOX9, was similar to that of human-derived epithelial cells (h.KER).

[0176] In addition, it was confirmed that human induced pluripotent stem cell-derived epithelial organoids showed increased expression of keratin, including genes related to keratinized epithelial cells (INVOLUCRIN (IVL), LORICRIN (LOR), FILAGGRIN (FLG)) and interfollicular epithelial cell-related genes (KRT10, KRT1), compared to conventional human-derived epithelial cells.

[0177] In other words, the expression of epithelial cell markers increased with each passage or maturation stage, and it was confirmed that stable expression was maintained after 5 passages.

[0178] Example 2: Establishment of Optimal Culture Conditions for Skin Epithelial Organoids

[0179] In order to establish optimal culture conditions for stably maintaining skin epithelial organoids after differentiation, as shown in Figure 3A, media (#1) containing A83-01, EGF, WNT-3A, and KGF, media (#2) containing WNT-3A and KGF, and media (#3) containing KGF, Noggin, and R-spondin were prepared. In addition, samples cultured for 14 days using media #1 to #3, respectively, as culture media according to the method of Example 1 were used.

[0180] As shown in Figure 3, the cell growth rate of organoids cultured in medium (#1) containing A83-01, EGF, WNT-3A, and KGF was the highest (B, C), and it was confirmed that the expression of KRT10, KRT1, IVL, FLG, and LOR, which are genes related to epithelial differentiation and keratinized epithelial cells, was maintained at a high level (D, E).

[0181] Experimental Example 2. Subculture, freezing and thawing, and verification of differentiation degree of skin epithelial organoids

[0182] After treating organoids with trypLE at 37°C for 15 minutes to dissociate them into single cells, 1–2 x 10⁶ 90% GFR per 10 µL of matrigel 4 Cultured at cell density.

[0183] As shown in Fig. 4, the human pluripotent stem cell-derived skin epithelial organoid according to Example 2 dissociated into single cells, with 1–2 x 10⁶ 90% GFR per 10 µL of matrigel. 4 It was confirmed that the cells were cultured at a cell density and continued to proliferate for up to 14 days (A, B).

[0184] In addition, through RNA sequencing, a next-generation sequencing method, it was confirmed that the human pluripotent stem cell-derived skin epithelial organoid according to Example 2 underwent a keratinization process or differentiation into keratinized cells (C to E).

[0185] In addition, on the 4th or 5th day after subculturing the organoids, organoids (at least 10 Matrigel domes, 100 µl) were collected and frozen in a mixture of 90% FBS and 10% DMSO. The frozen organoids were rapidly thawed in a 37°C water bath, resuspended in 100 µl of 90% GFR Matrigel, distributed into 10 µl portions, solidified in a 37°C incubator, and then cultured.

[0186] As shown in Figure 4, stable expression of basal epithelial cell markers (p63, KRT5, KRT14) and keratinization-inducing markers (KRT1, KRT10, IVL, FLG, LOR) was confirmed in skin epithelial organoids that were frozen, thawed, and subcultured (F).

[0187] Example 3. Evaluation of in vitro epithelial tissue reorganization and differentiation into hair follicle structures of skin epithelial organoids

[0188] Epithelial stem cells are known to have the potential to differentiate into skin appendages such as hair follicles. The developed skin epithelial organoid contains stem cell populations.

[0189] Accordingly, the in vitro reorganization and differentiation into hair follicle structures of human pluripotent stem cell-derived skin epithelial organoids according to Example 2 were evaluated.

[0190] As shown in Fig. 5, when skin epithelial organoids were reorganized using an air-liquid interface culture (ALI culture) utilizing a transwell, it was confirmed that they structurally mimic skin epithelial tissue (A, B).

[0191] In addition, when human pluripotent stem cell-derived skin epithelial organoids according to Example 2 were mixed with mouse newborn dermal cells in a 1:1 ratio and cultured, it was confirmed that differentiation into a form mimicking the structure of a hair follicle was possible in vitro (C, D).

[0192] Example 4. Evaluation of the impact of (ultra)fine dust based on a skin epithelial organoid platform

[0193] We aimed to verify the utility of skin epithelial organoids as a model for studying the effects of (ultra)fine dust on skin tissue.

[0194] To this end, two days prior to treatment with (ultra)fine dust, skin epithelial organoids dissociated into single cells were cultured in a GFR matrigel dome. Subsequently, (ultra)fine dust was applied to the organoid medium at a concentration of 100 μg / ml on days 0, 2, and 4, and on day 5 of (ultra)fine dust treatment, the organoids were collected and mRNA expression levels were confirmed by qRT-PCR.

[0195] As shown in Figure 6A, skin epithelial organoids dissociated into single cells before treatment with (ultra)fine dust were cultured in a GFR matrigel dome for 2 days, and then treated with (ultra)fine dust starting from the 2nd day, and the effects of fine dust were evaluated on the 7th day.

[0196] As shown in Figure 6, it was confirmed that organoid growth was inhibited, as the longer the (ultra)fine dust treatment time, the smaller the organoid size and the inhibited expression of the growth factor marker KI67 (B, C). In addition, an increase in the expression of the p53 and p21 cell cycle inhibitor genes was confirmed (C).

[0197] In addition, it was confirmed that as the treatment time with (ultra)fine dust increased, the expression of the basal stem cell marker p63 decreased significantly, and the expression of markers contributing to keratinization, such as KRT1, IVL, FLG, and LOR, increased, thereby confirming that (ultra)fine dust inhibited the expression of skin stem cell markers and induced skin keratinization (C).

[0198] Accordingly, it is determined that the skin epithelial organoid according to the present invention can be utilized as a skin epithelial model for evaluating skin diseases.

[0199] Meanwhile, as shown in Fig. 7A, skin epithelial organoids dissociated into single cells were cultured in a GFR matrigel dome for 7 days, and then treated with (ultra)fine dust at intervals (3 and 5 days).

[0200] Structural changes in the organoids were observed using phase-contrast microscopy, and as the concentration of (ultra)fine dust and treatment time increased, the size and number of organoids decreased, the maintenance of the spherical structure was inhibited, and internal vacuolization increased (Fig. 7B).

[0201] In addition, when the amount of ROS (reactive oxygen species) produced following treatment with 100 μg / mL of (ultra)fine dust was measured, intracellular ROS significantly increased as the treatment time increased (Fig. 7C). This suggests that exposure to fine dust induces oxidative stress in epidermal organoids.

[0202] In other words, it was confirmed that exposure to fine dust induces time-dependent oxidative stress, which inhibits the growth of organoids.

[0203] Next, the effects of (ultra)fine dust on the growth and proliferation of organoids were examined. Analysis of changes in organoid size and cell growth rates revealed that the longer the treatment time with 100 μg / mL of (ultra)fine dust, the smaller the organoid size became, and in particular, the cell growth rate decreased significantly starting from the 5th day of treatment (Fig. 7D).

[0204] In addition, it was confirmed that organoid growth was inhibited as the treatment time with (ultra)fine dust at 100 μg / mL increased, with a decrease in the expression of the growth factor marker KI67 (Fig. 7E, up), and cell damage was confirmed to increase with an increase in the expression of p-H2A.X, a marker associated with Double-Strand Breaks (DSB) (Fig. 7E, bottom). As the concentration and duration of (ultra)fine dust increased, along with the inhibition of KI67 expression, the expression of p53 and p21, genes reflecting cell stress, apoptosis, cell cycle arrest, and senescence, increased (Fig. 7F).

[0205] In addition, the presence of cell death caused by (ultra)fine dust was analyzed using immunofluorescence staining with cleaved Caspase-3, and a pattern of cell death induction was observed starting from the 3rd day of treatment (Fig. 8A). Flow cytometry based on Annexin V / PI staining also confirmed that both early and late apoptosis rates increased (Fig. 8B).

[0206] In other words, it was confirmed that fine dust treatment not only inhibits cell proliferation but also causes direct genetic damage and induces apoptosis.

[0207] Next, hierarchical clustering analysis was performed on genes showing changes in expression according to (ultra)fine dust treatment time, and a total of 6 clusters were classified based on similar expression patterns.

[0208] Specifically, Cluster 1-3 are gene groups whose expression increases with increasing treatment time, Cluster 1 mainly contained genes related to immune response, while Cluster 2 and Cluster 3 contained an abundance of genes related to apoptosis, keratinocyte differentiation, skin barrier formation, and cornified envelope formation.

[0209] Clusters 4-6 are gene groups whose expression decreases with increasing treatment time, and Cluster 4 included genes related to epidermal development, wound healing, and the WNT signaling pathway. Cluster 5 included genes related to oxidative stress, hypoxic response, and response to exogenous stimuli, while Cluster 6 consisted of genes related to skin morphogenesis, cell cycle and cell division, and the extracellular matrix (ECM) (Fig. 9).

[0210] As such, GSEA (Gene set enrichment analysis) was performed on gene groups exhibiting changes in expression patterns following (ultra)fine dust treatment. As a result, the expression of gene groups related to the cell cycle (G2 / M checkpoint) decreased in the group treated with fine dust. In addition, qRT-PCR confirmed that the expression levels of representative genes CENPF and AURKB decreased in a concentration-dependent manner (Fig. 9D).

[0211] On the other hand, the gene group related to immune response increased, and in particular, the expression of OAS1, OAS2, and OAS3 genes gradually increased, confirming that the immune response of the skin epithelial organoid was activated (Fig. 9E). In addition, the gene group related to apoptosis also showed increased expression as the (ultra)fine dust treatment time increased (Fig. 9F).

[0212] In other words, it was confirmed that various biological response pathways of organoids are simultaneously regulated by fine dust treatment.

[0213] Next, analysis by immunofluorescence staining revealed that the expression of basal stem cell markers △Np63 and KRT15 decreased significantly as the treatment time with (ultra)fine dust increased (Fig. 10). On the other hand, the expression of differentiation markers KRT5, KRT14, and KRT1, as well as the IVL, FLG, LOR, and SPRR families of markers contributing to keratinization, was found to increase (Figs. 11 and 12).

[0214] In other words, it was confirmed that fine dust disrupts epidermal homeostasis by promoting the differentiation and keratinization processes of stem cells.

[0215] In summary, the aforementioned experiments confirmed that treatment with (ultra)fine dust induces various pathological changes in skin epithelial organoids, including structural changes, growth inhibition, activation of apoptosis, inhibition of the cell cycle, and activation of immune responses. This demonstrates that the human skin epithelial organoid model of the present invention can systematically reproduce skin tissue responses to external harmful environmental stimuli at the morphological, molecular, and transcriptomic levels, suggesting that it is a model applicable in various fields such as environmental hazardous substance toxicity assessment and skin disease research.

[0216] Example 5. Preparation of transgenic skin epithelial organoids through genetic modification and applications thereof

[0217] An inducible CRISPR expression system was introduced into skin epithelial organoids differentiated from human pluripotent stem cells. To this end, stable genetic modification was induced using a lentivirus.

[0218] Specifically, organoids on the 3rd day of culture after subculturing were isolated from Matrigel and infected with a lentivirus containing polybrene for 6 hours. After infection, the cells were harvested, washed twice with DMEM / F12 medium containing 5% FBS, and 10 μL of DMEM / F12 medium and GFR Matrigel were mixed in a 1:9 ratio and plated in dome shapes. Culture was initially carried out in the first maturation medium, then switched to the second maturation medium (Y-27632 (10 μM) added to the first maturation medium) the following day, and the Y-27632 was removed the next day.

[0219] The transformation status of the organoids was confirmed through RFP expression. After 7 days had passed since infection, observation using a fluorescence microscope confirmed that most organoids contained RFP-positive cells, and subsequently, RFP-expressing cells were selected through FACS analysis. The selected RFP-positive cells were then plated in dome shapes in 10 μL portions of a mixture of DMEM / F12 medium and GFR Matrigel at a ratio of 1:9, and cultured under the same conditions.

[0220] The genetically modified skin epithelial organoids obtained through the above process maintained stable RFP expression even after cryopreservation, and RFP-positive cells were continuously maintained even after more than 5 passages (Fig. 13).

[0221] Therefore, through the above experiment, it was confirmed that a transgenic organoid capable of long-term stable gene expression can be manufactured. In addition, the Inducible CRISPRi organoid system according to the present embodiment can be utilized for selective gene expression inhibition and subsequent molecular mechanism analysis by additionally introducing gRNA for target genes.

Claims

1. a) A step of differentiating pluripotent stem cells into ectoderm by treating them with a differentiation medium containing a BMP activator, FGF family, TGF-β inhibitor, and extracellular matrix; b) A step of differentiating the ectoderm into cranial neural crest-like cells by treating the ectoderm with a differentiation medium containing a BMP inhibitor and the FGF family; c) a step of inducing differentiation of skin epithelial organoids by adding culture medium to cranial neural crest-like cells; d) a step of maturing skin epithelial organoids by treating them with a first maturation medium containing a ROCK inhibitor, a TGF-β inhibitor, a Wnt activator, and a ligand for a tyrosine kinase receptor; and e) a step of culturing the matured skin epithelial organoid by subculturing it at least 5 times in a second maturation medium containing a TGF-β inhibitor, a Wnt activator, and a ligand for a tyrosine kinase receptor; comprising a method for producing a skin epithelial organoid.

2. A method for preparing a skin epithelial organoid according to claim 1, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

3. A method for manufacturing a skin epithelial organoid according to claim 1, wherein the FGF family is any one selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, and FGF10.

4. A method for preparing a skin epithelial organoid according to claim 1, wherein the TGF-β inhibitor is any one selected from the group consisting of A83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-36494 and SJN-2511.

5. A method for preparing a skin epithelial organoid according to claim 1, wherein the BMP inhibitor is any one selected from the group consisting of Noggin, Dorsomorphin, DMH-1, DMH-2, K02288, LDN-193189, LDN-212854, LDN-214117 and ML347.

6. A method for preparing a skin epithelial organoid according to claim 1, wherein the maturation of step d) is performed under a first maturation medium, and said first maturation medium comprises Y-27632, WNT-3A, EGF, KGF and A83-01.

7. A method for preparing a skin epithelial organoid according to claim 1, wherein the Wnt activator is any one selected from the group consisting of WNT-3A, R-spondin 1, R-spondin 2, R-spondin 3 and R-spondin 4.

8. A method for preparing a skin epithelial organoid according to claim 1, wherein the ligand of the tyrosine kinase receptor is any one selected from the group consisting of NRG1 (Neuregulin β1), HRG1 (Heregulin β1), epidermal growth factor (EGF), transforming growth factor-α (TGF-α), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF), and keratinocyte growth factor (KGF).

9. A method for preparing a skin epithelial organoid according to claim 1, wherein the second maturation medium of step e) comprises A83-01, EGF, WNT-3A, and KGF.

10. A method for producing a skin epithelial organoid according to claim 1, wherein the organoid expresses one or more genes selected from the group consisting of P63, KRT5, KRT14, KRT1, KRT10, IVL, FLG, and LOR.

11. A method for producing a skin epithelial organoid according to claim 1, wherein the subculture of step e) is performed at intervals of 5 to 10 days.

12. A method for producing a hair follicle organoid comprising the step of mixing and culturing a skin epithelial organoid according to any one of claims 1 to 11; and dermal cells.

13. A method for manufacturing a hair follicle organoid comprising the step of mixing and culturing the skin epithelial organoid and dermal cells in a ratio of 0.5 to 2:0.5 to 2 according to claim 12. 14.i) A step of differentiating pluripotent stem cells into ectoderm by treating them with a differentiation medium containing a BMP activator, FGF family, TGF-β inhibitor, and extracellular matrix; ii) A step of differentiating the ectoderm into cranial neural crest-like cells by treating it with a differentiation medium containing a BMP inhibitor and the FGF family; iii) a step of inducing differentiation of skin epithelial organoids by adding culture medium to cranial neural crest-like cells; iv) a step of maturing skin epithelial organoids by treating them with a first maturation medium containing a ROCK inhibitor, a TGF-β inhibitor, a Wnt activator, and a ligand for a tyrosine kinase receptor; and v) a step of reorganizing a skin epithelial organoid on top of a transwell through an air-liquid interface culture; a method for preparing skin epithelial tissue comprising 15. An in vitro skin mimic model obtained by the method of any one of claims 1 to 11.

16. A method for screening a skin disease treatment agent, comprising the step of treating an in vitro skin mimic model according to paragraph 15 with a test substance.

17. A screening method for therapeutic agents according to claim 16, wherein the skin disease is any one selected from skin disease caused by fine dust, dermatitis, skin cancer, and contact dermatitis.

18. A screening method for therapeutic agents according to claim 17, wherein the skin disease caused by fine dust is characterized by a decrease in the expression of the P63 gene or an increase in the expression of any one gene selected from the group consisting of KRT1, IVL, FLG, and LOR.

19. A screening method for a skin regeneration drug comprising the step of treating an in vitro skin mimic model according to paragraph 15 with a test substance.

20. A method for evaluating fine dust comprising the step of treating fine dust on an in vitro skin mimic model according to paragraph 15.

21. A method for evaluating skin toxicity comprising the step of treating a sample in an in vitro skin mimic model according to paragraph 15.

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