A chemically defined 2D culture method for culturing intestinal stem cell aggregates derived from 3D intestinal organoids

Abstract

The present invention relates to a method for two-dimensionally culturing intestinal stem cell aggregates in a medium having a chemically defined composition and uses thereof, and to a method for differentiating said intestinal stem cell aggregates into 2.5-dimensional intestinal epithelial cells and uses thereof.

Description

[Technical field]

[0001] The present invention relates to a method for two-dimensionally culturing intestinal stem cell aggregates in a medium having a chemically defined composition and uses thereof, and also to a method for differentiating said intestinal stem cell aggregates into 2.5-dimensional intestinal epithelial cells and uses thereof. [Background technology]

[0002] Intestinal organoids are a cell model system capable of modeling human intestinal development and disease, and are attracting attention as an excellent cell model that can be used in a variety of fields, including research into intestinal physiology / pathological functions, development of therapeutic agents for the treatment of intestinal diseases, and evaluation of the efficacy and toxicity of new drug candidates.

[0003] In addition, organoids are being garnered attention as a regenerative therapeutic agent that can be transplanted into intestines that are damaged or underdeveloped due to intestinal diseases to regenerate affected areas. Intestinal organoids are composed of various cells, including intestinal stem cells, which are the core component of regeneration, and have been confirmed to be able to take root in affected areas and efficiently regenerate damaged tissues, increasing the possibility of their development as a therapeutic agent.

[0004] Recently, various groups have repeatedly confirmed the phenomenon of regeneration of damaged intestinal tissue by transplanting human intestinal organoids into models of damaged intestinal tissue, and it has been confirmed that transplanted intestinal organoids are in fact capable of regenerating the structure and function of intestinal tissue at a high level.

[0005] However, in order for intestinal organoids to be utilized as a cell model system for various purposes or as a cell therapeutic agent for the regeneration of damaged intestinal tissue, it is necessary to mass-culture intestinal organoids in a homogeneous state in which the morphology and function can be adjusted within a predetermined range, and it is necessary to develop a culture system that allows easy long-term culture and cryopreservation.

[0006] Currently, intestinal organoids are cultured using a 3D culture method using Matrigel, which is produced by isolating extracellular matrix produced from sarcoma transplanted into animals. However, this culture method has the disadvantages of low homogeneity due to the large difference in morphology and function of intestinal organoids between placements, difficulty in mass culture, impossibility of long-term culture and frozen storage, and high culture costs.

[0007] Therefore, there is a need to develop a new intestinal organoid culture method that allows for long-term culture and frozen storage of homogeneous intestinal organoids, enables a stable supply of cells through mass culture, and allows for development as a cell model system or cell therapeutic agent with little difference between configurations.

[0008] Under these circumstances, the present inventors developed a method for culturing cells that allows simple and rapid cell culture by isolating only intestinal stem cells from 3D intestinal organoids derived from human total differentiation potential stem cells and culturing them in a culture medium with a defined chemical composition, and that allows for long-term stable mass culture through subculture, as well as frozen storage and thawing. As a result, the present inventors found that it is possible to provide homogeneous intestinal stem cells and various applications, and thus completed the present invention. DISCLOSURE OF THEINVENTION [Problem to be solved by the invention]

[0009] The present invention aims to provide a method for culturing intestinal stem cell aggregates, in which single cells or small cell clusters isolated from three-dimensional intestinal organoids derived from all-potential stem cells are two-dimensionally cultured in a culture medium with a defined chemical composition.

[0010] Another object of the present invention is to provide a method for producing intestinal epithelial cells, which comprises culturing the intestinal stem cell aggregates in a differentiation medium having a defined chemical composition by an air-liquid interface culture method.

[0011] A further object of the present invention is to provide uses of the above-mentioned intestinal stem cell aggregates and / or intestinal epithelial cells. [Means for solving the problem]

[0012] In the following, in order to avoid confusion, the description of the overlapping contents will be omitted. In other words, the contents of the invention are not limited to the following contents alone, and the contents of the invention should be interpreted based on the contents of the invention as a whole.

[0013] The terms used in this application are merely used to describe certain embodiments and are not intended to limit the present invention. A singular expression includes a plural expression unless the context clearly indicates otherwise. In this application, the terms "include" or "have" specify the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, and should be understood not to preclude the presence or additional possibility of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0014] Whenever an amount, concentration, or other value or parameter is given herein by recitation of a range, a preferred range or an upper preferred value and a lower preferred value, that should be understood to specifically disclose all ranges formed to any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether a range is otherwise disclosed.

[0015] Where a range of numerical values ​​is recited herein, unless otherwise specified, it is intended that the endpoints of the range and the scope of the invention within that range are not limited to the specific values ​​recited when defining the range.

[0016] Fully competent stem cells As used herein, the term "stem cell" refers to a cell that has the ability to continue proliferation into an undifferentiated state, i.e., the ability to self-renew, and has the differentiation potential to differentiate into various types of specific cells that make up tissues or organs.

[0017] As used herein, the term "pluripotent stem cell (PSC)", also commonly known as PS cell, includes any cell that can differentiate into almost any cell, i.e., a cell derived from any of the three germ layers (germline epithelium), including endoderm (inner stomach wall, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, urogenital tract, and ectoderm (epithelial tissue and nervous system). PSCs can be derived from embryonic stem cells (including embryonic germ cells) or can be the progeny of totipotent cells obtained by inducing non-pluripotent cells, e.g., adult somatic cells, by forcing the expression of certain genes.

[0018] As used herein, the term "induced pluripotent stem cell (iPSC)", also commonly abbreviated as iPS cell, refers to a type of pluripotent stem cell that is artificially induced from a normally non-pluripotent cell, e.g., an adult somatic cell, by inducing "forced" expression of certain genes.

[0019] As used herein, the term "embryonic stem cell (ESC), also commonly abbreviated as ES cell, refers to a cell that is pluripotent and derived from the inner cell mass of an early-stage embryo, the blastocyst. For purposes of the present invention, the term "ESC" is also sometimes used broadly to include embryonic germ cells.

[0020] Basal medium The medium used in the methods of the present invention is any basal medium suitable for animal or human cells, subject to the limitations provided herein.

[0021] Basal media for the culture of animal or human cells typically contain a number of components necessary to support the maintenance of cultured cells. Suitable combinations of components can be readily formulated by the skilled artisan in light of the following: Basal media for use in the present invention generally comprise a nutrient solution containing standard cell culture components, such as amino acids, vitamins, lipid supplements, inorganic salts, a carbon energy source, and a buffering agent, as described in more detail in the literature and above. In some embodiments, the culture medium is further supplemented with one or more standard cell culture components selected from, for example, amino acids, vitamins, liquid supplements, inorganic salts, a carbon energy source, and a buffering agent.

[0022] A skilled person can understand from common general knowledge the types of culture media that can be used as the basal medium in the differentiation medium of the present invention. Potentially suitable cell culture media are commercially available and include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Knockout-DMEM (KO-DMEM), Glasgow Minimum Essential Medium (G-MEM), Basal Eagle Medium (BME), DMEM / Ham's F12, Advanced DMEM / Ham's F12, Iscove's Modified Dulbecco's Media and Minimum Essential Medium (MEM), Ham's F-10, Ham's F-12, Medium 199, RPMI 1640 medium, and KnockOut Serum replacement XenoFree medium.

[0023] More specifically, it may be selected from the group consisting of RPMI 1640, DMEM (Dulbecco's Modified Eagle Medium), Ham's F12, DMEM / F12, DMEM / F12 and Advanced DMEM / F12.

[0024] For example, the basal medium can be DMEM / F12, Advanced DMEM / F12 and / or RPMI 1640. Optionally, Advanced DMEM / F12 or Advanced RPMI, optimized for serum-free culture and already containing insulin, is used.

[0025] Method for culturing intestinal stem cell aggregates The present invention provides a method for culturing intestinal stem cell aggregates, in which single cells or small cell clusters isolated from 3D intestinal organoids derived from all differentiation potential stem cells are two-dimensionally cultured in a culture medium with a chemically defined composition.

[0026] Specifically, the method includes the steps of (a) dissociating the 3D intestinal organoids derived from all potent stem cells into single cells or small cell clusters; (b) two-dimensionally culturing the single cells or small cell clusters in a culture medium containing a WNT / R-spondin activator, an activator of the prostaglandin signaling pathway, and a receptor tyrosine kinase ligand.

[0027] As used herein, "organoid" refers to a three-dimensional collection of one or more cell types that mimics the appearance or actual structure or function of a surface area of ​​a tissue or organ.

[0028] The 3D intestinal organoids of the present invention are derived from omnipotent stem cells, (a) culturing the full differentiation potential stem cells in a medium containing one or more selected from the group consisting of Nodal, Activin A, Activin B, BMP4, CHIR99021, WNT3A and bFGF, thereby differentiating the full differentiation potential stem cells into definitive endoderm; (b) culturing the definitive endoderm in a medium containing any one or more GSK3 inhibitors selected from the group consisting of BIO (6-bromoindirubin-3'-oxime), SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), GSK-3β inhibitor VII (α,4-dibromoacetophenone), L803-mts (Myr-N-GKEAPPAPPQSpP-NH2) and CHIR99021; and fibroblast growth factor (FGF), and differentiating the definitive endoderm into a three-dimensional hindgut spheroid; (c) culturing the 3D hindgut spheroids in a medium containing a BMP inhibitor; a WNT / R-spondin activator; a receptor tyrosine kinase ligand; and one or more factors selected from the group consisting of IL-2, IL-22, IL-6, IL-1β, IL-11, EGF, OSM, NRG-1, IL-10 and colivelin, thereby producing a 3D intestinal organoid.

[0029] Differentiation of intestinal organoids from all-potential stem cells involves the following steps: 1) differentiation of all-potential stem cells into definitive endoderm (DE) cells, 2) differentiation of definitive endoderm (DE) cells into hindgut (HG), and 3) differentiation of hindgut (HG) into (mature) intestinal organoids.

[0030] 1) Differentiation of full-potential stem cells into definitive endoderm (DE) cells One or more growth factors are used during the differentiation process of full-potential stem cells into definitive endoderm (DE) cells.

[0031] Specifically, growth factors that can affect Activin A / Nodal signaling activation or Phosphatidylinositol 3-kinase (PI3K) signaling inhibition can be used.

[0032] For example, the one or more growth factors used in the differentiation process can include growth factors in the TGF-β superfamily. The one or more growth factors can include the Nodal / Activin and / or BMP subgroups of growth factors in the TGF-beta superfamily.

[0033] In some aspects, the one or more growth factors are selected from the group consisting of Nodal, Activin A, Activin B, BMP4, CHIR99021, WNT3A, bFGF, or any combination of these growth factors.

[0034] In some aspects, the full differentiation potential stem cells are treated with one or more growth factors for 6 hours or more; 12 hours or more; 18 hours or more; 24 hours or more; 36 hours or more; 48 hours or more; 60 hours or more; 72 hours or more; 84 hours or more; 96 hours or more; 120 hours or more; 150 hours or more; 180 hours or more; or 240 hours or more. In some aspects, the embryonic stem cells or iPSCs are treated with the one or more growth factors at a concentration of 10ng / ml or more; 20ng / ml or more; 50ng / ml or more; 75ng / ml or more; 100ng / ml or more; 120ng / ml or more; 150ng / ml or more; 200ng / ml or more; 500ng / ml or more; 1,000ng / ml or more; 1,200ng / ml or more; 1,500ng / ml or more; 2,000ng / ml or more; 5,000ng / ml or more; 7,000ng / ml or more; 10,000ng / ml or more; or 15,000ng / ml or more. In some aspects, the concentration of the growth factor is maintained at a predetermined level during the treatment process. The concentration of the growth factor can be changed during the treatment process.

[0035] That is, the method may include a step of treating all-potential stem cells with any one or more selected from the group consisting of Nodal, Activin A, Activin B, BMP4, CHIR99021, bFGF, and WNT3A, and differentiating them into definitive endoderm cells. More specifically, the method may include a step of treating embryonic stem cells or induced pluripotent stem cells with any one selected from the group consisting of Activin A, BMP4, CHIR99021, bFGF, and WNT3A, and differentiating them into definitive endoderm cells. More specifically, the method may include a step of treating embryonic stem cells or induced pluripotent stem cells with Activin A, and differentiating them into definitive endoderm cells.

[0036] Although not limited thereto, the factor can be used at, for example, 1 ng / ml to 1,000 ng / ml, preferably 10 ng / ml to 500 ng / ml.

[0037] According to one embodiment of the present invention, Activin A can be used to differentiate omnipotent stem cells into definitive endoderm (DE) cells. In such differentiation, for example, but not limited to, 10 ng / ml to 500 ng / ml of Activin A can be used. More specifically, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 ng / ml of Activin A can be used. More specifically, but not limited to, the cells can be cultured for 6 hours to 120 hours, or 12 hours to 100 hours.

[0038] According to one embodiment of the present invention, the growth factors are optionally suspended in a medium containing fetal bovine serum (FBS) at various concentrations, such as 0.1, 0.2, 0.3, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0% of any fetal bovine serum (FBS or FCS) suitable for growth.

[0039] If necessary, the cells can be cultured in contact with an extracellular matrix. The extracellular matrix includes all of the substances described herein and can be applied to the present differentiation method.

[0040] 2) Differentiation of definitive endoderm (DE) cells into 3D hindgut (HG) spheroids After undergoing the process of producing definitive endoderm induced by the factors mentioned above, preferably induced by Activin, more preferably Activin A, the definitive endoderm (DE) can undergo a differentiation step into the hindgut (HG) by FGF / Wnt signaling.

[0041] FGF and Wnt ligands can direct differentiation of DE developed into iPSCs or ESCs into the hindgut.

[0042] Differentiation into the hindgut or midgut can be performed by three-dimensional culture, spheroid culture, etc., as necessary. For example, differentiation into the hindgut or midgut can be performed on a micropatterned substrate / plate (e.g., Aggrewell, EZSPHERE) and produced in the form of an aggregation (e.g., spheroid), or can be performed by three-dimensional culture (e.g., Suspension) in a bioreactor. In the case of serum-free differentiation, B-27 can be added instead of fetal bovine serum (FBS or FCS). In addition, the culture medium may further contain, but is not limited to, any one or more selected from the group consisting of Activin A, FGF, bFGF, BMP-4, LY294002 (PI3K inhibitor), CHIR99021 (GSK3 inhibitor) and retinoic acid.

[0043] Altering the expression of any Wnt signaling protein in combination with any FGF ligand can result in directed differentiation as described herein.

[0044] Regulators / activators of Wnt signaling pathway include Wnt1, Wnt2, Wnt2b, Wnt3, WNT3A, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11 and Wnt16.In some aspects, the regulation of pathway can be achieved by using small molecule regulators or protein regulators that activate the above-mentioned pathway or proteins that activate the pathway.For example, small molecule regulators of Wnt pathway include, but are not limited to, lithium chloride; 2-amino-4,6-doubly substituted pyrimidine (hetero)arylpyrimidine; IQ1; QS11; NSC668036; DCA beta-catenin; 2-amino-4-[3,4-(methylenedioxy)-benzyl-amino]-6-(3-methoxyphenyl)pyrimidine. Exemplary natural inhibitors of Wnt signaling include, but are not limited to, Dkk1, SFRP protein and FrzB. In some aspects, exogenous molecules include, but are not limited to, small molecules such as WAY-316606; SB-216763; or BIO (6-bromoindirubin-3'-oxime).

[0045] Also included are molecules or proteins that inhibit GSK3, which activate the Wnt signaling pathway.Exemplary GSK3 inhibitors include, but are not limited to, LY2090314, BIO (6-bromoindirubin-3'-oxime), SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), GSK-3β inhibitor VII (α,4-dibromoacetophenone), L803-mts (Myr-n-GKEAPPAPPQSpP-NH 2 ) and CHIR99021.

[0046] Specifically, it includes CHIRon / CHIR99021, which inhibits GSK3. The GSK3 inhibitor can be administered in an amount of about 0.1 μM to about 100 μM, or about 0.2 μM to about 50 μM, or about 0.3 μM to about 10 μM.

[0047] Fibroblast growth factors (FGFs) are a group of growth factors involved in angiogenesis, wound healing and embryonic development. In some aspects, one skilled in the art can understand that any FGF can be used with a protein derived from the Wnt signaling pathway.

[0048] In some embodiments, the FGF signaling pathway is activated by contacting the cell with one or more molecules selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22 and FGF23.

[0049] Specifically, definitive endoderm was cultured with BIO (6-indirubin'-3'-oxime), SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), GSK-3β inhibitor VII (α,4-dibromoacetophenone), L803-mts (Myr-N-GKEAPPAPPQSpP-NH 2 ) and CHIR99021; and fibroblast growth factor (FGF), and a step of differentiating the cells into three-dimensional hindgut spheroids.

[0050] In some aspects, DE cells are treated with one or more modulators of a signaling pathway described herein for 6 hours or more; 12 hours or more; 18 hours or more; 24 hours or more; 36 hours or more; 48 hours or more; 60 hours or more; 72 hours or more; 84 hours or more; 96 hours or more; 120 hours or more; 150 hours or more; 180 hours or more; 200 hours or more, 240 hours or more; 270 hours or more; 300 hours or more; 350 hours or more; 400 hours or more; 500 hours or more; 600 hours or more; 700 hours or more; 800 hours or more; 900 hours or more; 1,000 hours or more; 1,200 hours or more; or 1,500 hours or more.

[0051] In some aspects, DE cells are treated with one or more molecules of the FGF signaling pathway described herein at a concentration of 10 ng / ml or more; 20 ng / ml or more; 50 ng / ml or more; 75 ng / ml or more; 100 ng / ml or more; 120 ng / ml or more; 150 ng / ml or more; 200 ng / ml or more; 500 ng / ml or more; 1,000 ng / ml or more; 1,200 ng / ml or more; 1,500 ng / ml or more; 2,000 ng / ml or more; 5,000 ng / ml or more; 7,000 ng / ml or more; 10,000 ng / ml or more; or 15,000 ng / ml or more.

[0052] In some aspects, the concentration of the signaling molecule is maintained constant during the process. In other aspects, the concentration of the signaling pathway molecule is altered during the process.

[0053] In some aspects, the signaling molecules according to the present invention are suspended in DMEM medium.

[0054] In some aspects, signaling molecules according to the present invention are suspended in a medium containing fetal bovine serum (FBS or FCS).

[0055] It is applicable to any known molecule in the signaling pathways described herein, alone or in combination, including any molecule in the Wnt and FGF signaling pathways.

[0056] That is, it includes a step of differentiating definitive endoderm cells into the hindgut by treating with FGF and a Wnt ligand, more specifically, it includes a step of differentiating definitive endoderm cells into the hindgut by treating with FGF and CHIR99021.

[0057] According to one embodiment of the present invention, the differentiation of definitive endoderm (DE) to hindgut can be achieved by a GSK3 inhibitor and fibroblast growth factor. More specifically, the differentiation to hindgut can be achieved by treating with CHIR99021 and FGF4 in 3D culture.

[0058] In such differentiation, for example, but not limited to, 100ng / ml, 120ng / ml, 150ng / ml, 200ng / ml, 500ng / ml, 1,000ng / ml, or 1,200ng / ml of FGF4 may be used, specifically, 100ng / ml to 1,200ng / ml, more specifically, 120 to 1,000ng / ml.

[0059] Also, CHIR99021 can be used in an amount of about 0.3 μM to about 10 μM. If necessary, it is suspended in a medium containing fetal bovine serum (FBS). For example, any fetal bovine serum (FBS or FCS) suitable for growth can be included, such as 0.1, 0.2, 0.3, 0.5, 1.0, 2.0, 3.0, 4.0, or 5.0%.

[0060] More specifically, but not limited to, the culture may be for 24 hours or more, 36 hours or more, 48 hours or more, 60 hours or more, 72 hours or more, 84 hours or more, 96 hours or more, 120 hours or more, 150 hours or more, or 180 hours or more, preferably for 24 hours to 360 hours, more preferably for 36 hours to 240 hours.

[0061] If necessary, a BMP activator can be added to the culture to regulate the BMP signal. The BMP activator can be one of BMP2, BMP4 or all of them, a small molecule that activates the BMP pathway, and / or a protein that activates the BMP pathway.

[0062] If necessary, the cells can be cultured in contact with an extracellular matrix. The extracellular matrix includes all of the substances described herein and can be applied to the present differentiation method.

[0063] 3) Differentiation of 3D hindgut (HG) spheroids into 3D intestinal organoids The 3D intestinal organoid of the present invention is a mature intestinal organoid, and refers to an intestinal organoid in which the genes necessary for digestive function, transport system, immune function and host defense that the adult small intestine has are expressed.Specifically, the adult small intestine has unique characteristics, including the enhanced expression of small intestinal stem cell marker genes, digestive function, transport system, extensive immune function and host defense genes.In particular, the appropriate expression and activity of transporters involved in physiological and pharmacodynamic roles are prerequisites for normal small intestinal functions such as drug absorption, distribution and excretion.

[0064] In particular, the 3D intestinal organoid according to the present invention has improved efficacy in terms of engraftment. More specifically, this may be due to the high expression of vascular endothelial-related factors possessed by the matrix cell layer present around the intestinal epithelial cell layer constituting the organoid, and various secretory factors that are characteristics of matrix cells.

[0065] In addition, the organoids exhibit the characteristic of a developed budding structure present on the intestinal organoids, and show a higher development of goblet cells, and show high efficiency in terms of differentiation ability of intestinal stem cells.

[0066] The differentiation of 3D hindgut (HG) spheroids into 3D intestinal organoids involves the essential functions of BMP inhibitors, WNT / R-spondin activators and receptor tyrosine kinase ligands.

[0067] This may further include one or more additional components selected from ROCK inhibitors, B27, N-acetyl-L-cysteine ​​(NAC), N2, nicotinamide, and gastrin, IGF-1, heregulin-1β, bFGF, A-83-01, and / or SB202190.

[0068] BMP inhibitors are agonists that bind to BMP molecules to form complexes. The inhibitors can be agonists that bind to BMP receptors and prevent the binding of BMP ligands to the receptor, for example, antibodies that bind to the receptor. BMP inhibitors can be proteins or small molecules and can be natural, modified and / or partially or fully synthetic. BMP inhibitors can be Noggin, DAN, or DAN-like proteins, including CERBERUS and GREMLIN (R&D Systems). A preferred BMP inhibitor is Noggin. Noggin can be used at any suitable concentration. It can contain about 10 ng / ml to about 100 ng / ml of Noggin. For example, the culture medium can contain about 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml or more of Noggin. Preferably, it can contain about 10 to 100 ng / ml of Noggin.

[0069] The WNT / R-spondin activator may preferably be R-spondin1, R-spondin2, R-spondin3 or R-spondin4. The culture medium may contain 50ng / ml, 100ng / ml, 200ng / ml, 300ng / ml, 500ng / ml, 600ng / ml, 700ng / ml, 800ng / ml, 900ng / ml, 1μg / ml, 1.5μg / ml or 2μg / ml or more. Preferably, the culture medium may contain about 50-800ng / ml of R-spondin1.

[0070] The receptor tyrosine kinase ligand is, for example, a mitogenic growth factor selected from the growth factors consisting of epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF) and epidermal growth factor (KGF).

[0071] The preferred is EGF. EGF is a potent mitogen for various cultured ectodermal and mesodermal cells and has sufficient effects on differentiation of certain cells and some fibroblasts in cell cultures in vivo and in vitro. The preferred concentration is 10, 20, 25, 30, 40, 45, or 50ng / ml, 100ng / ml, 200ng / ml, 300ng / ml, 500ng / ml, 600ng / ml, 700ng / ml or more. The more preferred concentration is 50ng / ml or more and 300ng / ml or less.

[0072] Factors added other than the essential composition of the BMP inhibitor, WNT / R-spondin activator and receptor tyrosine kinase ligand as described above can be interpreted as further including the addition of any known factor of intestinal organoids.

[0073] Preferably, depending on the form of differentiation desired, one or more additional components selected from the group consisting of ROCK inhibitor, B27, N-acetyl-L-cysteine ​​(NAC), N2, nicotinamide, gastrin, IGF-1, heregulin-1β, bFGF, A-83-01 and SB202190 may be included.

[0074] The ROCK inhibitor is preferably selected from R-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), 5-(1,4-diazepan-1-ylsulfonyl)isoquinoline (fasudil or HA1077), and (S)-(+)-2-methyl 1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride (H-1 152).

[0075] N-Acetyl-L-cysteine ​​(NAC), N2, nicotinamide, gastrin, IGF-1, heregulin-1β, bFGF, A-83-01 and / or SB202190 may be added as needed, for example, to improve organoid culture efficiency and lifespan, regulate cell proliferation, aid DNA stability, etc.

[0076] Preferably, B27 can be further included. More specifically, the B27 supplement is "B27 supplement minus vitamin A" (also referred to herein as "B27 without vitamin A" or "B27 wo VitA"; available from Invitrogen, Carlsbad, Calif., USA; www.invitrogen.com; currently catalog number 12587010; and from PAA Laboratories GmbH, Pasching, Austria; www.paa.com; catalog number F01-002; [Brewer et al. (1993) J Neurosci Res. 35(5):567-76]).

[0077] In some embodiments, the B27 supplement may be substituted 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.

[0078] If necessary, they can be cultured in contact with an extracellular matrix during differentiation of 3D hindgut (Hindgut; HG) spheroids into 3D intestinal organoids.

[0079] In some embodiments, the extracellular matrix is ​​a three-dimensional matrix. In some embodiments, the hindgut is embedded in an extracellular matrix. The culture medium of the present invention can diffuse into the three-dimensional extracellular matrix.

[0080] The matters relating to extracellular matrices 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.

[0081] The extracellular matrix includes, but is not limited to, fibrin, laminin, collagen and / or alginate. Examples of extracellular matrix-producing cells are chondrocytes, which mainly produce collagen and proteoglycan, fibroblasts, which mainly produce type IV collagen, laminin, epileptiform procollagen and fibronectin, and colon myofibroblasts, which mainly produce collagen (types I, III and V), chondroitin sulfate proteoglycan, hyaluronic acid, fibronectin and tenascin-C. These are "naturally produced extracellular matrices". Naturally produced extracellular matrices can be commercially provided. Examples of commercially available extracellular matrices include extracellular matrix proteins (Invitrogen) and basement membrane preparations from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (e.g., Cultrex® basement membrane extract (Trevigen, Inc.), type I collagen (Invitrogen), Vitrogel® (TheWell Bioscience Inc.), Geltrex (ThermoFisher) or Matrigel™ (BD Biosciences)).

[0082] In addition to the factors mentioned in the differentiation factors for organoids, differentiation of 3D hindgut (HG) spheroids into 3D intestinal organoids essentially involves treatment with cytokines secreted in co-culture with T-lymphocytes (i.e., treatment with cytokines secreted by T-lymphocytes) and / or treatment with STAT3 and mTOR signaling pathway activators, which are used in co-culture to mimic the in vivo intestinal environment.

[0083] More specifically, the cytokine secreted by the T-lymphocytes can be one or more selected from the group consisting of IL-2 (interleukin-2), IL-22 (interleukin-22), IL-6 (interleukin-6), IL-1β (interleukin-1β), IL-11 (interleukin-11), EGF (epidermal growth factor), OSM (oncostatin M) and IL-10 (interleukin-10), and more specifically, can be IL-2.

[0084] In addition to the factors mentioned above for differentiation into organoids, intestinal organoids can be matured by treating with STAT3 and mTOR signaling pathway activators. For example, intestinal organoids can be matured by treating with colivelin.

[0085] In addition, NRG-1 (Neuregulin-1) can be treated to induce maturation of intestinal organoids.

[0086] That is, the 3D intestinal organoids can be produced by the steps of culturing 3D hindgut spheroids in a medium containing a BMP inhibitor; a WNT / R-spondin activator; a receptor tyrosine kinase ligand; and one or more factors selected from the group consisting of IL-2, IL-22, IL-6, IL-1β, IL-11, EGF, OSM, NRG-1, IL-10 and colivelin, to produce the 3D intestinal organoids.

[0087] That is, by treating the hindgut with one or more cytokines selected from the group consisting of IL-2, IL-22, IL-6, IL-1β, IL-11, EGF, OSM and IL-10 for immediate intestinal maturation, and / or colivelin as medium components, the degree of maturation of organoids is significantly enhanced.

[0088] That is, one or more cytokines selected from the group consisting of IL-2, IL-22, IL-6, IL-1β, IL-11, EGF, OSM, and IL-10, and / or colivelin may be included as a medium component. The preferred concentrations are 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0 ng / ml or more. More preferably, the concentration may be 0.3 to 2.0 ng / ml.

[0089] Dissociation of 3D intestinal organoids derived from total stem cells into single cells or small cell clusters In the present invention, the step of separating the 3-dimensional intestinal organoids derived from all differentiation potential stem cells into single cells or small cell clusters can be performed by treating with EDTA, trypsin, or all of these to separate the intestinal organoids.

[0090] More specifically, it is preferable to purify isolated single cells or small cell clusters by treating with EDTA, trypsin or all of these while removing the extracellular matrix remaining in the 3D intestinal organoids to the maximum extent possible.

[0091] During this process, the 3D intestinal organoids can be finely separated by pipetting or the like, if necessary, and more preferably, separated into nearly single cells.

[0092] The thus separated single cells or small cell clusters can be cultured for a while in a basal medium or washed, if necessary, and then cultured in the intestinal stem cell culture medium according to the present invention.

[0093] 2D culture in a culture medium containing a WNT / R-spondin activator, an activator of the prostaglandin signaling pathway, and a receptor tyrosine kinase ligand. The culture medium for producing intestinal stem cell aggregates used in the present invention is provided for two-dimensional culturing of intestinal stem cell aggregates, and essentially contains a WNT / R-spondin activator, an activator of the prostaglandin signaling pathway, and a receptor tyrosine kinase ligand.

[0094] The WNT / R-spondin activator plays a role in regulating the stemness and proliferation of 2D intestinal stem cells by activating the WNT signaling system, and may be any one or more selected from the group consisting of R-spondin1, R-spondin2, R-spondin3, R-spondin4, and R-spondin mimetics, preferably R-spondin1.

[0095] The culture medium may contain R-spondin 1 at a concentration of 50ng / ml, 100ng / ml, 200ng / ml, 300ng / ml, 500ng / ml, 600ng / ml, 700ng / ml, 800ng / ml, 900ng / ml, 1μg / ml, 1.5μg / ml or 2μg / ml or more. Preferably, the culture medium may contain about 50 to 800ng / ml of R-spondin 1.

[0096] The activator of the prostaglandin signaling pathway plays a role in regulating the proliferation of intestinal stem cell populations by activating the prostaglandin signaling system, and may be any one or more selected from the group consisting of arachidonic acid (AA), prostaglandin E2 (PGE2), prostaglandin G2 (PGG2), prostaglandin F2 (PGF2), prostaglandin H2 (PGH2) and prostaglandin D2 (PGD2), preferably prostaglandin E2 (PGE2).

[0097] As an activator of the prostaglandin signaling pathway, prostaglandin E2 can be administered preferably in an amount of about 0.1 μM to about 100 μM, or about 0.2 μM to about 50 μM, or about 0.3 μM to about 10 μM.

[0098] The receptor tyrosine kinase ligand plays a role in preventing cell death and regulating proliferation of intestinal stem cell populations, and may be any one selected from the group consisting of epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF) and epidermal growth factor (KGF), preferably epidermal growth factor (EGF).

[0099] Receptor tyrosine kinase ligands can be treated at concentrations, for example, of 10 ng / ml or more; 20 ng / ml or more; 50 ng / ml or more; 75 ng / ml or more; 100 ng / ml or more; 120 ng / ml or more; 150 ng / ml or more; 200 ng / ml or more; 500 ng / ml or more; 1,000 ng / ml or more; 1,200 ng / ml or more; 1,500 ng / ml or more; 2,000 ng / ml or more; 5,000 ng / ml or more; 7,000 ng / ml or more; 10,000 ng / ml or more; or 15,000 ng / ml or more.

[0100] The above mentioned combination of WNT / R-spondin activator, activator of the prostaglandin signaling pathway and receptor tyrosine kinase ligand is preferably R-spondin1, prostaglandin E2 (PGE2) and epidermal growth factor (EGF).

[0101] Such a combination of a WNT / R-spondin activator, an activator of the prostaglandin signaling pathway, and a receptor tyrosine kinase ligand is essential for maintaining stem cell potential and cell growth and division, and has the efficacy for stem cell enrichment culture and long-term culture.

[0102] Without being limited thereto, WNT / R-spondin activators can show improved effects on stemness and / or cell proliferation, prostaglandin signaling pathway activators can show improved efficacy on stemness and / or cell growth, and receptor tyrosine kinase ligands can show improved efficacy on cell survival and / or cell proliferation.

[0103] In addition to the above-mentioned essential components, the culture medium of the present invention may further contain one or more selected from the group consisting of B27, N-acetyl-L-cysteine ​​(NAC), nicotinamide, gastrin, a TGF-beta inhibitor, a Wnt signaling pathway activator, a BMP inhibitor, and a p38 inhibitor for stable long-term subculture.

[0104] The B27, N-acetyl-L-cysteine ​​(NAC) and nicotinamide can be added as components of the basal medium, and in particular, B27 can be replaced with a generic formulation containing one or more components 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.

[0105] The TGF-beta inhibitor may be any one selected from the group consisting of A-83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-36494 and SJN-251, and is preferably A-83-01.

[0106] The Wnt signaling pathway activator may be any one selected from the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, WNT3A, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11 and Wnt16, and may be preferably WNT3A.

[0107] The BMP inhibitor may be Noggin, Dorsomorphin, DMH1, or LDN-193189, and preferably Noggin.

[0108] The p38 inhibitor may be any one selected from the group consisting of SB202190, SB203580, SB239063, SB706504, BIR796, JX401, EO1428, RWJ67657, SCIO469, VX745, TAK715, ML3403, DBM1285 and PH797804, and may be preferably SB202190.

[0109] The concentration of each of these additional components may be appropriately adjusted within the general range typically used in culture media.

[0110] In addition, in order to prevent cell loss during subculture, a ROCK inhibitor, a Notch activator, or all of these may be further included in the culture medium at an early stage of culture, for example, immediately after the start of culture, for 1 day, 2 days, 3 days, 4 days, or 5 days.

[0111] More specifically, in the initial stage of the step of 2-dimensionally culturing the single cell or small cell cluster in the culture medium containing WNT / R-spondin activator, prostaglandin signaling pathway activator and receptor tyrosine kinase ligand, the culture medium can further contain ROCK inhibitor, Notch activator or all of them. This can be immediately after starting the culture, 1 day, 2 days, 3 days, 4 days or 5 days.

[0112] The ROCK (Rho-associated protein kinase) inhibitor plays a role in inhibiting the activity of serine / threonine kinase acting as a target protein for Rho (Rho A, Rho B and Rho c), and can be R-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), 5-(1,4-diazepan-1-ylsulfonyl)isoquinoline (fasudil or HA1077), and (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride (H-1 152). More preferably, it may be R-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632).

[0113] Such Y-27632 can be administered in an amount of, for example, about 0.1 μM to about 100 μM, or about 0.1 μM to about 50 μM.

[0114] The Notch activator means a protein or a small molecule compound that activates the Notch pathway function, and can be preferably Jagged-1 (JAG 1).

[0115] Such Jagged-1 (JAG 1) can be administered in an amount of, for example, about 0.1 μM to about 100 μM, or about 0.2 μM to about 50 μM, or about 0.3 μM to about 10 μM.

[0116] The intestinal stem cell aggregates according to the present invention can be cultured on feeder cells or on a plate coated with an extracellular matrix, as required. The extracellular matrix is ​​as mentioned above.

[0117] The intestinal stem cell aggregates according to the present invention may be cultured for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days, 2 weeks, 3 weeks or more, but are not limited thereto, and subculture may be performed as necessary.

[0118] In the present invention, the term "intestinal stem cell" refers to an undifferentiated cell derived from intestinal epithelial tissue, which has the ability to self-replicate and has the differentiation potential to differentiate into various types of specific cells present in the intestinal epithelium. The intestinal stem cell can differentiate into, for example, intestinal stem cells, intestinal progenitor cells, intestinal epithelial cells, intestinal goblet cells, intestinal endocrine cells, Paneth cells, etc.

[0119] In the present invention, the term "intestinal stem cell aggregate" refers to a state in which intestinal stem cells are densely packed, and may be in a tissue state, a cell cluster, or a single cell state.

[0120] The intestinal stem cell population of the present invention may be characterized by exhibiting an enhanced expression level of one or more markers selected from the group consisting of LGR5, CD44, SOX9, LRIG1, LYZ, AXIN2, CTNNB, and MKI67, and may be characterized by expressing one or more markers selected from the group consisting of LDHB, EIF3E, SOX9, and SHH.

[0121] More specifically, the intestinal stem cell aggregates may show increased expression levels of LGR5, CD44, SOX9, LRIG1, LYZ, AXIN2, CTNNB or MKI67 compared to a control group cultured in a culture medium not containing a WNT / R-spondin activator. The increased expression levels of LGR5, CD44, SOX9, LRIG1, LYZ, AXIN2, CTNNB or MKI67 as intestinal stem cell markers indicate superior stemness and self-renewal maintenance functions.

[0122] Alternatively, the intestinal stem cells can be characterized by expression of LDHB, EIF3E, SOX9 or SHH, which are specific markers of intestinal stem cells and progenitor cells, by analyzing gene expression patterns by single cell RNA sequencing.

[0123] The intestinal stem cell aggregate of the present invention may be characterized in that 80, 81, 82, 83, 84, 85, 86, 87, 88, 88, 89, or 90% or more of the cells constituting the aggregate are intestinal stem cells or progenitor cells. More specifically, the intestinal stem cell aggregate may be characterized in that 80, 81, 82, 83, 84, 85, 86, 87, 88, 88, 89, or 90% or more of the total cells in the aggregate are cells including S phase cells, LGR5+ stem cells, and early intestinal cells (Enterocytes).

[0124] The S phase cells refer to cells that are in the S phase, a stage in which DNA is replicated, among cells that divide according to the cell cycle.

[0125] The LGR5+ stem cells refer to stem cells that express LGR5, which is an indicator of the activity of adult stem cells, and the LGR5 is a target gene of the Wnt signal and a factor that acts as a receptor for R-spondin that amplifies the Wnt signal.

[0126] The early enterocytes are precursor cells of the epithelial cells that surround the small and large intestine, and include early enterocyte 1 and early enterocyte 2.

[0127] In addition, the cells can be frozen or thawed under freezing conditions as necessary, and used. Even under such organ subculture conditions and / or freezing and thawing conditions, the characteristics of the cells can be maintained and the cells can be cultured.

[0128] Accordingly, the present invention provides a method for producing a 3D intestinal organoid comprising the steps of: (a) dissociating a 3D intestinal organoid derived from a total potent stem cell into single cells or small cell clusters; (b) culturing the single cells or small cell clusters in a two-dimensional culture medium containing a WNT / R-spondin activator, an activator of the prostaglandin signaling pathway, and a receptor tyrosine kinase ligand to produce intestinal stem cell aggregates; and (c) freezing the produced intestinal stem cell aggregates.

[0129] The intestinal stem cell aggregates produced by step (b) may be centrifuged, if necessary, and resuspended at a predetermined concentration (cells / mL).

[0130] The freezing medium may further include any one selected from the group consisting of, for example, dimethyl sulfoxide (DMSO); glucose; 1,2-propanediol; ethylene glycol; glycerol; formamide; ethanediol or butane-2,3-diol; hydroxyethyl starch (HES), dextran, sucrose, trehalose, lactose, raffinose, ribitol, mannitol, and polyvinylpyrrolidone (PVP).

[0131] Preferably, the cells are suspended in a freezing medium that is commercially available or suitable for freezing and thawing the cells. TM The medium can be Cell Culture Freezing Medium (Gibco).

[0132] Depending on the case, the cell suspension medium for freezing is selected from CHB medium, CS10 medium or CS5 medium. CHB medium is a cell suspension medium containing 50% (v / v) fetal bovine serum (FBS), 40% (v / v) RPMI cell culture medium and 10% (v / v) dimethyl sulfoxide (DMSO). CS10 medium (BioLife Solutions, Inc., Bothell, WA) is a cell culture medium containing 10% (v / v) DMSO and is essentially free of animal components or serum. CS5 (BioLife Solutions, Inc.) medium is a cell culture medium containing 5% (v / v) DMSO and is essentially free of animal components or serum. Such cells can be placed in a vial and cryogenically frozen. A temperature sufficient to freeze the composition is about -80°C to about -190°C.

[0133] Such cryopreserved cells can be thawed by a commonly known thawing method as needed and used as an intestinal stem cell aggregate. Furthermore, the number of cells can be increased by subculture or the like as needed.

[0134] The present invention also provides an intestinal stem cell aggregate produced by the above culture method.

[0135] Method for producing intestinal epithelial cells The present invention provides a method for producing intestinal epithelial cells, which comprises culturing the intestinal stem cell aggregates in a differentiation medium having a defined chemical composition by an air-liquid interface culture method.

[0136] The present invention provides a method for producing intestinal epithelial cells, comprising the step of culturing intestinal stem cell aggregates in a differentiation medium containing an activator of the prostaglandin signaling pathway, a receptor tyrosine kinase ligand, a p38 inhibitor, a WNT / R-spondin activator and nicotinamide by an air-liquid interface culture method.

[0137] More specifically, the method includes (a) dissociating the 3D intestinal organoids derived from all potent stem cells into single cells or small cell clusters; (b) culturing the single cells or small cell clusters in a two-dimensional culture medium containing a WNT / R-spondin activator, an activator of the prostaglandin signaling pathway, and a receptor tyrosine kinase ligand to produce intestinal stem cell aggregates; and (c) culturing the intestinal stem cell aggregates by air-liquid interface culture in a differentiation medium containing an activator of the prostaglandin signaling pathway, a receptor tyrosine kinase ligand, a p38 inhibitor, a WNT / R-spondin activator and nicotinamide.

[0138] The matters relating to the all-potential stem cells, organoids and intestinal stem cells mentioned above include the descriptions mentioned above, and the description of the overlapping contents will be omitted in order to avoid excessive complexity of this specification.

[0139] In addition, since the culture medium in step (b) includes the above-mentioned descriptions regarding the WNT / R-spondin activator, the activator of the prostaglandin signaling pathway, and the receptor tyrosine kinase ligand, the redundant descriptions are omitted to avoid excessive complexity of this specification.

[0140] In the present invention, "differentiation medium" refers to a cell growth medium that causes undifferentiated stem cells to develop into cells having some or all of the characteristics of differentiated cells when cultured in the medium, and includes a basal medium.

[0141] In the present invention, the differentiation medium optimized for producing intestinal epithelial cells comprises an activator of the prostaglandin signaling pathway, a receptor tyrosine kinase ligand, a p38 inhibitor, a WNT / R-spondin activator and nicotinamide.

[0142] In the present invention, the "air-liquid interface culture method" can be cultured in a partially open culture vessel or a culture vessel partially filled with medium, but is not limited thereto. For example, the surface of the cells or organoids can be exposed to air. The gas can be air, and is not limited to compositions and mixtures of gases found in the surrounding environment. Specifically, the present invention contemplates and includes gas mixtures having a composition different from the surrounding environment, for example, mixtures enriched with a particular component or mixtures in which a particular component has been depleted or removed.

[0143] When cells are cultured at an air-liquid interface, the cells can be cultured on the porous substrate such that the cells are in contact with air on the top side of the porous substrate and in contact with cell culture medium on the bottom side. For example, a sufficient volume of medium can be added to the bottom of a culture vessel that includes a porous substrate (e.g., a filter insert) so that the medium contacts the bottom surface of the cells present on the porous substrate but does not encapsulate or submerge the cells. A suitable porous substrate can be formed of any material that does not adversely affect cell growth and differentiation. Illustratively, the porous substrate is made of a polymer such as polyethylene terephthalate (PET), polyester, or polycarbonate. A suitable porous substrate can be coated or uncoated. Examples of commercially available extracellular matrices include extracellular matrix proteins (Invitrogen) and basement membrane preparations from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (e.g., Cultrex® basement membrane extract (Trevigen, Inc.), type I collagen (Invitrogen), Vitrogel® (TheWell Bioscience Inc.) or Matrigel™ (BD Biosciences)), which can be coated with the cells.

[0144] That is, intestinal stem cells can be cultured under a transwell coated with an extracellular matrix. Any of the extracellular matrices mentioned above can be used as the extracellular matrix.

[0145] Preferably, the extracellular matrix can be Matrigel. The porosity of the substrate must be sufficient to maintain cell viability and promote cell differentiation.

[0146] Suitable substrates have pore sizes 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, 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 μm to about 3.0 μm, preferably about 0.4 μm, and about 50 million to about 120 million pores / cm. 2 Approximately 60 million to 110 million pores / cm 2 , about 70 million to 100 million pores / cm 2 , preferably about 80 million to about 100 million pores / cm 2 , about 90 million to 100 million pores / cm 2 , more preferably about 100 million pores / cm 2 The filter insert has a pore density of

[0147] It may be advantageous to change or renew the medium daily or every other day. Cells grown on top of the porous substrate are generally not single cells; rather, the cells are in the form of sheets or exist as aggregate clusters of cells. Cells cultured at an air-liquid interface can experience much higher oxygen tensions than cells submerged in the medium.

[0148] The intestinal epithelial cells of the present invention may be characterized by exhibiting enhanced expression levels of any one or more markers selected from the group consisting of VIL1, ECAD, FABP1, KRT20, LCT, LYZ and MUC2.

[0149] More specifically, the intestinal epithelial cells may be characterized by decreased expression of at least one of intestinal stem cell markers LGR5, CD44, MKI67, SOX9, ASCL2, OLFM4, AXIN2, or CTNNB, while increased expression of intestinal epithelial cell markers VIL1, ECAD, FABP1, KRT20, LCT, LYZ, or MUC2.

[0150] Additionally, the intestinal epithelial cells may exhibit enhanced expression levels of AKR1B15, DHRS11, GALNT4, GALNT5, DHRS3, RDH10, AADAC, NR1I2, SULTE1, DOUX2, FABP1, SLC6A20, SLC43A1 and / or CLDN3 compared to intestinal stem cells.

[0151] The intestinal epithelial cells can include small intestinal cells, mucus-secreting cells, hormone-secreting cells and Paneth cells.

[0152] The production of intestinal epithelial cells according to the present invention can be carried out for, but is not limited to, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days, 2 weeks, 3 weeks or more.

[0153] The present invention provides intestinal epithelial cells produced by the above-mentioned method for producing intestinal epithelial cells.

[0154] Purpose 1) Medical Use The present invention provides a method for treating patients suffering from or at risk of developing intestinal diseases by using the intestinal stem cells or intestinal epithelial cells as a cell therapy agent.

[0155] The present invention provides a tissue therapeutic agent comprising an intestinal stem cell population and / or an intestinal epithelial cell.

[0156] The present invention provides pharmaceutical compositions comprising intestinal stem cell populations and / or intestinal epithelial cells.

[0157] The present invention provides a pharmaceutical composition for preventing or treating an intestinal disease, comprising an intestinal stem cell population and / or an intestinal epithelial cell.

[0158] The present invention provides a pharmaceutical composition for supporting intestinal transplantation, comprising intestinal stem cell aggregates and / or intestinal epithelial cells.

[0159] The present invention provides a pharmaceutical composition for preventing or treating intestinal diseases, comprising an intestinal stem cell aggregate and / or an intestinal epithelial cell; and a small intestinal organoid.

[0160] Intestinal stem cell populations and / or intestinal epithelial cells can be used therapeutically as bioengineering techniques to restore cell or tissue function.

[0161] For example, the therapeutic agent or pharmaceutical composition can be used as a transplant material and can be applied to the treatment of various intestinal diseases, particularly as a material for regenerating and reconstructing damaged (including dysfunctional) intestinal tissue.

[0162] Thus, the pharmaceutical composition according to the present invention can be a tissue treatment agent.

[0163] Preferably, the cells may be an intestinal stem cell population.

[0164] In the present invention, the intestinal disease may be any one or more intestinal diseases selected from the group consisting of leaky gut syndrome, short bowel syndrome, post-irritable bowel disease, Crohn's disease, ulcerative colitis, intestinal Behcet's disease, infectious enteritis, ischemic enteropathy, and radiation enteritis.

[0165] The present invention relates to intestinal stem cell aggregates that can be used in pharmaceutical compositions for intestinal transplantation support for intestinal transplantation of small intestinal organoids.

[0166] The prerequisite for transplantation efficiency and regenerative therapy efficiency is to rapidly engraft at the transplanted site and supply blood to the transplanted cells and tissues by angiogenesis. Conventional small intestinal organoids or intestinal stem cells have a low engraftment rate during intestinal transplantation and insufficient angiogenesis, resulting in very low initial transplantation efficiency. The intestinal stem cell aggregate according to the present invention can be used as an adjuvant to improve the engraftment rate and angiogenesis efficiency during such intestinal transplantation and improve the efficacy of intestinal transplantation.

[0167] The composition also contains intestinal stem cell aggregates and small intestinal organoids and can be used as a composition for treating intestinal diseases.

[0168] In such a transplant, any one or more biodegradable supports selected from the group consisting of fibrin, laminin, collagen, gelatin, chitosan, alginate, hyaluronic acid, dextran, polylactic acid, poly(glycolic acid) (PGA), poly(lacticcoglycolic acid) (PLGA), poly-ε-(caprolactone), polyanhydride, polyorthoester, polyvinyl alcohol, polyethylene glycol, polyurethane, polyacrylic acid, poly-N-isopropylacrylamide, poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) copolymers, copolymers thereof, and mixtures thereof may be used together with the transplant.

[0169] That is, the cells to be the subject of the above-mentioned transplant material can be used for transplantation either as is or embedded in the above-mentioned support.

[0170] In addition, dimethyl sulfoxide (DMSO) or the like can be added for the purpose of protecting the cells, antibiotics or the like can be added for the purpose of preventing bacterial contamination, and various components (vitamins, cytokines, growth factors, steroids, etc.) can be added to the transplant material of the present invention for the purpose of activating, proliferating, or inducing differentiation of the cells.

[0171] The transplantation material of the present invention can also be used to construct an in vivo experimental system. For example, the transplantation material mentioned above can be transplanted into experimental animals such as mice, rats, guinea pigs, hamsters, pigs, cynomolgus monkeys, rhesus monkeys, and chimpanzees to produce humanized animals (human intestinal models). Such humanized animals are particularly useful for experiments such as pharmacokinetics and toxicity tests, and are expected to contribute to research on the influence of the first-pass effect on oral drugs and drug-induced enteritis.

[0172] In the present invention, prevention means any action that inhibits or delays the onset of intestinal diseases by administration of the composition, and treatment means any action that improves or ameliorates the symptoms of intestinal diseases by administration of the composition.

[0173] The composition of the present invention contains 1.0×10 5 pieces~1.0×10 10 pcs, preferably 1.0 x 10 6 pieces~1.0×10 9 The cell may include cells.

[0174] The pharmaceutical composition of the present invention can be formulated into various dosage forms such as solutions and suspensions by conventional methods.

[0175] The pharmaceutical composition of the present invention can be administered by a conventional method in the pharmaceutical field, by formulating it into a pharmaceutical preparation of a unit dosage form suitable for administration into the body of a patient, and the preparation contains an effective dosage amount by one or several administrations. As a dosage form suitable for such a purpose, parenteral administration preparations such as injections, infusions, and implants are preferable. In addition, the pharmaceutical composition can contain a pharmaceutical acceptable ordinary inert carrier and diluent. The pharmaceutical acceptable carrier and diluent can be biologically and physiologically compatible with the intestinal stem cell aggregates and the recipient of the transplant. The diluent can include, but is not limited to, saline, a water-soluble buffer, a solvent and / or a dispersion media. In addition, for example, in the case of an injection, a preservative, a pain-relieving agent, a solubilizer, or a stabilizer, and in the case of a preparation for local administration, a base, an excipient, a lubricant, or a preservative, can be further included.

[0176] The compositions of the invention can be used in an unfrozen state or frozen for later use. If freezing is required, standard cryopreservatives (e.g., DMSO, glycerol, Epilife Cell Freezing Medium (Cascade Biologics)) can be added to the cell population prior to freezing.

[0177] In addition, it can be implanted and administered using administration methods commonly used in the art, preferably, but not limited to, direct engraftment or transplantation at the diseased site of a patient in need of treatment. In addition, the administration can be non-surgical administration using a catheter, or surgical administration such as injection or transplantation after incision of the diseased site. The dosage is 5×10 5 ~10 8 / 60 kg adult or 5 x 10 5 ~10 8However, it should be understood that the actual dosage of the active ingredient must be determined in consideration of various relevant factors such as the disease to be treated, the severity of the disease, the administration route, the weight, age and sex of the patient, and therefore, the dosage does not limit the scope of the present invention in any way.

[0178] The present invention also provides a pharmaceutical composition comprising an intestinal stem cell aggregate for use in preventing or treating intestinal diseases.

[0179] The present invention also provides the use of intestinal stem cell populations in the manufacture of a medicament for use in the prevention or treatment of intestinal disease.

[0180] A method for treating an intestinal disorder is provided, comprising administering an intestinal stem cell population to a subject in need thereof.

[0181] By subject is meant humans and any animal capable of developing or contracting a kidney disease, including monkeys, cows, horses, sheep, pigs, chickens, turkeys, quail, cats, dogs, mice, rats, rabbits or guinea pigs.

[0182] The present invention also provides a pharmaceutical composition comprising an intestinal stem cell aggregate for use in intestinal transplantation support.

[0183] The present invention also provides the use of intestinal stem cell populations in the manufacture of a medicament for use in intestinal transplant support.

[0184] A method of assisted intestinal transplantation is provided, comprising administering an intestinal stem cell population to a subject in need thereof.

[0185] The present invention also provides a pharmaceutical composition comprising an intestinal stem cell aggregate for use in intestinal transplantation support.

[0186] The present invention also provides the use of intestinal stem cell populations in the manufacture of a medicament for use in intestinal transplant support.

[0187] A method of assisted intestinal transplantation is provided, comprising administering an intestinal stem cell population to a subject in need thereof.

[0188] The present invention also provides pharmaceutical compositions comprising intestinal stem cell aggregates and small intestinal organoids for use in preventing or treating intestinal diseases.

[0189] The present invention also provides the use of intestinal stem cell aggregates; and small intestinal organoids; in the manufacture of a medicament for use in preventing or treating intestinal disease.

[0190] Methods for treating intestinal disease are provided, comprising administering to a subject in need thereof intestinal stem cell populations; and small intestinal organoids.

[0191] 2) Model The present invention also provides an intestinal stem cell and / or intestinal epithelial cell model.

[0192] Specifically, intestinal stem cells can be used directly, or intestinal epithelial cells differentiated from the intestinal stem cells can be used as a model.

[0193] The intestinal stem cells according to the present invention can be used as a cell therapy agent for regenerative therapy of damaged intestinal tissue, and can help provide better results in research and clinical applications on tissue regeneration. More specifically, since the intestinal stem cells according to the present invention are composed of stem cell aggregates that are capable of engrafting in tissue and play a key role in tissue regeneration, they are not only highly likely to engraft in damaged tissue, but can also rapidly restore damaged tissue through direct regeneration.

[0194] In addition, it can be used for various evaluations, such as screening for the discovery of therapeutic agents important for regulating the regenerative and division ability of stem cells and evaluating the toxicity of drugs.

[0195] If necessary, the intestinal stem cells according to the present invention can also be used as a system model for developing an intestinal stem cell line, for example, a stem cell line by gene editing.

[0196] By stem cell line is meant cells that are capable of becoming fully differentiated and differentiating into other cell types with specific specialized functions or cells that can be maintained in an undifferentiated state.

[0197] The intestinal stem cells according to the present invention can be transformed to produce cell lines using, for example, lentivirus or CRISPR-Cas9 gene editing techniques, and the transformed cell lines provide various research methods, such as large-scale drug screening and efficacy evaluation, and real-time cell function tracking.

[0198] In addition, the fluorescent protein may be labeled with any one selected from the group consisting of green fluorescent protein (GFP), blue fluorescent protein (CFP), yellow fluorescent protein (YFP) and red fluorescent protein (DsRed), or the target cell may be transformed and used for analyzing the characteristics of the target cell.

[0199] In particular, it is possible to provide novel cell lines that can be monitored in real time by transformation with fluorescent proteins or the like.

[0200] The present invention provides an intestinal epithelial cell model comprising the intestinal epithelial cell. Preferably, the intestinal epithelium model can be a small intestinal epithelium model.

[0201] In the intestinal epithelial cell model comprising intestinal epithelial cells differentiated from the intestinal stem cell aggregate of the present invention, the intestinal epithelial cells may be intestinal epithelial cells having a crypt-villus structure. More preferably, the intestinal epithelial cells may be small intestinal epithelial cells. That is, the intestinal epithelial cell model may be an intestinal epithelial cell model having a crypt-villus structure.

[0202] The intestinal epithelial cell model can help provide more accurate results in research and clinical applications related to bacterial or viral infection and intestinal drug metabolism. More specifically, the intestinal epithelial cell model not only has the morphological and functional characteristics of living tissue, but also has high stability and reproducibility, and is highly applicable to evaluation of drug metabolism, bacterial or viral infection, microbial infection, etc.

[0203] In other words, it can be used as a platform for bacterial or viral infections, microbial infections, drug metabolic reactions, etc., and provides the ability to study symptoms related to bacterial, viral, and microbial infections, and to accurately predict drug side effects, stability, and interactions.

[0204] The intestinal epithelial cells produced by the production method of the present invention or an intestinal epithelial cell model containing the same can be used as an infectious disease model.

[0205] For example, the present invention can be used as a bacterial or viral infectious disease model or a microbial infectious disease model. Any known bacteria, viruses, or microorganisms can be used as the bacteria, viruses, or microorganisms. According to one embodiment of the present invention, the present invention can be used as a SARS-CoV-2 virus infectious disease model.

[0206] The present invention provides a method for screening therapeutic agents using the intestinal epithelial cell model according to the present invention.

[0207] That is, the present invention provides a drug screening method including the steps of: (a) infecting an intestinal epithelial cell model with bacteria or a virus; (b) treating the infected intestinal epithelial cell model with a drug; and (c) confirming a response to the drug treatment.

[0208] The present invention also provides a method for evaluating a drug, comprising the steps of (a) treating an intestinal epithelial cell model with a drug, and (b) evaluating the degree of absorption or bioavailability of the drug in the intestinal epithelial cell model of step (a).

[0209] Also provided is a method for providing an intestinal epithelial cell model for disease modeling, comprising the step of infecting the intestinal epithelial cell model with bacteria or viruses.

[0210] In the present invention, a step of treating the intestinal epithelial cell model with a test substance can be performed. In the present invention, the test substance is a substance predicted to prevent, improve or treat intestinal-related diseases, and for example, the drug candidate substance, test compound or test composition can include, but is not limited to, a low molecular weight compound, an antibody, an antisense nucleotide, a short interfering RNA, a short hairpin RNA, a nucleic acid, a protein, a peptide, and other extracts or natural products. In the present invention, if the expression of a biomarker protein or an mRNA that coats the biomarker protein increases or decreases after the treatment of the test substance compared to before the treatment, the test substance can be selected as a therapeutic agent for the disease.

[0211] Such diseases are, for example, bacterial or viral infectious diseases, microbial infectious diseases or gut-related diseases.

[0212] A bacterial or viral infectious disease or microbial infectious disease refers to any disease that can result from infection with a pathogenic bacterium, virus or microorganism.

[0213] In the present invention, the intestine-related disease includes, but is not limited to, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), Crohn's disease, ulcerative colitis (UC), short bowel syndrome, enterocolitis, and genetic intestinal diseases such as Hirschsprung's disease and Celiac disease.

[0214] All technical terms used in the present invention are used in the sense that they are commonly understood by those of ordinary skill in the art unless otherwise defined. In addition, although preferred methods and samples are described in this specification, similar or equivalent methods and samples are also included in the scope of the present invention. The contents of all publications referenced in this specification are incorporated herein by reference. Effect of the Invention

[0215] The intestinal stem cell culture method according to the present invention allows homogeneous intestinal stem cells to be cultured easily and quickly. In addition, the long-term subculture and cryopreservation / thawing process allows stable mass culture, and the cells can be cultured while maintaining their characteristics, allowing the construction of a highly reproducible intestinal stem cell culture system.

[0216] The intestinal stem cell model produced by the above culture system can be used as a new research model for research on human intestinal stem cells. In particular, intestinal stem cells derived from intestinal organoids formed from patient-tailored induced pluripotent stem cells are a cell source tailored to the patient and can be used as a cell source and research model for the development of regenerative therapeutic agents for intestinal diseases and the creation of artificial organs.

[0217] Furthermore, by using the method for producing intestinal epithelial cells according to the present invention, intestinal stem cells derived from intestinal organoids can be differentiated into intestinal epithelial cells, thereby obtaining a 2.5-dimensional intestinal epithelial cell model that has the diversity of intestinal cells and a structure similar to intestinal tissue.

[0218] The intestinal epithelial cell model is a model with increased enzymes for nutrition and substance metabolism, allowing for evaluation of the absorption and metabolism of various substances and serving as a barrier to separate the inside and outside of the body. It can also be used in modeling various diseases including infectious diseases, and can be used in various fields such as screening of therapeutic agents for disease treatment and evaluation of efficacy and toxicity of new drug candidates. [Brief description of the drawings]

[0219] [Figure 1] FIG. 1 is a schematic diagram of a method for isolating intestinal stem cell aggregates from 3D intestinal organoids derived from full-potential stem cells and culturing them in 2D. [Diagram 2] FIG. 1 shows cell morphology during 2D intestinal stem cell aggregate culture on feeder and Matrigel. [Diagram 3] FIG. 1 shows the morphology of cells when intestinal stem cell aggregates derived from 3D intestinal organoids derived from embryonic stem cells (hESC-hIO) were cultured in 2D on basement cells (feeder) and Matrigel. [Figure 4] This figure shows the results of screening various extracellular matrix coating conditions to maximize the engraftment ability of 2D intestinal stem cell aggregates. (a) This figure shows the cell morphology of 2D intestinal stem cell aggregates when 2D intestinal stem cells were cultured on culture vessels coated with 0.2% gelatin, 10 μg / ml Col Type I, 50 μg / ml Col Type I, 1% Matrigel, and 5% Matrigel. (b) This is a graph showing the results of calculating the surface area of ​​the cell colonies confirmed in (a) using the Image J program. [Diagram 5]Figures showing the results of screening to find the optimal culture medium composition for culturing 2-dimensional intestinal stem cell aggregates: (a) Colony size of intestinal stem cell aggregates confirmed by crystal violet (CV) staining when a single factor was removed from the components of the culture medium; (b) Graph showing the surface area calculation results using the Image J program for the colony size confirmed by crystal violet (CV) staining in (a). [Figure 6] FIG. 1 shows the morphology of intestinal stem cell aggregates when cultured in culture media from which essential factors (R-spondin 1, EGF, PGE2) have been removed, for culturing 2-dimensional intestinal stem cell aggregates. [Figure 7] The results indicate that WNT3A and R-spondin1, among the components of the culture medium, play an important role in maintaining the stemness and self-renewal of 2D intestinal stem cell aggregates by activating the WNT signaling pathway. (a) qPCR confirms that expression of marker genes related to stemness and self-renewal of 2D intestinal stem cell aggregates is reduced when WNT signaling is suppressed by removing WNT3A and R-spondin1. (b) Immunofluorescent staining of EdU and KI67 marker proteins confirms that proliferation of 2D intestinal stem cell aggregates is reduced when WNT signaling is suppressed by removing WNT3A and R-spondin1. [Figure 8] FIG. 1 shows the morphology of 2D intestinal stem cell aggregates whose growth was inhibited upon suppression of the WNT signaling pathway by removal of WNT ligands (WNT3A or R-spondin1) or treatment with WNT inhibitors (WNT-C59 or XAV939). [Figure 9]FIG. 1 shows the morphology of 2D intestinal stem cell aggregates in which cell proliferation was inhibited upon suppression of the EGFR signaling pathway by removal of EGF ligand or treatment with an EGF inhibitor (PD0325901), and the results of a cell viability / death (Calcein-AM (live) / Etidium homodimer1 (dead)) assay. [Figure 10] The results show that the growth of 2D intestinal stem cell aggregates is inhibited when the PGE2 signaling system is suppressed by removing the PGE2 ligand or by treatment with a PGE2 inhibitor (EP2i or EP4i). (a) This is a diagram showing the morphology of 2D intestinal stem cell aggregates whose growth was inhibited when the PGE2 signaling system was suppressed by removing the PGE2 ligand or by treatment with a PGE2 inhibitor (EP2i or EP4i). (b) This is a graph showing that of the PGE2 receptors (PTGER1 to PTGER4) and PGE2 synthetic enzymes (PTGES), PTGER2 and PTGER4 are specifically expressed in 2D intestinal stem cells. [Figure 11] The results show that stable long-term culture is possible by subculturing 2D intestinal stem cell aggregates. (a) Morphology of 2D intestinal stem cell aggregates at P0, P1, P3, P5, P10, P20, and P30. (b) Graph showing increase in total cell number during the subculturing process. [Figure 12] When the remaining factors other than the essential constituent factors of the culture medium in Figure 6 (R-spondin 1, EGF, PGE2) were removed from the culture medium, there was no effect on the growth of 2D intestinal stem cells at P0, but the cell engraftment and proliferation ability were reduced during subculture. [Figure 13] This figure shows that cell engraftment ability is increased by treatment with a NOTCH activator (Jagged-1 or Valproic acid) or a ROCK inhibitor (Y-27632) during subculture of 2-dimensional intestinal stem cells. (a) This figure shows the morphology of 2-dimensional intestinal stem cell aggregates engrafted when treated with a NOTCH activator and a ROCK inhibitor alone or simultaneously. (b) This is a graph showing the results of measuring the number of cells in the 2-dimensional intestinal stem cell aggregates engrafted in (a) using a Countess III cell counter. [Figure 14] Figure 1 shows the morphological characteristics of cells upon culturing 2D intestinal stem cell aggregates under optimized culture conditions: (a) 2D intestinal stem cell aggregates cultured under optimized culture conditions are composed of a monolayer; (b) 100% viability of 2D intestinal stem cell aggregates cultured under optimized culture conditions. [Figure 15] This figure shows that 2D intestinal stem cells can be frozen and thawed under optimized culture conditions, and that highly reproducible culture with little difference between arrangements is possible. (a) This figure shows the cell morphology on days 2, 4, and 8 after culturing thawed 2D intestinal stem cell aggregates after frozen storage. (b) This figure shows that 2D intestinal stem cell aggregates can be cultured for a long period with little difference between arrangements. [Figure 16] FIG. 1 shows a schematic of the analytical process for analyzing the cellular composition and characteristics of 2D intestinal stem cell aggregates by single cell RNA sequencing (scRNA-seq). [Figure 17] FIG. 1 shows that 2D intestinal stem cell aggregates contain abundant cells that display epithelial versus stromal cell characteristics, as determined by epithelial and stromal cell marker gene expression analysis. [Figure 18] Figure 1 shows that the results of genome analysis of 2D intestinal stem cell aggregates show similar characteristics to intestinal epithelial cells of 6-8 week fetuses. (a) A heatmap showing the results of comparing the expression of marker genes between 2D intestinal stem cell aggregates and human intestinal epithelial cells from fetuses to adults. (b) A dendrogram showing the results of hierarchical clustering of the results in (a) according to the similarity of marker gene expression patterns. [Figure 19]Figure 1 shows the types and composition of cells constituting a 2D intestinal stem cell aggregate based on the results of single-cell transcript analysis. (a) A figure showing the types and distribution of cells constituting a 2D intestinal stem cell aggregate on a UMAP based on the results of single-cell transcript analysis. (b) A diagram showing the ratio of cells constituting a 2D intestinal stem cell aggregate calculated based on the results of (a). [Figure 20] Figure 1 shows the results of a comparative analysis of single-cell transcriptome analysis of 2D intestinal stem cell aggregates with the results of a single-cell transcriptome analysis of human intestinal epithelium in a reference. (a) A UMAP showing the types and distribution of cells constituting the human intestinal epithelium based on the analysis of single-cell transcriptome reported in the reference. (b) The results of (a) and the analysis of single-cell transcriptome analysis of 2D intestinal stem cell aggregates are shown on the same UMAP, showing that the cellular composition of 2D intestinal stem cell aggregates is mostly composed of intestinal epithelial stem cells and progenitor cells. (c) A diagram showing the types and expression patterns of marker genes that are specifically expressed by the types of cells constituting the human intestinal epithelium based on the analysis of single-cell transcriptome. [Figure 21] FIG. 1 shows that 2D intestinal stem cell aggregates contain numerous cells that display characteristics of stem or progenitor cells, as determined by analysis of the expression patterns of stem cell-specifically expressed marker genes. [Figure 22] Figure 1 shows the results of immunofluorescence staining to confirm that the 2D intestinal stem cell aggregates are mainly composed of cells that exhibit the characteristics of stem cells and progenitor cells. (a) Immunofluorescence staining for stem cell-specific marker proteins LDHB, EIF3E, SOX9, and KI67 confirms that most of the cells in the 2D intestinal stem cell aggregates are stem cells or progenitor cells. (b) No expression of MUC2 and CHGA, marker proteins for secretory cells among the differentiated cells that make up the intestinal epithelium, confirms that the 2D intestinal stem cell aggregates do not differentiate into secretory cells. [Figure 23]This is a schematic diagram of a method for differentiating 2D intestinal stem cell aggregates into 2.5D intestinal epithelial cells by air-liquid interface culture. [Figure 24] This figure shows the morphology of cells differentiated after removing a single factor from the culture medium in order to find essential factors important for the differentiation of 2D intestinal stem cell aggregates into 2.5-dimensional intestinal epithelial cells using air-liquid interface culture. [Diagram 25] This figure confirms that 2-dimensional intestinal stem cell aggregates are normally differentiated into 2.5-dimensional intestinal epithelial cells by air-liquid interface culture in a minimal medium composed of essential factors. (a) This figure shows that all 2-dimensional intestinal stem cell aggregates differentiated into 2.5-dimensional intestinal epithelial cells by air-liquid interface culture in a culture medium (Full M) for 2-dimensional intestinal stem cell aggregates and a minimal medium (minimal M) composed of essential factors, show normal differentiation. (b) This figure shows that 2-dimensional intestinal stem cell aggregates derived from various types of full differentiation potential stem cells are differentiated into 2.5-dimensional intestinal epithelial cells by air-liquid interface culture in a minimal medium. (c) This figure shows that 2.5-dimensional intestinal epithelial cells differentiated by air-liquid interface culture can be cultured with high reproducibility with almost no difference between arrangements. [Figure 26] 1 is a graph showing that when 2D intestinal stem cell aggregates are differentiated into 2.5D intestinal epithelial cells by air-liquid interface culture, the expression of differentiated cell marker genes gradually increases as differentiation proceeds. [Figure 27]This figure confirms that a villus-like structure is formed and the expression of differentiated cell marker proteins increases during differentiation of 2D intestinal stem cell aggregates into 2.5D intestinal epithelial cells by air-liquid interface culture. (a) After differentiation of 2D intestinal stem cell aggregates into 2.5D intestinal epithelial cells by air-liquid interface culture, the cross sections of the cells were confirmed by H&E staining on days 4, 8, and 12, and the expression levels of marker proteins were confirmed by immunofluorescence staining. (b) This is a graph showing the results of measuring the thickness of the cell cross sections confirmed by H&E staining from (a) using the Image J program. (c) This is a graph confirming the barrier function of intestinal epithelial cells by measuring transepithelial electrical resistance (TEER) on days 4, 8, and 12 after differentiation of 2D intestinal stem cell aggregates into 2.5D intestinal epithelial cells by air-liquid interface culture. [Figure 28] This figure shows the results of principal component analysis (PCA) using transcript expression profiles to compare 2D intestinal stem cell aggregates and 2.5D intestinal epithelial cells differentiated by air-liquid interface culture with human PSCs, immature 3D intestinal organoids (Control hIO), mature 3D intestinal organoids (Mature hIO), functional intestinal epithelial cells (hIEC), and human intestinal epithelial tissue (hSI). [Figure 29] This figure shows gene clusters and representative genes whose expression patterns differ during differentiation into intestinal epithelial cells, as determined by transcriptomic analysis of 2D intestinal stem cell aggregates and 2.5D intestinal epithelial cells differentiated by air-liquid interface culture. (a) This is a diagram showing the types of gene clusters whose expression patterns increase or decrease during differentiation into 2.5D intestinal epithelial cells by air-liquid interface culture from 2D intestinal stem cell aggregates. (b) This is a graph showing the results of verifying the expression patterns of representative genes of the gene clusters discovered in (a) by qPCR experiments. [Diagram 30]FIG. 1 is a schematic diagram of the process of generating an intestinal stem cell line expressing a fluorescent protein by gene delivery using lentivirus. [Diagram 31] This is a diagram showing the cell morphology and expression of fluorescent protein at each step of the production of intestinal stem cell lines expressing fluorescent proteins. (a) This is a diagram showing the morphology and expression of fluorescent protein of intestinal stem cell lines expressing fluorescent proteins immediately after the delivery of fluorescent genes using lentivirus (after spin infection), after selection and expansion of clones using antibiotics, and then after selection and separation into single cells using trypsin (Trypsin-EDTA) to produce single-cell-derived cell lines, immediately after subculture (after cell seeding), and after culturing to a certain size or more (after expansion). (b) This is a diagram showing the morphology and expression of fluorescent protein of cells by date, in which only single clones were isolated and cultured after the single-cell-derived fluorescent protein-expressing clones produced in (a) were treated with collagenase and dispase (Collagenase type IV+Dispase) to separate them. [Diagram 32] The intestinal stem cell line prepared in Figure 31 was used to differentiate into 3D intestinal organoids and 2.5D intestinal epithelial cells by air-liquid interface culture, and the cell morphology and expression of fluorescent protein were confirmed. (a) This is a diagram showing the cell morphology and expression of fluorescent protein on the 6th, 14th, and 20th days after differentiation of an intestinal stem cell line expressing a fluorescent protein into a 3D intestinal organoid inside a Matrigel dome. (b) This is a diagram showing the morphology and expression of fluorescent protein in intestinal epithelial cells on the 8th day after differentiation of an intestinal stem cell line expressing a fluorescent protein into 2.5D intestinal epithelial cells by air-liquid interface culture. [Diagram 33] FIG. 1 is a schematic diagram of the intestinal disease modeling and intestinal stem cell transplantation experiment process to confirm the regenerative therapeutic effect of 2D intestinal stem cell aggregates and their possible use as a cell therapy agent. [Diagram 34]Figure 1 shows the process of transplanting two-dimensional intestinal stem cell aggregates using a mouse colonoscopy, and photographs of the condition of the mouse after transplantation. (a) A photograph of a mouse colonoscopy into which cells can be injected (left) and a photograph of the transplantation of two-dimensional intestinal stem cells using a mouse colonoscopy (right). (b) A photograph of the condition of a mouse transplanted with two-dimensional intestinal stem cell aggregates immediately after transplantation. [Diagram 35] FIG. 1 shows the change in body weight over time according to the dates after transplantation in mice transplanted with Matrigel or 2-dimensional intestinal stem cells. [Diagram 36] 1 shows charts and graphs depicting post-transplant survival rates of mice transplanted with Matrigel or 2D intestinal stem cells. [Figure 37] This figure shows the results of confirming the regenerative effect of intestinal epithelium using a mouse colonoscope before and after modeling intestinal epithelial damage using hot-EDTA, and immediately after, 3 days, and 14 days after transplantation of Matrigel or 2D intestinal stem cells into mice with induced intestinal damage. [Figure 38] Figure confirming that 2D intestinal stem cell aggregates had been engrafted onto the damaged intestinal epithelium. (a) Using IVIS equipment, it was confirmed that 2D intestinal stem cell aggregates labeled with DiR fluorescent dye had been engrafted onto the site of intestinal injury even 14 days after transplantation. (b) An intestinal stem cell line expressing a fluorescent protein (Green Fluorescent Protein; GFP) was transplanted onto the damaged intestinal epithelium, and the intestine was isolated 14 days later to confirm fluorescent protein expression, confirming that intestinal stem cells had been successfully engrafted onto the damaged intestinal epithelium. [Figure 39]This figure shows the regenerative ability of 2D intestinal stem cell aggregates by confirming that the damaged intestinal epithelium was normally regenerated by 2D intestinal stem cell aggregates engrafted at the site of damaged intestinal epithelium. (a) This figure shows that 14 days after transplantation of Matrigel or 2D intestinal stem cell aggregates at the site of damaged intestinal epithelium, the intestinal tissue was isolated and the morphology of the intestinal epithelial tissue was analyzed by H&E and AB-PAS staining, and it was found that the intestinal epithelial tissue was regenerated only when 2D intestinal stem cell aggregates were transplanted. (b) This figure shows that 14 days after transplantation of an intestinal stem cell line expressing a fluorescent protein at the site of damaged intestinal epithelium, the intestine was isolated and the expression of the fluorescent protein was confirmed, and it was confirmed that the fluorescent protein was expressed throughout the crypt-villus structure, confirming that the intestine was regenerated by the intestinal stem cell line. [Diagram 40] Schematic diagram of the entire process of disease modeling using SARS-CoV-2 virus infection of 2.5D intestinal epithelial cells differentiated from immature or mature 2D intestinal stem cell aggregates using air-liquid interface culture. [Diagram 41] FIG. 1 shows the morphology of 2.5-dimensional intestinal epithelial cells differentiated from immature or mature 2-dimensional intestinal stem cell aggregates by air-liquid interface culture, observed 2, 4, 6, 8, and 10 days after the start of differentiation. [Diagram 42] FIG. 13 is a graph showing that intestinal epithelial cells differentiated from mature 2D intestinal stem cell aggregates have relatively higher expression of marker genes indicating intestinal maturity compared to intestinal epithelial cells differentiated from immature 2D intestinal stem cell aggregates. [Diagram 43]This is a diagram showing the results of confirming that intestinal epithelial cells differentiated from mature 2D intestinal stem cell aggregates, the expression of receptors important for SARS-CoV-2 infection is relatively higher than intestinal epithelial cells differentiated from immature 2D intestinal stem cell aggregates. (a) This is a graph showing the results of analyzing only the expression of receptors important for SARS-CoV-2 infection from the results of transcriptome analysis of intestinal epithelial cells differentiated from mature or immature 2D intestinal stem cell aggregates. (b) This is a diagram showing the results of confirming the expression level of ACE2 protein, which was confirmed to have increased expression in (a), among the receptors important for SARS-CoV-2 infection in intestinal epithelial cells differentiated from mature or immature 2D intestinal stem cell aggregates, using immunofluorescence staining. [Diagram 44] This graph shows the results of confirming viral transcripts in intestinal epithelial cells using qPCR, demonstrating that intestinal epithelial cells differentiated from mature 2D intestinal stem cell aggregates are more susceptible to SARS-CoV-2 infection than intestinal epithelial cells differentiated from immature 2D intestinal stem cell aggregates. EXAMPLES

[0220] The present invention will be described in more detail below with reference to examples. These examples are provided to more specifically explain the present invention, and the scope of the present invention is not limited to these examples.

[0221] Experimental example 1. Cell culture and production of iPSCs hPSCs (human pluripotent stem cells), including hESCs (human embryonic stem cells) and hiPSCs (human induced pluripotent stem cells), were cultured by a known method (Molecular carcinogenesis 55, 387-396 (2016), Proteomics 15, 2220-2229 (2015)). Non-insertion-type hiPSCs were reprogrammed by transfection using an episomal iPSC reprogramming vector (Cat. No. A14703. Invitrogen, Carlsbad, CA, USA) by electroporation using a known method.

[0222] Five days after electroporation, fibroblasts were plated onto Matrigel (BD Biosciences, San Diego, CA, USA)-coated 6-well plates at 1 × 10 5 The cells were plated at 100 cells / well and cultured in E8 medium (Stem Cell Technologies, Vancouver, Canada). After 3 weeks, hiPSC colonies were selected and expanded in cell number for subculture and further characterization.

[0223] Experimental example 2. Differentiation of hPSCs into intestinal organoids (hIOs) for the production of 3D intestinal organoids Human intestinal organoids (hIOs) were produced using a known method (Nature 470, 105-109 (2011)). To induce complete endoderm, hPSCs were cultured in Matrigel or ECMatrix. TMThe cells were plated on dishes coated with 100ng / ml Activin A (R&D Systems, Minneapolis, MN, USA) in RPMI 1640 medium with 0%, 0.2% and 2% deficient fetal bovine serum (dFBS, HyClone, Thermo Fisher Scientific Inc., Waltham, MA, USA) for 3 days, and then treated with 500ng / ml FGF4 (R&D Systems) and 3μM CHIR99021 (TOCRIS) in RPMI 1640 medium with 2% dFBS for 4–6 days to differentiate into 3D hindgut spheroids. From day 4 after induction into the hindgut, the spheroids were inserted into Matrigel (BD Biosciences) and cultured in hIO medium (2 mm L-glutamine, 1% penicillin-streptomycin, and 15 mm HEPES buffer in Advanced DMEM F12) containing 1X B27 (Invitrogen), 200-250 ng / ml R-spondin 1 (R&D Systems), 100 ng / ml EGF (R&D Systems), and 40-50 ng / ml Noggin (R&D Systems), and subcultured once every 10-14 days. For maturation of the intestinal organoids, they were cultured in hIO medium with 1 ng / ml interleukin 2 (IL-2, R&D Systems) for approximately 2 passages.

[0224] Experimental Example 3. Isolation and culture of intestinal stem cells from 3D intestinal organoids The 3D intestinal organoids were separated from the Matrigel dome, and the remaining Matrigel was removed as much as possible by pipetting. The separated organoids were placed in 1 ml of 0.25% trypsin-EDTA (TE, Invitrogen) and incubated in a 37°C water bath for approximately 5 minutes. After this, the organoids were gently pipetted less than 5 times to separate them into single cells and small clumps, and then basal media was added to make the total volume 10 ml. The cells fed to the centrifuge were left on a culture dish coated with feeder cells or 1% Matrigel (Corning). The intestinal stem cell culture medium (2 mm L-glutamine, 1% penicillin-streptomycin, and 15 mm HEPES buffer in Advanced DMEM) was prepared by mixing 200 ng / ml R-spondin 1 (R&D Systems), 100 ng / ml EGF (R&D Systems), and 2.5 μM prostaglandin E2 (Sigma-aldrich) as main components, and 1X B27 (Invitrogen), 80 ng / ml Noggin (R&D Systems), 10 nM [Leu15]-Gastrin I (Sigma-aldrich), 100 ng / ml human recombinant WNT3A (R&D Systems), 500 nM A-83-01 (Tocris), 10 μM SB202190 (Sigma-aldrich), 1 mM N-acetylcysteine ​​(Sigma-aldrich), and 10 mM nicotinamide (Sigma-aldrich) as supplementary components. F12) and subcultured once every 7–10 days. During the first 2 days of subculture, 1 μM Jagged-1 (Anaspec) and / or 2.5 μM Y-27632 (Tocirs) were added to the intestinal stem cell culture medium.

[0225] Experimental Example 4. Freezing and thawing of intestinal stem cells To freeze intestinal stem cells, they were washed once or twice with PBS 3 to 5 days after subculture, and similar to the subculture process, they were treated with TE at 37°C for about 5 minutes to separate them into single cells or small cell clusters, and then washed once with hIO medium (2 mm L-glutamine, 1% Penicillin-Streptomycin, and 15 mm HEPES buffer in Advanced DMEM F12). Then, they were frozen in freezing medium (Recovery TM The cells were thoroughly lysed by adding Cell Culture Freezing Medium (Gibco), and then a suspension was prepared, frozen, and stored long-term in an LN2 tank.

[0226] A warm 37℃ medium was prepared in advance for thawing frozen intestinal stem cells. After quickly thawing the frozen cells, they were washed once with intestinal stem cell culture medium and cultured in intestinal stem cell culture medium containing WNT / R-spondin activator, prostaglandin signal transduction activator, and receptor tyrosine kinase ligand on a pre-coated Matrigel culture dish. The culture medium was changed every two days, and intestinal stem cells were cultured for 3 to 7 days depending on the state of the cells when first thawed, and then subcultured in the same manner.

[0227] Experimental Example 5. Intestinal epithelial cell differentiation method using the air-liquid interface culture method Intestinal stem cells grown at 70-80% confluency were washed once or twice with PBS and then incubated in TE for 5-7 minutes in a 37°C incubator. Single-cell dissociated intestinal stem cells were collected and diluted with hIO medium. Cells were collected by centrifugation, the supernatant was removed, intestinal stem cell culture medium was added and mixed thoroughly, and the number of cells was measured using a Countess III cell counter (Thermo Scientific, Inc.). 2.5-3.5 × 10 cells were plated onto the insert of a 12-Transwell plate (Corning) coated with 1% Matrigel. 5After adding 100 cells, they were cultured in an incubator. When the cell confluency reached 100%, the culture medium in the upper layer was completely removed, and the medium in the lower layer was replaced with differentiation medium (2mm L-glutamine, 1% Penicillin-Streptomycin, and 15mm HEPES buffer in Advanced DMEM F12) containing 200ng / ml R-spondin1 (R&D Systems), 100ng / ml EGF (R&D Systems), 2.5μM Prostaglandin E2 (Sigma-aldrich), 10μM SB202190 (Sigma-aldrich), and 10mM Nicotinamide (Sigma-aldrich). After that, the surface of the upper layer was washed with PBS or hIO medium every two days, and the lower layer was replaced with new differentiation medium and cultured for about 8 to 12 days.

[0228] Experimental Example 6. Cell viability measurement method To measure the viability of 2D intestinal stem cell aggregates cultured on 1% Matrigel-coated culture dishes, we used a kit (LIVE / DEAD Viability / Cytotoxicity Kit, Invitrogen) that can distinguish and stain live and dead cells. Live cells were stained with calcein-AM, and dead cells were stained with ethidium homodimer-1. The stained cells were observed under a fluorescence microscope (Olympus).

[0229] Experimental Example 7. Cell growth rate measurement method 2D intestinal stem cell aggregates were plated on a culture dish coated with 1% Matrigel and then cultured for 2 to 7 days. The culture medium was then removed, and the cells were washed 1 to 2 times with PBS (Sigma-Aldrich), followed by addition of TE (Invitrogen) to separate the cells. After centrifugation, media was added to separate the cells into single cells, and the number of cells was measured using a CountessIII cell counter (Thermo Scientific, Inc.).

[0230] Experimental Example 8. Crystal violet (CV) staining method Intestinal stem cell aggregates were fixed with 4% paraformaldehyde (PFA) and stained with 0.02% crystal violet solution (Sigma-Aldrich) for 10 min at room temperature. They were then washed three times with sterile water and images were taken. Colony size of intestinal stem cell aggregates was analyzed using Image J software (National Institute of Health).

[0231] Experimental example 9. Quantitative real-time RT-PCR (qRT-PCR) Total RNA was extracted from cells using the RNeasy kit (Qiagen) and reverse transcribed using the Superscript III cDNA synthesis kit (Invitrogen). qRT-PCR was performed using the 7500 Fast Real-time PCR system (Applied Biosystems, Foster City, CA, USA) as described previously (Cho et al., Oncotarget 6, 23837-23844, 2015). All experiments were performed in triplicate, and the C values ​​of each target gene were calculated using software provided by the manufacturer. The base sequences of the primers used are shown in Table 1.

[0232] [Table 1] JPEG2025506031000003.jpg229170 JPEG2025506031000004.jpg157170

[0233] Experimental example 10. Cell and tissue immunofluorescence Immunofluorescence was performed according to a known method (Kwak et al., Biochemical and biophysical research Communications 457, 554-560, 2015). Specifically, 2D intestinal stem cell aggregates and differentiated intestinal epithelial cells or intestinal tissue were fixed with 4% paraformaldehyde (PFA) and permeabilized with PBS containing 0.1% Triton X-100.

[0234] After cryoprotection of differentiated intestinal epithelial cells or intestinal tissues with sucrose, the membrane of the insert well was cut and placed vertically in optimal cutting temperature (OCT) compound (Sakura Finetek, Tokyo, Japan) and then frozen. Cryosections were then cut at 10 μM using a cryostat microtome at -20°C and permeabilized with PBS containing 0.1% Triton X-100 for immunofluorescence examination.

[0235] After blocking with 4% BSA, the cells were reacted with primary antibodies overnight at 4°C. Then, they were reacted with secondary antibodies for 1 hour at room temperature. The primary antibodies used are listed in Table 2. DAPI was added to visualize nuclei. Slides were observed using an EVOS FL Auto2 (ThermoFisher) and an Axiovert 200M microscope (Carl Zeiss, Göttingen, Germany) or a fluorescence microscope (IX51, Olympus, Japan).

[0236] [Table 2]

[0237] Experimental example 11. Single cell RNA sequencing After washing the 2D intestinal stem cell aggregates 2-3 times with PBS (Sigma-Aldrich), 0.25% TE (Invitrogen) was added and cells were separated for more than 10 minutes, and then single cells were separated using a 40 μm cell strainer (BD Bioscience). After dilution in PBS containing 0.04% BSA, cell number and viability were measured using a Countess III cell counter (Thermo Scientific, Inc.). Chromium Next GEM Single Cell 3' reagent kit v3.1 (10X Genomics) was used to construct a transcript library. Briefly, cells were diluted onto Chromium Next GEM Chip G to create a transcript library of approximately 5,000 single cells, and then approximately 60,000 base sequences per cell were analyzed using a Novaseq 6000 sequencer (Illumina).

[0238] Example 12. RNA Sequencing and RNA Quantification For RNA sequencing and quantification, RNA samples were prepared with an RNA Integrity Number (RIN) value of 7.5 or higher using the Agilent 2100 Bioanalyzer system (Agilent Biotechnologies, Palo Alto, USA), and mRNA libraries were prepared using the Illumina TruSeq kit. Sequencing was performed using Illumina HiSeq2500 machines (Illumina, San Diego, CA, USA). Sequencing quality was determined using the FastQC package, and reads with a trimmed length of 50 bases or less were excluded. Mapping was then performed using HISAT2 (v2.0.5), and human genome information was obtained from hg19. Differentially expressed genes (DEGs) between samples were analyzed using Cuffquant and Cuffnorm (Cufflinks v2.2.1).

[0239] Experimental Example 13. Bioinformatic Analysis For single-cell transcriptome sequencing results analysis, the initial data was processed and a gene expression matrix was constructed using 10X Genomics software CellRanger (version 3.1). Furthermore, for comparative analysis of transcriptomes, the results of the analysis of single-cell transcriptomes from fetal and adult intestinal epithelial tissues reported in references (Elmentaite et al. 2020) were used. The integrated data was normalized and selected using Scanpy package v1.8, and clustering and cell type annotation were performed using the primary processed data. Next, data integration was performed using Spearman's correlation, and the composition ratio was calculated for each cell.

[0240] Bioinformatics analysis was performed using IPA analysis software (Ingenuity systems, Redwood City, CA, USA), the PANTHER (Protein ANalysis THrough Evolutionary Relationships, http: / / www.pantherdb.org) database and DAVID Bioinformatics Resources 6.7 (http: / / david.abcc.ncifcrf.gov). Functionally grouped gene ontology (GO) / pathways were analyzed using the Cytoscape software platform (version 3.3.0, http: / / www.cytoscape.org / what_is_cytoscape.html) with the ClueGO plug-in (Version 2.2.5, http: / / apps.cytoscape.org / apps / cluego).

[0241] Experimental Example 14. Creation of fluorescent protein expressing cell lines by lentivirus infection To generate intestinal stem cell lines expressing green fluorescent protein (eGFP), lentivirus expressing EF-1α-Gene X-IRES2-eGFP-IRES-Puro was purchased from GeneCopoeia (MD, USA). Approximately 2–4 × 10 5 The 2D intestinal stem cells were centrifuged at 2,500 rpm for 90 minutes in medium containing lentivirus and 8 μg / ml polybrene, and then cultured for 48 hours after adding additional medium. For clonal selection, the cells were cultured in a culture medium containing 1 μg / ml puromycin until only colonies expressing fluorescent proteins remained.

[0242] To ensure that the clones were derived from single cells, the cells were isolated into single cells through TE treatment, plated at a low density, and cultured until the colonies reached a desired size. To harvest the whole colonies, collagenase type IV and dispase were mixed for 5 minutes and the single colonies were isolated by pipetting. The isolated single colonies were transferred to new plates and cultured until they grew to a sufficient size. To confirm the differentiation potential of the intestinal stem cell lines expressing fluorescent proteins, they were differentiated into 3D organoids in Matrigel domes or into 2.5D intestinal epithelial cells using the air-liquid interface culture method.

[0243] Experimental Example 15: 2D intestinal stem cell aggregate transplantation experiment using colonoscopy To confirm the tissue regeneration ability of the 2D intestinal stem cell aggregates, we created an intestinal epithelial injury model using hot-EDTA in male NIG mice (NOD / SCID deleted IL2Rg gene, 6-12 weeks old; GHBio, Daejeon, Korea). 62D intestinal stem cells were transplanted using a colonoscopic injector (Image 1 Hub HD H3-Z; D-Light C; Rigid HOPKINS telescope; Karl Storz, Tuttlingen, Germany; and optimised injector; Vetcom, Gwacheon, Korea) (Matrigel transplantation group, n=3; intestinal stem cell aggregate transplantation group, n=5). After transplantation, the anus was blocked with Vetbond Tissue Adhesive (3M, MN, USA) for 6 to 12 hours. Next, the transplantation site was monitored using a colonoscope on days 0, 3 and 14, and finally, on day 14, intestinal tissue was isolated from euthanized mice to confirm regenerative ability.

[0244] Experimental Example 16. Tissue analysis using a fluorescent stereomicroscope To confirm that intestinal stem cells had engrafted into the damaged intestinal epithelium, bright field and fluorescent images of the intestine of mice 14 days after transplantation were taken using a stereomicroscope (SZX16, Olympus, Japan).

[0245] Experimental Example 17. Histological (Hematoxylin & Eosin, H&E) staining experiment For histopathological analysis, intestinal tissues or intestinal epithelial cells were cryoprotected with sucrose, and the membrane of the insert well was cut and placed vertically in optimal cutting temperature (OCT) compound (Sakura Finetek, Tokyo, Japan) and then frozen. Frozen sections were then cut at 10 μM using a cryostat microtome at -20°C, attached to glass slides, and stained with H&E by the published method. Slides were observed under a light microscope (BX53F, Olympus, Japan).

[0246] Experimental Example 18: Transepithelial electrical resistance (TEER) measurement experiment To confirm the barrier functionality of differentiated intestinal epithelial cells, transepithelial electrical resistance was measured using an epithelial tissue volt / ohmmeter (EVOM, WPI, FL, USA). After washing the upper and lower layers of the transwell in which the intestinal epithelial cells were cultured with PBS, fresh culture medium was added, and electrodes were immersed in the upper and lower layers one by one, and the TEER value was measured.

[0247] Experimental Example 19. SARS-CoV-2 virus infection experiment The differentiated intestinal epithelial cells were infected with SARS-CoV-2 virus produced from Vero cells at a multiplicity of infection (MOI) of 0.01 or 0.001 for 1 hour. After that, the medium containing the virus was carefully removed, and fresh culture medium was added and the cells were further cultured for 72 hours. The cells were then harvested for RNA isolation and purification to detect the virus that had infected the intestinal epithelial cells.

[0248] Example 20. Statistical analysis All results are expressed as mean ± standard error of the mean (sem) and all experiments were performed in at least triplicate. P values ​​were determined using two-tailed t-tests or observational ANOVA. All analyses for statistical significance were calculated relative to the control group unless otherwise noted.

[0249] Example 1. Isolation and culture of intestinal stem cell aggregates from 3D intestinal organoids We have developed a new method to isolate and enrich intestinal stem cells from 3D intestinal organoids in order to easily and quickly mass-cultivate highly pure intestinal stem cells (Figure 1). The newly developed intestinal stem cell culture technique allows for 2D culture of intestinal stem cell aggregates on feeder cells or on plates coated with 1% Matrigel, and makes it possible to isolate and culture only intestinal stem cells from 3D intestinal organoids derived from various types of pan-differentiation potential stem cell lines (Figures 2-3).

[0250] In order to maximize the survival rate of intestinal stem cell aggregates, coating tests were conducted using various coating materials, and it was confirmed that the highest survival rate was observed when 1% Matrigel was coated (Figure 4). However, excellent survival rates were also observed when gelatin, collagen, etc. were used, confirming that intestinal stem cell aggregates can be easily cultured even without heterologous components.

[0251] Example 2. Development of culture medium with optimized composition for culturing 2-dimensional intestinal stem cell aggregates To minimize performance differences between 2D intestinal stem cell aggregate configurations, we screened to eliminate the use of factors with unclear chemical compositions and to identify new culture medium compositions composed of factors with well-defined composition and volume.

[0252] As a result, WNT / R-spondin activators, activators of the prostaglandin signaling pathway, and receptor tyrosine kinase ligands were identified as factors that play essential roles in the survival of intestinal stem cell aggregates. In particular, in relation to these signaling systems, it was confirmed that the combination of R-spondin 1, PGE2, and EGF was the most suitable combination of factors for the survival of 2D intestinal stem cell aggregates (Figures 5-6).

[0253] In order to specifically confirm the roles of the above essential factors, we examined more specifically the difference in effect depending on the presence or absence of each factor.

[0254] Among the essential factors, R-spondin 1 has been shown to regulate the stemness and proliferation of 2D intestinal stem cells by activating the WNT signaling pathway (Figures 7 to 8).

[0255] In addition, in the case of EGF, it has been revealed that activation of the EGF-EGFR signaling pathway prevents cell death in intestinal stem cell aggregates and regulates their proliferation (Figure 9).

[0256] Finally, in the case of PGE2, activation of the PGE2-EP2 / 4 signaling pathway has been shown to regulate the proliferation of intestinal stem cell populations (Figure 10).

[0257] Example 3. Establishment of subculture and cryopreservation method for mass culture of 2-dimensional intestinal stem cell aggregates To improve the utility of the 2D intestinal stem cell aggregates, we tested whether stable long-term culture, mass culture, and freezing and thawing were possible. First, we confirmed that stable subculture was possible for more than 30 times in the optimized medium, and mass proliferation was possible without cell loss (Figure 11).

[0258] On the other hand, the remaining factors other than the essential factors in the culture medium composition did not affect the engraftment and initial proliferation of 2D intestinal stem cell aggregates. However, for long-term subculture, we confirmed that long-term culture was more efficient when factors such as B27, Noggin, Gastrin, WNT3a, A-83-01, SB202190, n-acetylcysteine, and nicotinamide were present (Figure 12).

[0259] Furthermore, to prevent cell loss during subculture, we sought additional factors important for subculture. As a result, we confirmed that subculture efficiency increased when Jagged-1 or valproic acid, which activates the Notch signaling system, or Y-27632, which inhibits ROCK activity, was added, and that subculture efficiency was maximized when the two signals were simultaneously regulated (Figure 13). Under the optimized conditions, the 2D intestinal stem cell aggregates were composed of a monolayer, showed a 100% survival rate, and were confirmed to be capable of stable culture (Figure 14), could be frozen and thawed (Figure 15(a)), and were confirmed to be capable of long-term subculture without differences between arrangements (Figure 15(b)).

[0260] Example 4. Method for analysis of single cell transcriptomes (scRNA-seq) for characterization of 2D intestinal stem cell aggregates To characterize the 2D intestinal stem cell aggregates isolated and cultured from the 3D intestinal organoids, we performed single-cell transcriptomic sequence analysis and designed a new analytical method to analyze transcript expression patterns and compare them with previous study results reported in references (Figure 16).

[0261] Example 5. Validation of cellular characteristics and composition of 2D intestinal stem cell aggregates using single cell transcriptome analysis Based on the results of single-cell transcriptome analysis, the characteristics of the 2D intestinal stem cell aggregates were analyzed, and it was confirmed that the 2D intestinal stem cell aggregates mostly exhibited characteristics of intestinal epithelial cells, with very few cells exhibiting characteristics of intestinal stromal cells based on marker gene expression analysis (Figure 17). The 2D intestinal stem cell aggregates exhibiting such characteristics are most similar to human fetal intestinal epithelium, as compared with the results of single-cell transcriptome analysis of human intestinal epithelial tissues at different developmental stages reported in the reference literature (Figure 18).

[0262] Furthermore, it was confirmed that the cells that make up the 2D intestinal stem cell aggregates are primarily composed of stem cells and progenitor cells, even among cells that exhibit the characteristics of intestinal epithelial cells, and that more than 90% of the total cells are stem cells and progenitor cells (Figure 19). When the analysis results of the 2D intestinal stem cell aggregates were compared with the analysis results of single-cell transcriptomes of fetal intestinal epithelial tissue in the reference literature, it was confirmed that most of the cells were distributed in groups where intestinal stem cells and progenitor cells are distributed (Figure 20).

[0263] In addition, we confirmed the expression distribution of marker genes (LDHB, EIF3E, SOX9, SHH) known to be markers for intestinal stem cells and progenitor cells, and confirmed that all marker genes were expressed in most cells, verifying that most of the 2D intestinal stem cell aggregates exhibit characteristics of intestinal stem cells or progenitor cells (Figure 21).

[0264] Example 6. Validation of the cellular composition of 2D intestinal stem cell aggregates using immunofluorescence staining To confirm the characteristics of the 2D intestinal stem cell aggregates, we used immunofluorescence staining to confirm the cell composition. As a result, we were able to confirm that most of the cells expressed marker proteins for intestinal stem cells and progenitor cells (LDHB, EIF3E, SOX9), and that some of the cells were actively dividing (KI67+ cells) (Figure 22(a)). On the other hand, we were able to confirm that some of the cells expressed the marker protein for absorptive cells (FABP1) among differentiated cells, but no marker proteins for secretory cells (MUC2, CHGA) were observed at all (Figure 22(a)-(b)).

[0265] These results confirmed that the 2D intestinal stem cell aggregates were composed mostly of numerous stem and progenitor cells, with a very small proportion of differentiated cells present.

[0266] Example 7. Differentiation of 2D intestinal stem cell aggregates into intestinal epithelial cells using air-liquid interface culture method In order to differentiate 2D intestinal stem cell aggregates into highly functional intestinal epithelial cells, we developed a new differentiation method using air-liquid interface culture (Figure 23). Here, we performed media factor screening to construct the optimal medium for optimizing the differentiation of 2D intestinal stem cell aggregates into intestinal epithelial cells, uncovered five essential constituent factors, and were able to find the minimum medium composition (Figure 24).

[0267] Specifically, we confirmed that activators of the prostaglandin signaling pathway, receptor tyrosine kinase ligands, p38 inhibitors, WNT / R-spondin activators and nicotinamide are essential components for differentiation into intestinal epithelial cells. In particular, treatment with R-spondin 1, EGF, PGE2, SB202190 and nicotinamide was confirmed as essential factors that showed excellent effects on differentiation into intestinal epithelial cells, and this was used as the minimal medium composition.

[0268] It was confirmed that differentiation into intestinal epithelial cells in the above-mentioned minimal medium occurred in the same way as differentiation in the culture medium of 2D intestinal stem cell aggregates. Specifically, it was confirmed that all 2D intestinal stem cell aggregates derived from various pan-potent cell lines were successfully differentiated into intestinal epithelial cells (Figure 25).

[0269] Example 8. Analysis of intestinal epithelial cell characteristics by marker gene expression analysis To analyze the characteristics of intestinal epithelial cells differentiated by the air-liquid interface culture method, we confirmed the expression patterns of marker genes that are specifically expressed in intestinal stem cells and epithelial cells. When examining gene expression in intestinal epithelial cells by qPCR, we confirmed that some stem cell marker genes were less expressed, while the expression levels of most differentiated cell marker genes were increased (Figure 26).

[0270] In addition, after differentiation into intestinal epithelial cells, the cells were collected on days 4, 8, and 12 to confirm the cross-sectional morphology and expression of marker proteins. As a result, it was confirmed that a structure similar to a crypt-villus developed in the cross-section of the intestinal epithelial cells over time, increasing the height of the intestinal epithelium (Figure 27(a)-(b)). Furthermore, as the intestinal epithelial cells gradually developed over time, the expression level of the marker protein of the differentiated cells gradually increased proportionally (Figure 27(a)). Thus, as the differentiation degree of the intestinal epithelial cells increased over time, it was confirmed by measuring the TEER value that the barrier function of the intestinal epithelial cells also continued to increase (Figure 27(c)). Therefore, it was confirmed that the intestinal stem cell aggregates were successfully differentiated into intestinal epithelial cells by the air-liquid interface culture method.

[0271] Example 9. Characterization of intestinal stem cell populations and intestinal epithelial cells by comparative analysis of transcriptomes of various intestinal epithelial cells When comparing and analyzing the transcriptome and expression patterns of all-potential stem cells and the 3D intestinal organoids derived from them, functional intestinal epithelial cells, and actual human intestinal tissue, it was confirmed that the 2D intestinal stem cell aggregates were separated into cells that showed different characteristics from other cells (Figure 28). However, when they were differentiated into intestinal epithelial cells using the air-liquid interface culture method, it was confirmed that the transcriptome expression pattern changed to be similar to other intestinal epithelial cells, and in particular, it was confirmed that they were grouped most closely with functional intestinal epithelial cells (Figure 28). Here, when checking the gene group with the largest difference in expression between the 2D intestinal stem cell aggregates and intestinal epithelial cells, it was confirmed that the 2D intestinal stem cell aggregates had high expression of genes related to cell division, and the intestinal epithelial cells had high expression of metabolism-related genes (Figure 29). From these results, it was confirmed that the 2D intestinal stem cell aggregates showed the characteristics of stem cells that actively divide, and that intestinal epithelial cells can act as differentiated cells with the function of metabolizing various nutrients and substances.

[0272] Example 10. Development of intestinal stem cell lines expressing fluorescent proteins using lentiviruses To confirm the various uses of 2D intestinal stem cell aggregates, we confirmed whether it was possible to create gene-edited cell lines by introducing external genes (Figure 30). First, to introduce external genes, 2D intestinal stem cell aggregates were infected with lentivirus containing a fluorescent protein and the fluorescent protein was injected into the 2D intestinal stem cell aggregates. After that, positive selection using antibiotics was performed to select only cells expressing the fluorescent protein, and single clones derived from single cells were also created using the selected cells (Figure 31). To verify the functionality of the created single clones, they were differentiated into 3D organoids in Matrigel domes or into intestinal epithelial cells using the air-liquid interface culture method, and it was confirmed that they differentiated in the same way as normal cells (Figure 32). From these results, it was possible to verify that it is possible to create gene-edited cell lines using 2D intestinal stem cell aggregates.

[0273] Example 11: Experiment to verify the regenerative ability of 2D intestinal stem cell aggregates using a mouse model of intestinal epithelial tissue injury To verify the feasibility of using 2D intestinal stem cell aggregates as a cell therapy, an experiment was conducted in which 2D intestinal stem cell aggregates were transplanted into a mouse model with damaged intestinal epithelial tissue using hot-EDTA (Figure 33). To transplant 2D intestinal stem cell aggregates, a mouse colonoscope was used to transplant intestinal stem cell aggregates into the affected area (Figure 34). It was confirmed that mice transplanted with 2D intestinal stem cell aggregates had a faster weight recovery rate and a higher survival rate than the control group transplanted with only Matrigel (Figures 35-36). By observing the affected area before and after transplantation using a colonoscope, it was confirmed that the affected area recovered faster and the immune reaction was lower when 2D intestinal stem cell aggregates were transplanted (Figure 37). This efficacy was due to the successful engraftment of the 2D intestinal stem cell aggregates into the affected area (Figure 38) and the promotion of regeneration of damaged intestinal tissue (Figure 39). Therefore, it was confirmed that 2D intestinal stem cell aggregates can effectively regenerate the affected area when transplanted into damaged intestinal epithelial tissue, and are a biomaterial that can be used as a cell therapy for intestinal epithelial tissue regeneration treatment.

[0274] Example 12. Modeling SARS-CoV-2 infection using a 2.5-dimensional intestinal epithelial cell model To verify the utility of 2.5D intestinal epithelial cells derived from 2D intestinal stem cell aggregates, we performed infectious disease modeling using the SARS-CoV-2 virus (Figure 40). To confirm whether there is a difference in susceptibility to SARS-CoV-2 virus infection depending on the maturity of intestinal epithelial cells, we isolated 2D intestinal stem cell aggregates from immature / mature 3D intestinal organoids and produced immature / mature intestinal epithelial cells using the air-liquid interface culture method. After performing the air-liquid interface culture method, we monitored the cell morphology for 10 days and found no difference between immature and mature intestinal epithelial cells (Figure 41), but we confirmed a difference in the expression of maturity-related marker genes (Figure 42).

[0275] In particular, among the receptors important for SARS-CoV-2 virus infection, we confirmed that the expression level of ACE2 was higher in mature intestinal epithelial cells (Figure 43), and thus confirmed that SARS-CoV-2 infection is more sensitive in mature intestinal epithelial cells (Figure 44).

[0276] Based on these results, we confirmed that 2.5D intestinal epithelial cells derived from 2D intestinal stem cell aggregates can be used as a cell model for modeling various diseases, including infectious diseases.

Claims

1. (a) dissociating the total potent stem cell-derived three-dimensional intestinal organoids into single cells or small cell clusters; (b) two-dimensionally culturing the single cells or small cell clusters in a culture medium containing a WNT / R-spondin activator, an activator of the prostaglandin signaling pathway, and a receptor tyrosine kinase ligand.

2. The method for culturing intestinal stem cell aggregates according to claim 1, wherein the WNT / R-spondin activator is any one or more selected from the group consisting of R-spondin 1, R-spondin 2, R-spondin 3, R-spondin 4, and R-spondin mimetics.

3. The method for culturing intestinal stem cell aggregates according to claim 1, wherein the activator of the prostaglandin signaling pathway is one or more selected from the group consisting of arachidonic acid (AA), prostaglandin E2 (PGE2), prostaglandin G2 (PGG2), prostaglandin F2 (PGF2), prostaglandin H2 (PGH2) and prostaglandin D2 (PGD2).

4. The method for culturing intestinal stem cell aggregates according to claim 1, wherein the receptor tyrosine kinase ligand is any one selected from the group consisting of epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF), and epidermal growth factor (KGF).

5. The method for culturing intestinal stem cell aggregates according to claim 1, wherein the culture medium in step (b) further comprises one or more selected from the group consisting of B27, N-acetyl-L-cysteine ​​(NAC), nicotinamide, gastrin, a TGF-beta inhibitor, a WNT signaling pathway activator, a BMP inhibitor, and a p38 inhibitor.

6. The method for culturing intestinal stem cell aggregates according to claim 1 , wherein the culture medium in step (b) further comprises an initial culture medium, a ROCK inhibitor, a Notch activator, or all of these.

7. The method for culturing intestinal stem cell aggregates described in claim 1, wherein the intestinal stem cell aggregates exhibit enhanced expression levels of any one or more markers selected from the group consisting of LGR5, CD44, SOX9, LRIG1, LYZ, AXIN2, CTNNB and MKI67.

8. The method for culturing intestinal stem cell aggregates according to claim 1, wherein the intestinal stem cell aggregates express one or more markers selected from the group consisting of LDHB, EIF3E, SOX9 and SHH.

9. The method for culturing an intestinal stem cell aggregate according to claim 1, wherein the intestinal stem cell aggregate comprises 80% or more of cells including S phase cells, LGR5+ stem cells and early intestinal cells (Enterocytes) relative to the total cells of the aggregate.

10. 3D intestinal organoids derived from omnipotent stem cells (a) culturing the allopotent stem cells in a medium containing one or more selected from the group consisting of Nodal, Activin A, Activin B, BMP4, CHIR99021, WNT3A, and bFGF, and differentiating the allopotent stem cells into definitive endoderm; (b) culturing definitive endoderm in a medium containing one or more GSK3 inhibitors selected from the group consisting of BIO (6-bromoindyl'-3'-oxime), SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), GSK-3β inhibitor VII (α,4-dibromoacetophenone), L803-mts (Myr-N-GKEAPPAPPQSpP-NH2), and CHIR99021; and fibroblast growth factor (FGF), thereby differentiating the definitive endoderm into three-dimensional hindgut spheroids; (c) culturing three-dimensional hindgut spheroids in a medium containing a BMP inhibitor; a WNT / R-spondin activator; a receptor tyrosine kinase ligand; and one or more factors selected from the group consisting of IL-2, IL-22, IL-6, IL-1β, IL-11, EGF, OSM, NRG-1, IL-10, and colivelin, to produce three-dimensional intestinal organoids.

11. An intestinal stem cell aggregate produced by the culture method according to any one of claims 1 to 10.

12. (a) dissociating the total potent stem cell-derived three-dimensional intestinal organoids into single cells or small cell clusters; (b) two-dimensionally culturing the single cells or small cell clusters in a culture medium containing a WNT / R-spondin activator, an activator of the prostaglandin signaling pathway, and a receptor tyrosine kinase ligand to produce intestinal stem cell aggregates; (c) culturing the intestinal stem cell aggregates by air-liquid interface culture in a differentiation medium containing an activator of the prostaglandin signaling pathway, a receptor tyrosine kinase ligand, a p38 inhibitor, a WNT / R-spondin activator, and nicotinamide.

13. The method for producing intestinal epithelial cells according to claim 12, wherein the air-liquid interface culture method involves culturing intestinal stem cells under a transwell coated with an extracellular matrix.

14. The method for producing intestinal epithelial cells according to claim 12, wherein the WNT / R-spondin activator is any one or more selected from the group consisting of R-spondin 1, R-spondin 2, R-spondin 3, R-spondin 4, and R-spondin mimetics.

15. The method for producing intestinal epithelial cells according to claim 12, wherein the activator of the prostaglandin signaling pathway is one or more selected from the group consisting of arachidonic acid (AA), prostaglandin E2 (PGE2), prostaglandin G2 (PGG2), prostaglandin F2 (PGF2), prostaglandin H2 (PGH2) and prostaglandin D2 (PGD2).

16. 13. The method for producing intestinal epithelial cells according to claim 12, wherein the receptor tyrosine kinase ligand is any one selected from the group consisting of epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF), and epidermal growth factor (KGF).

17. 13. The method for producing intestinal epithelial cells according to claim 12, wherein the p38 inhibitor is any one selected from the group consisting of SB202190, SB203580, SB239063, SB706504, BIR796, JX401, EO1428, RWJ67657, SCIO469, VX745, TAK715, ML3403, DBM1285 and PH797804.

18. The method for producing intestinal epithelial cells according to claim 12, wherein the intestinal epithelial cells exhibit enhanced expression levels of one or more markers selected from the group consisting of VIL1, ECAD, FABP1, KRT20, LCT, LYZ, and MUC2.

19. The method for producing intestinal epithelial cells according to claim 12, wherein the intestinal epithelial cells exhibit enhanced expression levels of one or more markers selected from the group consisting of AKR1B15, DHRS11, GALNT4, GALNT5, DHRS3, RDH10, AADAC, NR1I2, SULTE1, DOUX2, FABP1, SLC6A20, SLC43A1, and CLDN3.

20. The method for producing intestinal epithelial cells according to claim 12 , wherein the intestinal epithelial cells include small intestinal cells, mucus-secreting cells, hormone-secreting cells, and Paneth cells.

21. An intestinal epithelial cell produced by the production method according to any one of claims 12 to 20.

22. An intestinal epithelial cell model comprising the intestinal epithelial cell of claim 21.

23. The intestinal epithelial cell model according to claim 22, wherein the intestinal epithelial cell model has a crypt-villus structure.

24. (a) infecting the intestinal epithelial cell model of claim 22 with bacteria or viruses; (b) treating the infected intestinal epithelial cell model with a drug; (c) confirming a response to drug treatment.

25. (a) treating the intestinal epithelial cell model according to claim 22 with a drug; (b) a step of evaluating the absorption or bioavailability of the drug in the intestinal epithelial cell model of step (a).

26. A method for providing an intestinal epithelial cell model for disease modeling, comprising the step of infecting the intestinal epithelial cell model of claim 22 with bacteria or viruses.

27. A tissue therapeutic agent comprising the intestinal stem cell aggregate of claim 11.

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