A method for producing alveolar organoids using alveolar cell-derived iPSCs(induced pluripotent stem cells)

Abstract

The present invention relates to a method for producing alveolar organoids using induced pluripotent stem cells derived from alveolar cells. The alveolar organoids produced according to the present invention are differentiated from human alveolar cell-derived induced pluripotent stem cells and can efficiently differentiate into alveolar organoids due to the epigenetic memory of alveolar cells. It is expected that this can be usefully utilized for research on the pathogenesis of respiratory diseases and for screening to discover therapeutic drugs.

Description

Technology Field

[0001] The present invention relates to a method for producing alveolar organoids using human alveolar cell-derived induced pluripotent stem cells, etc. Background Technology

[0003] Organoids are mini-organs created by culturing or recombining cells isolated from stem cells or organ cells. They are characterized by their ability to differentiate into actual tissues as closely as possible, based on the fact that they possess structures and functions very similar to the corresponding organs.

[0004] Recently, research on organoids as a form of regenerative medicine or cell therapy has been actively underway. While attempts to analyze actual drug metabolic responses are primarily conducted in animal experiments, the genetic structures of humans and animals are not 100% identical; consequently, substances that elicit no reaction in animals can be fatal to humans, raising concerns regarding the infringement of animal rights. Furthermore, since cells cultured in two dimensions are immortalized, their drug metabolic capabilities differ from those of actual organs, making accurate experiments difficult. Additionally, while the most effective method for evaluating drug metabolism is to directly utilize human organ cells, there are limitations in obtaining these tissues, and obtaining large quantities is challenging due to the difficulty of in vitro proliferation.

[0005] Meanwhile, recent research findings indicate a correlation between increased concentrations of fine dust, a source of air pollution, and increased mortality rates associated with lung diseases, including cancer. Furthermore, while lung disease is known to be the third leading cause of death worldwide, the mechanisms for restoring damaged lungs remain unknown.

[0006] Although the lungs are structurally complex and contain a diverse range of cell types compared to other organs, they have not yet been the subject of much research. In particular, alveolar type 2 cells (AT2 cells) make up about 7% of the alveolar epithelial cells in lung tissue (The Formation of Pulmonary Alveoli; Stephen E. McGowan; in The Lung (Second Edition), 2014), and they play a role in preventing collapse by producing surfactant proteins to maintain lung function and surface tension.

[0007] However, when type 2 alveolar cells are cultured in 2D, they take on a phenotype similar to that of type 1 alveolar cells, making culture difficult, and it is difficult to maintain type 2 alveolar cells in human lung tissue. Prior art literature

[0009] Republic of Korea Registered Patent No. 10-2043603 The problem to be solved

[0010] Accordingly, the inventors produced an alveolar organoid using human alveolar cell-derived induced pluripotent stem cells and developed a culture medium suitable for producing alveolar organoids.

[0011] Accordingly, the object of the present invention is to provide a method for producing alveolar organoids from alveolar cell-derived induced pluripotent stem cells.

[0012] Another objective of the present invention is to provide an alveolar organoid produced by the method of producing an alveolar organoid according to the present invention.

[0013] Another objective of the present invention is to provide a kit for producing alveolar organoids.

[0014] Another objective of the present invention is to provide a composition for producing alveolar organoids.

[0015] Another objective of the present invention is to provide a tissue therapeutic agent comprising an alveolar organoid according to the present invention.

[0016] Another objective of the present invention is to provide a method for screening therapeutic agents for lung-related diseases using alveolar organoids according to the present invention.

[0018] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below. means of solving the problem

[0020] To achieve the objective of the present invention, the present invention provides a method for producing an alveolar organoid from induced pluripotent stem cells derived from alveolar cells, comprising the following steps:

[0021] a) A step of inducing differentiation into endoderm cells by culturing alveolar cell-derived induced pluripotent stem cells in a medium for inducing endoderm cell differentiation;

[0022] b) A step of inducing differentiation of the endoderm cells from step a) into anterior foregut endoderm cells in AFM (Anterior Foregut Endoderm Media) medium;

[0023] c) a step of inducing differentiation of the anterior full-length endoderm cells of step b) into alveolar progenitors in a progenitor differentiation medium; and

[0024] d) A step of culturing the alveolar precursor of step c) above in AM (Alveolar Media).

[0025] In one embodiment of the present invention, the alveolar cells may be type 2 alveolar cells, but are not limited thereto.

[0026] In another embodiment of the present invention, the AFM medium may include, but is not limited to, one or more selected from the group consisting of Ham's F12, dexamethasone, IBMX (3-Isobutyl-methylxanthine), B27 supplement, N2 supplement, BSA (Bovine serum albumin), HEPES (Hydroxyethyl piperazine Ethane Sulfonicacid), calcium chloride (CaCl2), 8-Br-cAMP, Recombinant Human KGF, Recombinant Human FGF-10, Anti Antibiotic-Antimycotic, Ascobic acid, Glutamax, Recombinant Human BMP4, and CHIR99021.

[0027] In another embodiment of the present invention, the AM medium may comprise one or more selected from the group consisting of Ham's F12, dexamethasone, IBMX (3-Isobutyl-methylxanthine), B27 supplement, N2 supplement, BSA (Bovine serum albumin), HEPES (Hydroxyethyl piperazine Ethane Sulfonicacid), calcium chloride (CaCl2), 8-Br-cAMP, Recombinant Human KGF, Recombinant Human FGF-10, and Anti Antibiotic-Antimycotic, but is not limited thereto.

[0028] In another embodiment of the present invention, steps b) to d) may be performed by a three-dimensional culture method, but are not limited thereto.

[0029] In addition, the present invention provides a kit for producing alveolar organoids comprising a medium for inducing endoderm cell differentiation, an AFM (Anterior Foregut Endoderm Media) medium, a precursor differentiation medium, and an AM (Alveolar Media) medium.

[0030] In one embodiment of the present invention, the kit may further include, but is not limited to, instructions describing a method for producing alveolar organoids from alveolar cell-derived induced pluripotent stem cells according to the present invention.

[0031] In addition, the present invention provides a composition for producing alveolar organoids.

[0032] In addition, the present invention provides an alveolar organoid produced by the method of producing an alveolar organoid according to the present invention.

[0033] In addition, the present invention provides a tissue therapeutic agent comprising an alveolar organoid according to the present invention.

[0034] In addition, the present invention provides a method for preventing or treating lung-related diseases, comprising the step of administering and / or transplanting an alveolar organoid according to the present invention to an individual in need thereof.

[0035] In addition, the present invention provides a use of the alveolar organoid according to the present invention for the prevention or treatment of lung-related diseases.

[0036] In addition, the present invention provides a use for manufacturing a lung-related disease treatment using an alveolar organoid according to the present invention.

[0037] In addition, the present invention provides a method for screening therapeutic agents for lung-related diseases using alveolar organoids according to the present invention.

[0038] In one embodiment of the present invention, the method may include, but is not limited to, the step of treating the organoid with a test substance; and the step of selecting the test substance as a therapeutic agent for a lung-related disease if, after treatment with the test substance, the expression of a biomarker protein for a lung-related disease or mRNA encoding the same increases or decreases compared to before treatment.

[0039] In another embodiment of the present invention, the lung-related disease may be selected from the group consisting of cystic fibrosis, respiratory distress syndrome, acute respiratory distress syndrome, pulmonary tuberculosis, cough, bronchial asthma, cough based on increased airway hyperresponsiveness, influenza syndrome, cough suppression, airway hyperresponsiveness, tuberculosis disease, asthma, chronic obstructive pulmonary disease, emphysema, pulmonary fibrosis, idiopathic pulmonary fibrosis, reversible airway obstruction, adult respiratory disease syndrome, pigeon breeder's disease, farmer's lung, bronchopulmonary dysplasia, airway disease, emphysema, allergic bronchopulmonary aspergillosis, allergic bronchitis bronchiectasis, occupational asthma, reactive airway disease syndrome, interstitial lung disease, and parasitic lung disease, but is not limited thereto. Effects of the invention

[0041] The alveolar organoid produced according to the present invention is differentiated from human alveolar cell-derived induced pluripotent stem cells and can efficiently differentiate into an alveolar organoid due to the epigenetic memory of the alveolar cells; therefore, it is expected to be usefully utilized for research on the pathogenesis of respiratory diseases and screening for the discovery of therapeutic drugs. Brief explanation of the drawing

[0043] Figure 1 is a diagram showing the process of manufacturing induced pluripotent stem cells (iPSCs) by reprogramming human alveolar cells (AT2 cells). Figure 2 shows the results of analyzing the protein characteristics of induced pluripotent stem cells (iPSCs) produced by reprogramming human alveolar cells (AT2 cells) using immunofluorescence staining. The figure (left) shows the relative expression levels of the iPSC undifferentiation markers Oct3 / 4, NANOG, and LIN28 in AT2 cells, and the figure (right) shows the results of observing the expression of Oct3 / 4, SSEA4, TRA-1-60, and TRA-1-81 using a confocal microscope. Figure 3 is a diagram showing the results of observing the differentiation process of alveolar organoids from induced pluripotent stem cells (iPSCs) produced by reprogramming human alveolar cells (AT2 cells) using an optical microscope. Figure 4 is a diagram showing a schematic protocol for differentiating alveolar organoids from induced pluripotent stem cells (iPSCs) produced by reprogramming human alveolar cells (AT2 cells). Figure 5 is a diagram showing the results of confirming whether induced pluripotent stem cells (iPSCs) produced by reprogramming human alveolar cells (AT2 cells) differentiated into the endoderm using immunofluorescence staining, and confirming whether they differentiated normally into the endoderm using endoderm characteristic factors SOX17 and FOXA2. Figure 6 is a diagram showing the results of observing the differentiation process of alveolar organoids from induced pluripotent stem cells (iPSCs) produced by reprogramming human alveolar cells (AT2 cells) using immunofluorescence staining. Specific details for implementing the invention

[0044] The present invention provides a method for producing alveolar organoids from alveolar cell-derived induced pluripotent stem cells comprising the following steps:

[0045] a) A step of inducing differentiation into endoderm cells by culturing alveolar cell-derived induced pluripotent stem cells in a medium for inducing endoderm cell differentiation;

[0046] b) A step of inducing differentiation of the endoderm cells from step a) into anterior foregut endoderm cells in AFM (Anterior Foregut Endoderm Media) medium;

[0047] c) a step of inducing differentiation of the anterior full-length endoderm cells of step b) into alveolar progenitors in a progenitor differentiation medium; and

[0048] d) A step of culturing the alveolar precursor of step c) above in AM (Alveolar Media).

[0049] In the present invention, the term "organoid" refers to a three-dimensional aggregate of one or more cell types that mimics the superficial appearance or actual structure or function of a tissue or organ.

[0050] According to one embodiment of the present invention, the alveolar cells may be type 2 alveolar cells (AT2 cells), but are not limited thereto.

[0051] The above type 2 alveolar cells (AT2 cells) may be isolated from lung tissue, subcultured after isolation, or commercially purchased, but are not limited thereto.

[0052] In the present invention, "lung tissue" includes all lung tissue structures, and related tissues including but not limited to veins, arteries, blood vessels, capillaries, and cells of a type that are part of or related to said structures; lung and pleural tissues; and may include, but not limited to, vascular smooth muscle, perivascular cells, and vascular endothelial systems and / or phenotypes. The lung tissue may be isolated from mammals, such as humans, mice, rats, guinea pigs, rabbits, monkeys, pigs, horses, cattle, sheep, antelopes, dogs, or cats, but is not limited thereto. Preferably, it may be human. Methods for obtaining such lung tissue are well known in the art.

[0053] In this invention, “differentiation” refers to the phenomenon in which the structure or function of cells becomes specialized during proliferation and growth; that is, it means that the form or function of cells, tissues, etc., changes in order to perform the tasks assigned to each.

[0054] The term “induced pluripotent stem cells (iPSCs)” as used in the present invention refers to cells induced by artificially performing a dedifferentiation process (reprogramming) on ​​adult cells that have already completed differentiation, and which possess pluripotency. The induced pluripotent stem cells can differentiate into various organ cells, such as the brain and heart. In the present invention, the induced pluripotent stem cells may be cells obtained by dedifferentiating human type 2 alveolar cells, but the type of tissue is not particularly limited as long as it is of human origin.

[0055] According to one embodiment of the present invention, the dedifferentiation may use a Sendai virus, but is not limited thereto.

[0056] In the present invention, "media" refers to a medium capable of supporting differentiation induction and survival of a target cell (including tissues or organoids) in vitro, and includes all conventional media used in the industry suitable for cell culture. The medium and culture conditions can be selected according to the type of cell.

[0057] In the present invention, “medium for inducing differentiation of endoderm cells” refers to a culture medium that promotes the differentiation of induced pluripotent stem cells (iPSCs) into endoderm cells. According to one embodiment of the present invention, the STEMdiff™Definitive Endoderm Kit (STEMCELL #05110) was used, but any medium capable of inducing differentiation of iPSCs into lung endoderm cells may be used without limitation.

[0058] The above STEMdiff™Definitive Endoderm Kit may be characterized by including 100 mL of Endoderm Basal Medium, 0.35 mL of Definitive Endoderm Supplement MR (100), and 1.1 mL of Definitive Endoderm Supplement CJ (100).

[0059] According to one embodiment of the present invention, step a) may involve adding MR supplement + CJ supplement and culturing for 2 days, and then adding only CJ supplement and culturing for 1 to 5 days, 1 to 3 days, 1 to 2 days, 2 to 4 days, or 2 to 3 days.

[0060] The term “endoderm” as used in this invention refers to the innermost germ layer among the three germ layers (ectoderm, mesoderm, and endoderm) formed as a result of gastrulation, when cells migrate into the inside of the gastrula. It differentiates into respiratory and digestive organs such as the lungs, liver, and pancreas. Therefore, in order to promote the differentiation of induced pluripotent stem cells into alveolar organoids in this invention, efficient differentiation into endodermous cells is essential as a prerequisite (Tissue Engineering and Regenerative Medicine 2007, vol.4, no.2, pp. 142-149).

[0061] According to one embodiment of the present invention, the endodermous cells may express endoderm characteristic factors SOX17 and FOXA2, but are not limited thereto.

[0062] In the present invention, the AFM (Anterior Foregut endoderm Media) medium refers to a culture medium that promotes the differentiation of endoderm cells into anterior full-length endoderm cells, and may include, but is not limited to, one or more selected from the group consisting of Ham's F12, dexamethasone, IBMX (3-Isobutyl-methylxanthine), B27 supplement, N2 supplement, BSA (Bovine serum albumin), HEPES (Hydroxyethyl piperazine Ethane Sulfonicacid), calcium chloride (CaCl2), 8-Br-cAMP, Recombinant Human KGF, Recombinant Human FGF-10, Anti Antibiotic-Antimycotic, Ascobic acid, Glutamax, Recombinant Human BMP-4, and CHIR99021. Specifically, based on 500 mL of Ham's F12, the AFM medium comprises 50 to 150 μM of IBMX, 0.5 to 3% of B27 supplement, 0.1 to 2% of N2 supplement, 20 to 70 nM of dexamethasone, 0.1 to 2% of BSA, 0.5 to 1.5 mM of calcium chloride (CaCl2), 10 to 20 mM of HEPES, 10 to 200 μM of 8-BrcAMP, 1 to 20 ng / ml of Recombinant Human KGF, 1 to 20 ng / ml of Recombinant Human FGF-10, 0.1 to 2% of Anti-Antibiotic-Antimycotic, 20 to 100 μg / ml of Ascobic acid, 0.5 to 5 mM of Glutamax, 1 to 20 ng / ml of Recombinant Human BMP-4, and / or CHIR99021 may contain 1 to 10 μM, but is not limited thereto.

[0063] The culture period of step b) above is not particularly limited and may be performed, for example, for 1 to 8 days, 1 to 5 days, 1 to 3 days, 1 to 2 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 3 to 7 days, 3 to 4 days, 4 to 7 days, 4 to 5 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, but is not limited thereto.

[0064] The term "precursor differentiation medium" used in the present invention refers to a culture medium that promotes the differentiation of anterior full-length endoderm cells into precursor cells, and according to one embodiment of the present invention, SAGM medium may be used.

[0065] The above “SAGM medium” or “bovine airway growth medium” means a growth medium containing one or more of hydrocortisone, epidermal growth factor, epinephrine, transferrin, insulin, retinoic acid, triiodothyronine, and bovine serum albumin-fatty acid-free.

[0066] In the present invention, “progenitor” refers to a cell that is configured to differentiate into a specific type of cell or form a specific type of tissue, and is in a state where partial differentiation has occurred from a stem cell; the stem cell can differentiate into a final adult cell through the progenitor stage.

[0067] In the present invention, the culture period of step c) is not particularly limited and may be performed, for example, for 1 to 8 days, 1 to 5 days, 1 to 3 days, 1 to 2 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 3 to 7 days, 3 to 4 days, 4 to 7 days, 4 to 5 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, but is not limited thereto.

[0068] According to one embodiment of the present invention, the alveolar precursor may express EPCAM, an epithelial cell factor.

[0069] In the present invention, the culture period of step d) is not particularly limited, but preferably may be cultured for up to 90 days, and if the cell density becomes too high during the culture period, the process of separating the cells using Accutase and re-embedding may be repeated, but is not limited thereto.

[0070] The term “AM (Alveolar Media) medium” used in the present invention refers to a culture medium that promotes the differentiation of precursor cells into final adult cells. In the present invention, the AM medium may comprise, but is not limited to, one or more selected from the group consisting of Ham's F12, dexamethasone, IBMX (3-Isobutyl-methylxanthine), B27 supplement, N2 supplement*, BSA (Bovine serum albumin), HEPES (Hydroxyethyl piperazine Ethane Sulfonicacid), calcium chloride (CaCl2), 8-Br-cAMP, Recombinant Human KGF, Recombinant Human FGF-10, and Anti Antibiotic-Antimycotic. Specifically, the AM medium may comprise, but is not limited to, 50 to 150 μM of IBMX, 0.5 to 3% of B27 supplement, 0.1 to 2% of N2 supplement, 20 to 70 nM of dexamethasone, 0.1 to 2% of BSA, 0.5 to 1.5 mM of calcium chloride (CaCl2), 10 to 20 mM of HEPES, 10 to 200 μM of 8-BrcAMP, 1 to 20 ng / ml of Recombinant Human KGF, 1 to 20 ng / ml of Recombinant Human FGF-10, and / or 0.1 to 2% of Anti Antibiotic-Antimycotic based on 500 mL of Ham's F12.

[0071] In the present invention, steps b) to d) may be performed by a three-dimensional culture method. Here, the specific conditions for the three-dimensional culture are not particularly limited and may be performed by methods generally practiced in the art, but, for example, may be performed using an adhesive material selected from the group consisting of polyester, polyalkylene, polyfluorochloroethylene, polyvinyl chloride, polystyrene, polysulfone, cellulose acetate, glass fiber, ceramic particles, matrigel, extracellular matrix components (e.g., fibronectin, chondronectin, laminin), collagen, poly L-lactic acid, and inert metal fibers, and preferably may be performed using matrigel.

[0073] In addition, the present invention provides, in another aspect of the present invention, a kit for producing alveolar organoids comprising a medium for inducing endoderm cell differentiation, an AFM (Anterior Foregut Endoderm Media) medium, a precursor differentiation medium, and an AM (Alveolar Media) medium.

[0074] In the present invention, the kit may further include, but is not limited to, instructions describing a method for producing alveolar organoids from alveolar cell-derived induced pluripotent stem cells according to the present invention.

[0075] The above instructions may be attached to the container or packaged independently of the container.

[0076] In addition, in addition to the culture medium included in the above kit, any composition known in the art as necessary for differentiating alveolar cell-derived induced pluripotent stem cells into alveolar organoids may be included without limitation.

[0077] Additionally, the kit may additionally include a culture dish, and the culture dish may be a culture dish for suspension culture or a culture dish for adherent culture.

[0078] The above-mentioned attached culture dish may be coated with a polypeptide, and the polypeptide may be, for example, vitronectin (VTN), laminine, fibronectin, poly ornithine, or Matrigel™.

[0080] In addition, the present invention provides a composition for producing alveolar organoids in another aspect of the present invention.

[0081] The above composition for producing alveolar organoids may have the same composition as the above AFM medium or AM medium and may include, but is not limited to, one or more selected from the group consisting of Ham's F12, dexamethasone, IBMX (3-Isobutyl-methylxanthine), B27 supplement, N2 supplement, BSA (Bovine serum albumin), HEPES (Hydroxyethyl piperazine Ethane Sulfonicacid), calcium chloride (CaCl2), 8-Br-cAMP, Recombinant Human KGF, Recombinant Human FGF-10, Anti Antibiotic-Antimycotic, Ascobic acid, Glutamax, Recombinant Human BMP4, and CHIR99021.

[0082] In the present invention, the composition may further include a small airway growth medium (SAGM), but is not limited thereto.

[0083] As used in the present invention, the term “organoid production” includes all activities capable of generating or maintaining an organoid. For example, it may involve differentiating cells isolated from a cell or a specific tissue into tissue or organ cells having a specific function, and / or making the organoid survive, grow, or proliferate.

[0085] In addition, the present invention provides an alveolar organoid produced by the method according to the present invention as another aspect of the present invention. In the present invention, since the alveolar organoid is produced using induced pluripotent stem cells derived from alveolar cells, it can be efficiently differentiated into an alveolar organoid due to the epigenetic memory of the alveolar cells.

[0086] According to one embodiment of the present invention, the alveolar organoid may contain both type 2 alveolar cells and type 1 alveolar cells, but is not limited thereto.

[0088] In addition, the present invention may provide a tissue therapeutic agent comprising the alveolar organoid in another aspect of the present invention, wherein the tissue may be lung tissue, but is not limited thereto.

[0089] In addition, the present invention provides a method for preventing or treating lung-related diseases, comprising the step of administering and / or transplanting the alveolar organoid to an individual in need of it, in another aspect of the present invention.

[0090] In addition, the present invention provides a use of the alveolar organoid according to the present invention for the prevention or treatment of lung-related diseases.

[0091] In addition, the present invention provides a use for manufacturing a lung-related disease treatment using an alveolar organoid according to the present invention.

[0092] In the present invention, “individual” may be a human or non-human mammal that has developed or may develop a lung-related disease. Non-human mammals include, for example, livestock and pet mammals such as sheep, cattle, pigs, dogs, cats, and murines. Preferably, the individual is a human.

[0093] The above “lung-related diseases” may include, for example, but not limited to, cystic fibrosis, respiratory distress syndrome, acute respiratory distress syndrome, pulmonary tuberculosis, cough, bronchial asthma, cough based on increased airway hyperresponsiveness, influenza syndrome, cough suppression, airway hyperresponsiveness, tuberculosis disease, asthma, chronic obstructive pulmonary disease, emphysema, pulmonary fibrosis, idiopathic pulmonary fibrosis, reversible airway obstruction, adult respiratory disease syndrome, pigeon breeder’s disease, farmer’s lung, bronchopulmonary dysplasia, airway disease, emphysema, allergic bronchopulmonary aspergillosis, allergic bronchitis bronchiectasis, occupational asthma, reactive airway disease syndrome, interstitial lung disease, and parasitic lung disease.

[0094] In the present invention, "administration" means introducing the pharmaceutical composition of the present invention to a patient by any appropriate method, and the route of administration of the composition of the present invention may be administered via various oral or parenteral routes as long as it can reach the target tissue.

[0095] In the present invention, “prevention” refers to any act of delaying lung-related diseases through the administration or transplantation of a tissue therapeutic agent or alveolar organoid according to the present invention, “treatment” refers to any act of improving or beneficially altering the symptoms of lung-related diseases through the administration or transplantation of a tissue therapeutic agent or alveolar organoid according to the present invention, and “improvement” refers to any act of reducing parameters related to lung-related diseases, such as the severity of symptoms, through the administration or transplantation of a tissue therapeutic agent or alveolar organoid according to the present invention.

[0096] The tissue therapeutic agent of the present invention may be a pharmaceutical composition comprising the alveolar organoid as an active ingredient, and the pharmaceutical composition may further comprise a suitable carrier, excipient, and diluent commonly used in the manufacture of pharmaceutical compositions.

[0097] In the present invention, "carrier" is also called a vehicle and refers to a compound that facilitates the addition of proteins or peptides into cells or tissues. For example, dimethyl sulfoxide (DMSO) is a commonly used carrier that facilitates the introduction of many organic substances into the cells or tissues of living organisms.

[0098] In the present invention, "diluent" is defined as a compound that is diluted in water, which not only stabilizes the biologically active form of a target protein or peptide but also causes the protein or peptide to dissolve. Salts dissolved in a buffer solution are used as diluents in the field. A commonly used buffer solution is a phosphate-buffered saline solution, as this mimics the salt state of human body fluids. Since buffer salts can control the pH of the solution at low concentrations, it is rare for the buffer diluent to alter the biological activity of the compound.

[0099] In addition, the pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, and sterile injectable solutions according to conventional methods, and carriers, excipients, and diluents that may be included in the composition may include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulating, the product is prepared using diluents or excipients such as commonly used fillers, fillers, binders, wetting agents, decomposers, and surfactants. Solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules, and these solid dosage forms are prepared by mixing at least one excipient with the above compound, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid dosage forms for oral administration include suspensions, liquids, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives, in addition to commonly used simple diluents like water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As bases for suppositories, witepsol, macrogol, tween 61, cacao oil, laurin oil, glycerogelatin, etc. may be used.

[0100] The pharmaceutical composition of the present invention may be administered orally or parenterally, preferably parenterally, and in the case of parenteral administration, may be administered by intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, local administration, transdermal administration, etc.

[0101] Suitable dosages of the pharmaceutical composition of the present invention can be prescribed in various ways depending on factors such as the formulation method, mode of administration, patient's age, body weight, sex, pathological condition, food, time of administration, route of administration, excretion rate, and response sensitivity.

[0102] The pharmaceutical composition of the present invention may be prepared in a unit volume form or contained in a large volume container by formulation using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily carried out by a person skilled in the art to which the invention belongs. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0104] In addition, the present invention provides, in another aspect of the present invention, a method for screening therapeutic agents for lung-related diseases using an alveolar organoid according to the present invention.

[0105] In the present invention, the method for screening the lung-related disease treatment agent comprises the step of treating the organoid with a test substance; and

[0106] If, after treatment with the above-mentioned test substance, the expression of a biomarker protein for a lung-related disease or mRNA encoding the same increases or decreases compared to before treatment, the method may include a step of selecting the above-mentioned test substance as a therapeutic agent for a lung-related disease.

[0107] In the present invention, the substance to be tested is a substance predicted to prevent, improve, or treat lung-related diseases, and for example, the drug candidate substance, the compound to be tested, or the composition to be tested may include, but is not limited to, small molecule compounds, antibodies, antisense nucleotides, short interfering RNA, short hairpin RNA, nucleic acids, proteins, peptides, other extracts, or natural products.

[0109] The terms used in this invention have been selected based on currently widely used general terms, taking into account their functions within the invention; however, these terms may vary depending on the intent of those skilled in the art, case law, the emergence of new technologies, etc. Additionally, in specific cases, terms have been arbitrarily selected by the applicant, and in such cases, their meanings will be described in detail in the relevant description of the invention. Therefore, the terms used in this invention should be defined not merely by their names, but based on their meanings and the overall content of the invention.

[0110] Terms such as "first," "second," etc., may be used to describe various components, but said components should not be limited by said terms. These terms are used solely for the purpose of distinguishing one component from another. For example, without departing from the scope of the present invention, the first component may be named the second component, and similarly, the second component may be named the first component. The term "and / or" includes a combination of a plurality of related described items or any of a plurality of related described items.

[0111] Throughout the specification of the present invention, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components. Throughout the specification of the present invention, terms such as "approximately" and "substantially" are used to mean at or near the stated value when inherent manufacturing and material tolerances are presented in the said sense, and are used to prevent unscrupulous infringers from unfairly exploiting the disclosures in which precise or absolute values ​​are mentioned to aid in understanding the present invention.

[0112] Throughout the specification of the present invention, the term “combination thereof” included in the Markush-style expression means one or more mixtures or combinations selected from the group consisting of the components described in the Markush-style expression, and means including one or more selected from the group consisting of said components.

[0113] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention, and the scope of the invention is not limited by the following embodiments.

[0115] [Example]

[0116] Example 1. Isolation and culture of alveolar cells (AT2 cells)

[0117] The human lung tissue used in this experiment was obtained during lung surgery and was used with the patient's prior consent. After collecting normal lung tissue, cells were isolated by transporting it on ice in an HBSS solution (Gibco, Gaithersberg, MD) containing an antibiotic-antifungal solution (Giboco, amphotericin B, penicillin, streptomycin). Specifically, to isolate human type 2 alveolar cells, normal lung tissue resected during surgery was washed with an HBSS solution containing an antibiotic-antifungal solution until no blood was visible. The blood-removed lung tissue was cut into small pieces of less than 0.5 mm³ using surgical scissors, placed in 20 ml of HBSS containing 0.375 ml of trypsin (10 KU / ml; sigma), 75 ml of elastase (10 U / ml), and 100 ml of collagenase (Type 1 collagenase, 60 mg / ml), and reacted in a shaking incubator at 37 ℃ for 30 minutes.

[0118] Then, the reacted solution was passed through a 40 mm strainer, collected in a 50 ml Falcon tube, and centrifuged at 1500 rpm for 10 minutes at room temperature. The supernatant was discarded, and 3 ml of Red Blood Cell Lysis Buffer (11814389001 Roche) was added and reacted at room temperature for 3 minutes. Then, the solution was centrifuged at 1500 rpm for 3 minutes at room temperature, and the cells were suspended in 10 ml of SAGM media (Lonza) (containing 10 μM ROCK inhibitor) and placed in a dish, and cultured in a 37 ℃, 5% CO2 incubator.

[0119] SAGM media was exchanged once every 2-3 days, and when AT2 cells grew to over 60%, cells were detached using the Animal Component-Free Cell Dissociation Kit (Catalog # 05426) and subcultured.

[0120] In addition, to confirm that the cultured alveolar cells were AT2 cells, lamellar bodies, which are characteristic markers of AT2 cells, were reacted with Lysotracker fluorescence (green) for 30 minutes and then observed with a fluorescence microscope.

[0121] As a result, as shown in Figure 1, it was confirmed that the cultured cells exhibited lamellar bodies around the nucleus, confirming that they were AT2 cells.

[0123] Example 2. Reprogramming of iPSCs (induced pluripotent stem cells) using Sendai virus

[0124] 2-1. Transduction using Sendai virus

[0125] To culture alveolar cells in a state suitable for transduction, human alveolar cells obtained in Example 1 were seeded in equal amounts into a 6-well plate, and the number of cells in one well was checked when the alveolar cells had grown to about 70 to 80% of the dish (2 wells or more).

[0126] 1 ml of SAGM media was mixed with Cytotune™ 2.0 Sendai mix (manufactured by Invitrogen) at MOI=5-5-3 (KOS MOI=5, hc-Myc MOI=5, hKlf4 MOI=3) and placed in a 6-well plate. After 24 hours, the mixture was replaced with new SAGM media, and the medium was changed every 2 to 3 days.

[0127] Subculture was performed on the 7th day after transduction using the above Cytotune™ 2.0 Sendai mix. The timing of the subculture may vary depending on the cell proliferation rate, and subculture can be performed when the cell density in the plate reaches approximately 70% to 80% after treatment with Sendai mix. After removing the SAGM media from the wells where the cells grew, the cells were washed with PBS and detached using TrypLE. The detached cells were collected in the SAGM media and centrifuged at 1500 rpm for 3 minutes. The supernatant was removed, and the cells were seeded into a pre-prepared cell culture plate. The cell culture plate was coated with vitronectin (manufactured by Gibco) diluted to a 1 / 100 ratio in PBS at room temperature for 1 hour, after which any remaining vitronectin was removed and the plate was washed with PBS. The cells were replaced with E8 medium within 7 days of seeding, and the medium was replaced daily thereafter while observing under a microscope whether colonies formed for 3 to 4 weeks.

[0128] As a result, it was confirmed that colonies formed after a certain period, and after a further period, the iPSC colonies grew in size. They grew to the point where the edges of the cells were distinguishable from the previously seeded cells, and the round parts of the iPSC colonies were observed to form with a three-dimensional appearance.

[0130] 2-2. Analysis of Protein Characteristics of Induced Pluripotent Stem Cells Prepared Using Sendai Virus

[0131] To confirm whether the induced pluripotent stem cells (iPSCs) prepared from the above human alveolar cells actually possess the properties of pluripotent stem cells, the expression of Oct3 / 4, SSEA-4, and NANOG was verified using immunofluorescence staining. For the staining process, the cells were first fixed using 4% paraformaldehyde, then washed with PBS, and blocked with 1% BSA solution. Primary antibodies against Oct3 / 4, SSEA4, TRA-1-60, TRA-1-81, LIN28, and NANOG (each manufactured by Santa Cruz Biotechnology) were treated and incubated at 4°C for 18 hours. After washing with PBS, fluorescently labeled secondary antibodies against Oct3 / 4, SSEA4, TRA-1-60, TRA-1-81, LIN28, and NANOG (Alexa Fluor 594-conjugates, manufactured by Santa Cruz Biotechnology) were treated and incubated at room temperature for 1 hour. After washing with PBS and mounting with mounting solution, expression was analyzed using confocal microscopy. DAPI (4',6-diamidino-2-phenylindole) was used for the visualization of intracellular nuclei through DNA staining.

[0132] As a result, as shown in Figure 2, it was confirmed that the iPSC undifferentiation markers Oct3 / 4, SSEA4, TRA-1-60, TRA-1-81, LIN28, and NANOG were expressed in induced pluripotent stem cells prepared from human alveolar cells.

[0134] Example 3. Subculture (passaging) and mass culture of induced pluripotent stem cells (iPSCs) prepared from human alveolar cells

[0135] Among the cells subcultured after performing transduction in 2-1 of Example 2 above, there are also cells that were not transduced by the virus, i.e., not reprogrammed. Therefore, to selectively culture only induced pluripotent stem cells, subculture was performed using the following method. A new cell culture dish was coated with vitronectin diluted with PBS at room temperature for 1 hour, after which the residual vitronectin was removed and washed with PBS. When the cells subcultured after performing transduction in 2-1 of Example 2 above were cultured for about 3 to 4 weeks, colonies were formed. At this time, the medium was removed from the cells, washed with PBS, and E8 medium supplemented with 10 μM ROCK inhibitor (manufactured by Sigma) was added. Then, while observing under a microscope, the cells were fragmented from the edges of the induced pluripotent stem cells using a 100 μl pipette tip. After adding E8 (w / 10 μM ROCK inhibitor) medium to a new cell culture dish, the fragmented induced pluripotent stem cells were harvested with E8 medium, placed in the new culture dish, and cultured in an incubator.

[0136] As a result, it was confirmed that on day 1, the size was small and the edges were attached in an elongated shape, but as time passed, the edges became rounded and the size of the colony also increased. In addition, by confirming the nucleolus present in the center of the cell, it was confirmed that the induced pluripotent stem cell colonies were dividing normally.

[0137] Next, to culture induced pluripotent stem cells in large quantities, subculture was performed using the following method. Colonies composed solely of induced pluripotent stem cells were obtained by subculturing the fragmented induced pluripotent stem cells in E8 medium, and these colonies were treated with ReLeSR™ (manufactured by STEMCELL Technologies), a cell isolation solution specifically for iPSCs, at room temperature for 1 minute. After removing the ReLeSR™ solution, the cells were harvested into E8 medium and centrifuged at 1500 rpm for 5 minutes. The supernatant was removed, and the pellet was suspended in E8 (w / 10 μM ROCK inhibitor) medium. The suspended cells were placed in a cell culture dish containing pre-prepared E8 (w / 10 μM ROCK inhibitor) medium and cultured in an incubator.

[0138] As a result, it was confirmed that the cell colonies proliferated on day 4 compared to day 1. As such, using the ReLeSR™ solution allows for separation into single cells, which can slow down the rate of colony proliferation.

[0140] Example 4. Differentiation of Induced Pluripotent Stem Cells (iPSCs) into Alveolar Organoids

[0141] As shown in Fig. 4, the AT2 cell-derived iPSC (AT2 iPSC) obtained in Example 3 was differentiated into an alveolar organoid to produce an alveolar organoid, and the specific method is as follows.

[0143] 4-1. Differentiation of AT2 iPSCs into Endoderm Cells

[0144] The AT2 iPSCs obtained in Example 3 above were cultured in a dish coated with probenectin, and the medium was replaced once a day. TeSR™-E8™ (STEMCELL #05990) medium was used. When the AT2 iPSCs in the dish reached a cell density of 70–80%, they were cultured using STEMdiff™Definitive Endoderm Kit (STEMCELL #05110) differentiation medium. According to the manufacturer's instructions, MR and CJ supplements were added to the medium and cultured for 2 days, after which only the CJ supplement was added and cultured for 2–4 days.

[0145] To confirm whether AT2 iPSCs differentiated into endoderm cells, the expression of endoderm characteristic factors SOX17 and FOXA2 was checked using the immunofluorescence staining method described in 2-2 of Example 2 above.

[0146] As a result, as shown in Figure 5, it was confirmed that the endoderm characteristic factors SOX17 and FOXA2 were expressed.

[0148] 4-2. Differentiation of Endodermal Cells into Alveolar Organoids

[0149] In Example 4-1 above, the differentiation of AT2 iPSCs into endodermal cells was performed using a two-dimensional culture method in a culture dish, and the subsequent process was carried out using Matrigel ® (Corning ® The process was carried out using a dome-shaped 3D culture method with Matrigel (#356231), and ® It was used at 60%.

[0150] To convert the endodermal cells of Example 4-1 into anterior foregut endoderm cells, which are precursors of lung and airway cells, they were cultured in AFM (Anterior Foregut Endoderm Media) medium for 7 days. Then, to induce differentiation into alveolar progenitors, they were cultured in SAGM™ (Lonza #CC-3118) medium for 7 days; after culture in SAGM medium was completed, they were continued in AM (Alveolar Media) medium for the maturation of the alveolar organoids. If the cell density of the spheroid became too high due to continuous cell division during culture in AM medium, Accutase ® (Corning ® After separating the spheroid using #25-058-CI, again Matrigel ® The cells were cultured for up to 90 days while repeating the re-dome process of embedding. Meanwhile, if the spheroid maintained a spherical shape, maintained an appropriate cell density, and a lumen was confirmed, the cells were continued to be cultured in AM medium without the re-dome process, and fluorescence staining was used to confirm whether the cells constituting the spheroid included alveolar epithelial cells. The specific compositions of the AFM medium and AM medium used are shown in Table 1 and Table 2, respectively.

[0151] AFM (Anterior Foregut endoderm Media) component Ham's F12(Gibco™#11765054) 500 mL Dexamethasone (Sigma-Aldrich #D4902) 50 nM 3-Isobutyl-1-methylxanthine(IBMX) (Sigma-Aldrich #I5879) 100 μM B27 supplement (Gibco™#17504004) 2% N2 supplement (Gibco™ #17502048) 1% 7.5% Bovine serum albumin Fraction ⅴ(Gibco™ #15260037) 1% HEPES (Gibco™ #15630080) 15 mM CaCl2(Sigma-Aldrich #21115) 0.8 mM 8-BrcAMP (Sigma-Aldrich #B7880) 100 μM Recombinant Human KGF (PEPROTECH #100-19) 10 ng / ml Recombinant Human FGF-10 (PEPROTECH #100-26) 10 ng / ml Anti-Antibiotic-Antimycotic (Gibco™ #15240096) 1% Ascobic acid (Sigma-Aldrich #A4544) 50 μg / ml Glutamax (Gibco™ #35050061) 2 mM Recombinant Human BMP-4 (rndsystems #314-BP) 10 ng / ml CHIR99021 (TOCRIS #4423) 3 μM

[0153] AM(Alveolar Media) component Ham's F12(Gibco™ #11765054) 500 mL Dexamethasone (Sigma-Aldrich #D4902) 50 nM 3-Isobutyl-1-methylxanthine(IBMX) (Sigma-Aldrich #I5879) 100 μM B27 supplement (Gibco™ #17504004) 2% N2 supplement (Gibco™ #17502048) 1% 7.5% Bovine serum albumin Fraction ⅴ(Gibco™ #15260037) 1% HEPES (Gibco™ #15630080) 15 mM CaCl2(Sigma-Aldrich #21115) 0.8 mM 8-BrcAMP (Sigma-Aldrich #B7880) 100 μM Recombinant Human KGF (PEPROTECH #100-19) 10 ng / ml Recombinant Human FGF-10 (PEPROTECH #100-26) 10 ng / ml Anti Antibiotic-Antimycotic(Gibco™ #15240096) 1%

[0155] 4-3. 폐포 오가노이드의 분화 특성 확인

[0156] Immunofluorescence staining was performed to confirm the characteristics of the differentiation process of alveolar organoids.

[0157] As a result, as shown in Figure 6, it was confirmed that the epithelial cell factor EPCAM was expressed during the early stages of differentiation, and as differentiation progressed, the expression of type 2 alveolar cells (AT2) and type 1 alveolar cells (AT1) appeared, and as differentiation progressed further, it was confirmed that an alveolar organoid containing AT2 cells and AT1 cells was formed inside the organoid.

[0159] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

Claim 1 a) a step of inducing differentiation into endoderm cells by culturing alveolar cell-derived induced pluripotent stem cells in a medium for inducing differentiation into endoderm cells; b) a step of inducing differentiation into anterior foregut endoderm cells in an AFM (Anterior Foregut Endoderm Media) medium; c) a step of inducing differentiation into alveolar progenitors in a progenitor differentiation medium; and d) a step of culturing the alveolar progenitors in an AM (Alveolar Media) medium, wherein the alveolar cells are type 2 alveolar cells and the AM medium comprises B27 supplement and N2 supplement. Claim 2 delete Claim 3 The method according to claim 1, wherein the AFM medium comprises one or more selected from the group consisting of Ham's F12, dexamethasone, IBMX (3-Isobutyl-methylxanthine), B27 supplement, N2 supplement, BSA (Bovine serum albumin), HEPES (Hydroxyethyl piperazine ethane sulfonic acid), calcium chloride (CaCl2), 8-Br-cAMP, Recombinant Human KGF, Recombinant Human FGF-10, Anti Antibiotic-Antimycotic, Ascobic acid, Glutamax, Recombinant Human BMP-4, and CHIR99021. Claim 4 The method according to claim 1, wherein the AM medium further comprises one or more selected from the group consisting of Ham's F12, dexamethasone, IBMX (3-Isobutyl-methylxanthine), BSA (Bovine serum albumin), HEPES (Hydroxyethyl piperazine ethane sulfonic acid), calcium chloride (CaCl2), 8-Br-cAMP, Recombinant Human KGF, Recombinant Human FGF-10, and Anti-Antibiotic-Antimycotic. Claim 5 A method according to claim 1, wherein steps b) to d) are performed by a three-dimensional culture method. Claim 6 An alveolar organoid produced by the method of any one of claims 1 and 3 to 5, wherein the alveolar organoid is characterized by being differentiated from type 2 alveolar cell-derived induced pluripotent stem cells. Claim 7 A kit for producing alveolar organoids comprising a medium for inducing endoderm cell differentiation, an AFM (Anterior Foregut endoderm Media) medium, a precursor differentiation medium, and an AM (Alveolar Media) medium, wherein the alveolar organoid is differentiated from induced pluripotent stem cells derived from type 2 alveolar cells, and the AM medium comprises a B27 supplement and an N2 supplement. Claim 8 A kit according to claim 7, further comprising instructions describing a method for producing alveolar organoids from alveolar cell-derived induced pluripotent stem cells according to claim 1.

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