Two-dimensional culture method with explicit chemical composition for culturing population of three-dimensional gut organoids-derived gut stem cells

Abstract

The invention relates to a method for two-dimensional culture of an intestinal stem cell population in a culture medium with definite chemical components and application of the intestinal stem cell population, and also relates to a method for differentiating the intestinal stem cell population into 2.5-dimensional intestinal epithelial cells and application of the 2.5-dimensional intestinal epithelial cells.

Description

Technical Field

[0001] The present invention relates to a method for two-dimensionally culturing an intestinal stem cell population in a culture medium with a defined chemical composition and its use, as well as a method for differentiating an intestinal stem cell population into 2.5-dimensional intestinal epithelial cells and its use. Background Art

[0002] Intestinal organoids are cell model systems that can simulate human intestinal development and diseases and are considered to be excellent cell models that can be used in various fields, such as the study of physiological / pathological functions of the intestine, the development of therapeutic agents for the treatment of intestinal diseases, and the evaluation of the efficacy and toxicity of new drug candidates.

[0003] In addition, intestinal organoids are attracting attention as therapeutic agents for regenerating lesions by transplanting them into the intestine that is damaged or dysplastic due to intestinal diseases. Intestinal organoids are composed of various cells including intestinal stem cells, which are key components of regeneration, and have been shown to effectively regenerate damaged tissues by transplanting intestinal organoids into lesions, making the possibility of development as therapeutic agents increasing.

[0004] Recently, it has been repeatedly demonstrated by several groups that human intestinal organoids transplanted into damaged intestinal tissue models are able to regenerate damaged intestinal tissue, and transplanted intestinal organoids have been shown to be able to regenerate the structure and function of actual intestinal tissue at a high level.

[0005] However, in order to use intestinal organoids as cell model systems for various purposes or as cell therapeutics for regenerating damaged intestinal tissues, homogeneous intestinal organoids whose shapes and functions can be adjusted within a certain range must be cultured in large quantities, and a culture system that facilitates long-term culture and cryopreservation is required.

[0006] Currently, intestinal organoids are cultured by three-dimensional culture using Matrigel, which is produced by isolating the extracellular matrix produced by sarcomas transplanted into animals. However, this culture has large differences in shape and function between batches of intestinal organoids, resulting in low homogeneity, difficulty in large-scale culture, inability to culture for a long time and cryopreservation, and high culture costs.

[0007] Therefore, it is necessary to develop a novel method for culturing intestinal organoids that can culture and cryopreserve homogeneous intestinal organoids for a long time and provide a stable supply of cells through large-scale culture, so as to develop intestinal organoids as cell model systems or cell therapeutic agents with less batch-to-batch variability.

[0008] In the above background, the present inventors have developed a method for culturing cells, which can be easily and quickly cultured by isolating only intestinal stem cells from three-dimensional intestinal organoids derived from human pluripotent stem cells and then culturing the intestinal stem cells in a chemically defined culture medium, and can be large-scale cultured, cryopreserved, and thawed through long-term stable subculture. Therefore, it was confirmed that homogeneous intestinal stem cells and various uses can be provided, thereby completing the present disclosure. Summary of the invention

[0009] Technical issues

[0010] An object of the present invention is to provide a method for culturing an intestinal stem cell population by two-dimensionally culturing single cells or small cell aggregates isolated from three-dimensional intestinal organoids derived from pluripotent stem cells in a chemically defined culture medium.

[0011] Another object of the present invention is to provide a method for producing intestinal epithelial cells, which method cultured intestinal stem cell populations in a chemically defined differentiation medium through air-liquid interface culture.

[0012] Another object of the present invention is to provide a use of the above-mentioned intestinal stem cell population and / or intestinal epithelial cells.

[0013] Technical Solution

[0014] Hereinafter, descriptions of repeated contents will be omitted to prevent any potential confusion caused by repetition. In other words, the contents of the present invention are not limited to the following contents and should be interpreted based on the overall contents of the present invention.

[0015] The terms used herein are only used for the purpose of describing specific embodiments and are not intended to limit the present disclosure. Singular expressions also include plural meanings as long as they do not have different meanings in the context. In addition, the terms "including", "having" and the like used herein are used to indicate the presence of features, numbers, steps, elements or combinations thereof, and they should not exclude the possibility of combining or adding one or more features, numbers, operations, elements or combinations thereof.

[0016] It should be understood that when an amount, concentration or other value or parameter used herein is given as a range, a preferred range or a list of preferred upper and lower limits, all ranges formed by any pair of any upper limits or preferred values ​​and any pair of any lower limits or preferred values ​​are specifically disclosed, regardless of whether the range is disclosed separately.

[0017] When numerical ranges are described herein, it is intended that the endpoints of the range and the scope of the disclosure within that range are not limited to the specific values ​​described when defining the range, unless otherwise indicated.

[0018] Pluripotent stem cells

[0019] As used herein, the term "stem cell" refers to a cell that has the ability to continue to proliferate into an undifferentiated cell, ie, the ability to self-replicate, and has the ability to differentiate into various specific cells constituting a tissue or an organ from all three germ layers.

[0020] As used herein, the term "pluripotent stem cell (PSC)" is generally referred to as a PS cell, and includes any cell that can differentiate into almost any cell, i.e., cells derived from any of the three germ layers (germ epithelium) including endoderm (stomach lining, gastrointestinal tract, lung), mesoderm (muscle, bone, blood, urogenital), and ectoderm (epithelial tissue and nervous system). PSCs may be derived from embryonic stem cells (including embryonic germ cells), or may be descendants of pluripotent cells obtained by forcibly expressing specific genes in non-pluripotent cells (such as adult somatic cells or fully differentiated cells).

[0021] As used herein, the term "induced pluripotent stem cells (iPSCs)" are often abbreviated as iPS cells, and refer to a type of pluripotent stem cells that are normally and artificially induced from non-pluripotent cells (eg, adult somatic cells) by inducing "forced" expression of specific genes.

[0022] As used herein, the term "embryonic stem cells (ESC)" is often abbreviated as ES cells and refers to cells derived from the inner cell mass of a blastocyst, which is a pluripotent early embryo. For the purposes of this disclosure, the term "ESC" is sometimes used broadly to include embryonic germ cells.

[0023] Basic culture medium

[0024] The culture medium used in the methods of the present disclosure is any basal medium suitable for animal or human cells, and the limitations provided herein apply to these basal media.

[0025] The basal medium used for animal or human cell culture usually contains many components necessary for the maintenance of supporting cultured cells. Taking into account the following, the technician can easily prepare the combination of suitable components. The basal medium used in the present disclosure usually includes a nutrient solution containing standard cell culture components as described in more detail above and in the literature, such as amino acids, vitamins, lipid supplements, inorganic salts, carbon energy sources and buffers. In some embodiments, the culture medium is further supplemented with one or more standard cell culture components selected from, for example, amino acids, vitamins, liquid supplements, inorganic salts, carbon energy sources and buffers.

[0026] Those skilled in the art will understand from common knowledge the types of culture media that can be used as the basal culture media in the differentiation medium of the present disclosure. Potentially suitable cell culture media include commercially available, but not limited to: Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), KnockOut-DMEM (KO-DMEM), Glasgow Minimum Essential Medium (G-MEM), Eagle's Basal Medium (BME), DMEM / Ham's F12 (Ham's F12), Advanced DMEM / Ham's F12, Iscove's Modified Dulbecco's Medium, Minimum Essential Medium (MEM), Ham's F-10, Ham's F-12, Medium 199, RPMI 1640 medium and serum-free replacement xeno-free medium (KnockOut Serum replacement XenoFree medium).

[0027] More specifically, the cell culture medium may be selected from the group consisting of RPMI 1640, Dulbecco's Modified Eagle's Medium (DMEM), Ham's F12, DMEM / F12, DMEM / F12, and Advanced DMEM / F12.

[0028] For example, the basal medium can be DMEM / F12, Advanced DMEM / F12, and / or RPMI 1640. Advanced DMEM / F12 or Advanced RPMI optimized for serum-free culture and already containing insulin is used as needed.

[0029] Methods for culturing intestinal stem cell populations

[0030] The present disclosure provides a method for culturing an intestinal stem cell population by two-dimensionally culturing single cells or small cell aggregates isolated from three-dimensional intestinal organoids derived from pluripotent stem cells in a chemically defined culture medium.

[0031] Specifically, a method for culturing an intestinal stem cell population is provided, the method comprising: (a) separating pluripotent stem cell-derived three-dimensional intestinal organoids into single cells or small cell clumps; and

[0032] (b) culturing the single cell or the small cell aggregate in two dimensions in a culture medium comprising a WNT / R-floorplate response protein activator, an activator of a prostaglandin signaling pathway, and a receptor tyrosine kinase ligand.

[0033] As used herein, the term "organoid" refers to a three-dimensional population of one or more cell types that mimics the physical appearance, actual structure, or function of a tissue or organ.

[0034] The three-dimensional intestinal organoids of the present invention are derived from pluripotent stem cells, wherein the three-dimensional intestinal organoids can be produced by the following steps:

[0035] (a) culturing pluripotent stem cells in a medium comprising any one or more selected from the group consisting of Nodal, activin A, activin B, BMP4, CHIR99021, Wnt3a and bFGF to differentiate the stem cells into definitive endoderm;

[0036] (b) in a mixture comprising a mixture 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-GKEAPPQSpP-NH 2 ) and CHIR99021; and culturing the definitive endoderm in a medium containing fibroblast growth factor (FGF) to differentiate the definitive endoderm into three-dimensional hindgut spheroids; and

[0037] (c) culturing the three-dimensional hindgut spheroids in a culture medium comprising a BMP inhibitor; a WNT / R-floor plate response protein activator; a receptor tyrosine kinase ligand; and any 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 colivin to produce three-dimensional intestinal organoids.

[0038] The differentiation of pluripotent stem cells into intestinal organoids undergoes: 1) the differentiation process of pluripotent stem cells into definitive endoderm (DE) cells, 2) the differentiation process of definitive endoderm (DE) cells into hindgut (HG), and 3) the differentiation process of hindgut (HG) into (mature) intestinal organoids.

[0039] 1) Steps for differentiating pluripotent stem cells into definitive endoderm (DE) cells

[0040] During differentiation of pluripotent stem cells into definitive endoderm (DE) cells, one or more growth factors are used.

[0041] In particular, growth factors that may affect activation of activin A / nodal signaling or inhibition of phosphatidylinositol 3-kinase (PI3K) signaling may be used.

[0042] For example, the one or more growth factors used in the differentiation process may include growth factors in the TGF-β superfamily. The one or more growth factors may include Nodal / Activin and / or BMP subgroups of growth factors in the TGF-β superfamily.

[0043] In some aspects, the one or more growth factors are selected from Nodal, activin A, activin B, BMP4, CHIR99021, Wnt3a, bFGF, or any combination of these growth factors.

[0044] In some aspects, the pluripotent 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, be 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 one or more growth factor processing embryonic stem cell or iPSC with concentration.In some aspects, in processing procedure, the concentration of growth factor is maintained at constant level.Can change the concentration of growth factor in processing procedure.

[0045] That is, the method may include treating pluripotent stem cells with any one or more selected from the group consisting of Nodal, activin A, activin B, BMP4, CHIR99021, bFGF and Wnt3a to differentiate pluripotent stem cells into definitive endoderm cells. More specifically, the method may include 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 to differentiate embryonic stem cells or induced pluripotent stem cells into definitive endoderm cells. More specifically, the method may include treating embryonic stem cells or induced pluripotent stem cells with activin A to differentiate embryonic stem cells or induced pluripotent stem cells into definitive endoderm cells.

[0046] Although the present disclosure is not limited thereto, the factor may be used in an amount of, for example, 1 ng / ml to 1,000 ng / ml, preferably 10 ng / ml to 500 ng / ml.

[0047] According to one embodiment of the present disclosure, activin A can be used to differentiate pluripotent stem cells into definitive endoderm (DE) cells. In differentiation, for example, 10 ng / ml to 500 ng / ml of activin A can be used, but the present disclosure is not limited thereto. 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, the factor can be cultured for 6 hours to 120 hours or 12 hours to 100 hours, but the present disclosure is not limited thereto.

[0048] According to one embodiment of the present disclosure, the growth factor is suspended in a culture medium containing fetal bovine serum (FBS) with different concentrations as required. For example, the culture medium can contain any fetal bovine serum (FBS or FCS) suitable for growth, such as 0.1%, 0.2%, 0.3%, 0.5%, 1.0%, 2.0%, 3.0%, 4.0% or 5.0%.

[0049] If desired, the cells can be cultured by contacting an extracellular matrix. The extracellular matrix includes all substances described herein and can be applied to the differentiation method of the present invention.

[0050] 2) Steps for differentiating definitive endoderm (DE) cells into three-dimensional HG spheroids

[0051] The definitive endoderm (DE) that has undergone the process of producing definitive endoderm induced by the above factors, preferably induced by activin A, more preferably induced by activin A, can undergo a step of differentiating into hindgut (HG) by FGF / Wnt signals.

[0052] FGF and Wnt ligands can direct the differentiation of DE developed from iPSCs or ESCs into the hindgut.

[0053] Differentiation into hindgut or mid-hindgut can be carried out by three-dimensional culture, spheroid culture, etc. as needed. For example, differentiation into hindgut or mid-hindgut can be carried out on a micropatterned matrix / plate (e.g., Aggrewell, EZSPHERE), and formed into aggregates (e.g., spheroids) or can be carried out by three-dimensional culture (e.g., suspension) in a bioreactor. For serum-free differentiation, B-27 can be added instead of fetal bovine serum (FBS or FCS). In addition, the culture medium can also include 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, but is not limited thereto.

[0054] Altered expression of any Wnt signaling protein in combination with any FGF ligand can result in the directed differentiation described herein.

[0055] Regulators / activators of the 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 pathway can be regulated by using small molecule regulators or protein regulators that activate the above pathways or proteins that activate the pathway. Examples of small molecule regulators of the Wnt pathway include, but are not limited to, lithium chloride; 2-amino-4,6-disubstituted pyrimidine (hetero) aryl pyrimidine; IQ1; QS11; NSC668036; DCA-β catenin; and 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 proteins, 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).

[0056] In addition, exogenous molecules include GSK3 inhibitory molecules or proteins that 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.

[0057] Specifically, GSK3 inhibitors include Chiron / CHIR99021 that inhibits GSK3. The amount of GSK3 inhibitor treatment can be 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.

[0058] Fibroblast growth factor (FGF) is a group of growth factors associated with angiogenesis, wound healing, and embryonic development. In some aspects, one of ordinary skill in the art will appreciate that any FGF can be used with proteins derived from the Wnt signaling pathway.

[0059] In some embodiments, the FGF signaling pathway is activated by contacting the cell with at least one molecule 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.

[0060] That is, in the presence of: selected from 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-GKEAPPQSpP-NH 2 ) and CHIR99021; and culturing the definitive endoderm in a medium containing fibroblast growth factor (FGF) to differentiate the definitive endoderm into three-dimensional hindgut spheroids.

[0061] 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; 1000 hours or more; 1,200 hours or more; or 500 hours or more.

[0062] 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.

[0063] In some aspects, the concentration of the signaling molecule remains constant during the treatment. In another aspect, the concentration of the molecule in the signaling pathway changes during the treatment.

[0064] In some aspects, the signaling molecules according to the present invention are suspended in DMEM medium.

[0065] In some aspects, the signaling molecules according to the invention are suspended in a culture medium comprising fetal bovine serum (FBS or FCS).

[0066] Any known molecules of the signaling pathways described herein may be used alone or in combination, including any molecules of the Wnt signaling pathway and the FGF signaling pathway.

[0067] That is, the method comprises the step of treating FGF and Wnt ligand to differentiate definitive endoderm cells into hindgut. More specifically, the method comprises the step of treating FGF and CHIR99021 to differentiate definitive endoderm cells into hindgut.

[0068] According to one embodiment of the present disclosure, differentiation of definitive endoderm (DE) to hindgut can be performed by GSK3 inhibitor and fibroblast growth factor. More specifically, by treating CHIR99021 and FGF4, differentiation can be performed to hindgut through three-dimensional culture.

[0069] In differentiation, for example, 100 ng / ml, 120 ng / ml, 150 ng / ml, 200 ng / ml, 500 ng / ml, 1,000 ng / ml or 1,200 ng / ml of FGF4 can be used, but the present disclosure is not limited thereto. Specifically, the concentration of FGF4 that can be used is 100 ng / ml to 1,200 ng / ml, more specifically, 120 ng / ml to 1,000 ng / ml.

[0070] In addition, the amount of CHIR99021 can be about 0.3uM to about 10uM. As needed, the factor is suspended in a culture medium comprising fetal bovine serum (FBS). For example, the culture medium can contain any fetal bovine serum (FBS or FCS) suitable for growth, such as 0.1%, 0.2%, 0.3%, 0.5%, 1.0%, 2.0%, 3.0%, 4.0% or 5.0%.

[0071] More specifically, the culture medium can be cultured for 24 hours or longer; 36 hours or longer; 48 hours or longer; 60 hours or longer; 72 hours or longer; 84 hours or longer; 96 hours or longer; 120 hours or longer; 150 hours or longer; 180 hours or longer, but the disclosure is not limited thereto. Preferably, the culture medium can be cultured for 24 hours to 360 hours, more preferably 36 hours to 240 hours.

[0072] If necessary, a BMP activator can be added and cultured to control BMP signaling. The BMP activator can be BMP2, one of BMP4, or both BMP2 and BMP4, a small molecule that activates the BMP pathway, and / or a protein that activates the BMP pathway.

[0073] If desired, the cells can be cultured by contacting an extracellular matrix. The extracellular matrix includes all substances described herein and can be applied to the differentiation method of the present invention.

[0074] 3) Steps for differentiating 3D hindgut (HG) spheroids into 3D intestinal organoids

[0075] The three-dimensional intestinal organoids of the present invention are mature intestinal organoids, which refer to intestinal organoids in which genes necessary for the digestive function, transport system, immune function and host defense of the adult small intestine are expressed. Specifically, the small intestine of an adult has unique characteristics, including enhanced expression of small intestinal stem cell marker genes and genes necessary for digestive function, transport system, immune function and host defense. In particular, the proper expression and activity of transport proteins involved in physiological and pharmacokinetic effects are prerequisites for normal small intestinal function, such as absorption, distribution and excretion of drugs.

[0076] In particular, the three-dimensional intestinal organoids according to the present disclosure have improved efficacy in transplantation. More specifically, this may be due to the high expression characteristics of vascular endothelial-related factors and various secretory factors that are characteristics of mesenchymal stromal cells in the mesenchymal stromal cell layer present around the intestinal epithelial cell layer constituting the organoid.

[0077] In addition, the organoids showed features of bud-like structure development present in intestinal organoids. In addition, the organoids showed increased goblet cell development, thereby demonstrating high efficiency in the differentiation capacity of intestinal stem cells.

[0078] BMP inhibitors, WNT / R-spondin activators, and receptor tyrosine kinase ligands are essential for the differentiation of 3D hindgut (HG) spheroids into 3D intestinal organoids.

[0079] In addition, one or more additional ingredients selected from the group consisting of ROCK inhibitor, B27, N-acetyl-L-cysteine ​​(NAC), N2, nicotinamide, gastrin, IGF-1, regulatory protein-1β, bFGF, A-83-01 and / or SB202190 may be included.

[0080] BMP inhibitors are agents that bind to BMP molecules to form a complex. Inhibitors can be agents that bind to BMP receptors and prevent BMP ligands from binding to receptors, such as antibodies that bind to receptors. BMP inhibitors can be proteins or small molecules, and can be natural, modified and / or partially or completely synthesized. 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. The culture medium can include about 10 ng / ml to about 100 ng / ml of Noggin. For example, the culture medium can include about 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, or 50 ng / ml or more of Noggin. Preferably, the culture medium can include about 10 ng / ml to 100 ng / ml of Noggin.

[0081] Preferably, the WNT / R-floor response protein activator can be R-floor response protein 1, R-floor response protein 2, R-floor response protein 3 or R-floor response protein 4. The culture medium can include a WNT / R-floor response protein activator 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 can include about 50ng / ml to about 800ng / ml of R-floor response protein 1.

[0082] The receptor tyrosine kinase ligand is, for example, a mitogenic growth factor selected from the group consisting of epidermal growth factor (EGF), transforming growth factor-α (TGF-α), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF).

[0083] Preferably, the receptor tyrosine kinase ligand is EGF. EGF is a potent mitogen for ectoderm and mesoderm cells of various cultures, and has sufficient effects on the differentiation of some fibroblasts in vivo and in vitro certain cells and cell cultures. Preferred concentrations are at least 10 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 40 ng / ml, 45 ng / ml or 50 ng / ml and 100 ng / ml, 200 ng / ml, 300 ng / ml, 500 ng / ml, 600 ng / ml and 700 ng / ml or more. More preferably, the concentration is greater than or equal to 50 ng / ml and less than or equal to 300 ng / ml.

[0084] In addition to the necessary components of BMP inhibitors, WNT / R-floor planar response protein activators, and receptor tyrosine kinase ligands as described above, additional factors can be construed to further include the addition of any known factors to intestinal organoids.

[0085] Preferably, depending on the desired differentiation form, one or more additional ingredients selected from the group consisting of ROCK inhibitor, B27, N-acetyl-L-cysteine ​​(NAC), N2, nicotinamide, gastrin, IGF-1, regulatory protein-1β, bFGF, A-83-01 and SB202190 may also be included.

[0086] 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-isoquinolyl)sulfonyl]-hexahydro-1H-1,4-diazepine Dihydrochloride (H-1152).

[0087] N-acetyl-L-cysteine ​​(NAC), N2, nicotinamide, gastrin, IGF-1, modulin-1β, bFGF, A-83-01 and / or SB202190 can be added as needed, for example, to improve the culture efficiency and lifespan of organoids, regulate cell proliferation, contribute to DNA stability, etc.

[0088] Preferably, B27 may also be included. More specifically, the B27 supplement is referred to as a "B27 supplement without vitamin A" (also referred to herein as "B27 without vitamin A" or "B27 wo VitA"; and can be found at Invitrogen (located in Carlsbad, California, USA); www.invitrogens.com; current catalog number 12587010; and PAA Laboratories GmbH (located in Pasching, Austria); www.paa.com; catalog number F01-002; and in the literature [Brewer et al. (1993) J Neurosci Res. 35(5):567-76]).

[0089] In some embodiments, the B27 supplement can be replaced with a generic formulation that includes one or more ingredients selected from the following list: biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-α-tocopherol (vitamin E), albumin, insulin, and transferrin.

[0090] If desired, cells can be cultured by contacting an extracellular matrix after differentiation of 3D hindgut (HG) spheroids into 3D intestinal organoids.

[0091] 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 disclosure can diffuse into the three-dimensional extracellular matrix.

[0092] The extracellular matrices disclosed in Polymer Hydrogel-Guided Organotypic and Organoid Culture (Adv Funct Mater, 2020, Valentina Magno et al.) and Engineered Extracellular Matrices for Organoid Culture (Int J Stem Cells. 2022 Feb 28; 15(1): 60-69) are incorporated herein by reference.

[0093] The extracellular matrix includes fibrin, laminin, collagen and / or alginate, but is not limited thereto. The example of the cell producing the extracellular matrix includes chondrocytes, which mainly produce collagen and proteoglycans; fibroblasts, which mainly produce type IV collagen, laminin, interstitial procollagen and fibronectin; and colon myofibroblasts, which mainly produce collagen (type I, type III and type V), chondroitin sulfate proteoglycans, hyaluronic acid, fibronectin and tenascin-C. These are "naturally produced extracellular matrices". Naturally produced extracellular matrix can be provided commercially. Examples of commercially available extracellular matrices include extracellular matrix proteins from Engelbreth-Holm-Swarm (EHS) murine sarcoma cells (Invitrogen) and basement membrane preparations (e.g., Cultrex (registered trademark) basement membrane extract (Trevigen, Inc.), type I collagen (Invitrogen), Vitrogel (registered trademark) (The Well Bioscience Inc.), Geltrex (ThermoFisher), or Matrigel (trademark) (BD Biosciences)).

[0094] In addition to the above-mentioned factors for differentiation into organoids, differentiation of three-dimensional hindgut (HG) spheroids into three-dimensional intestinal organoids mainly includes treatment with cytokines used in co-culture that simulates the in vivo intestinal environment or treatment with cytokines secreted in T lymphocyte co-culture (i.e., treatment with cytokines secreted by T lymphocytes), and / or treatment with STAT3 and mTOR signaling pathway activators.

[0095] More specifically, the cytokines secreted by 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), more specifically, IL-2.

[0096] In addition to the above-mentioned factors for differentiation into organoids, STAT3 and mTOR signaling pathway activators can be treated to perform maturation of intestinal organoids. For example, maturation of intestinal organoids can be performed by treatment with colivelin.

[0097] In addition, maturation of intestinal organoids can be enhanced by treatment with NRG-1 (Neuregulin-1).

[0098] That is, three-dimensional intestinal organoids can be produced by the following steps: culturing three-dimensional hindgut spheroids in a culture medium containing a BMP inhibitor; a WNT / R-floor plate response protein activator; a receptor tyrosine kinase ligand; and any 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.

[0099] That is, at least one cytokine selected from the group consisting of IL-2, IL-22, IL-6, IL-1β, IL-11, EGF, OSM and IL-10, and / or treatment of culture medium components with colivelin for intestinal maturation directly in the hindgut greatly enhances the maturation of organoids.

[0100] In other words, any 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 can be included as a medium component. The preferred concentration is at least 0.1 ng / ml, 0.2 ng / ml, 0.3 ng / ml, 0.4 ng / ml, 0.5 ng / ml, 0.6 ng / ml, 0.7 ng / ml, 0.8 ng / ml, 0.9 ng / ml, 1.0 ng / ml, 2.0 ng / ml, 3.0 ng / ml. More preferably, the concentration can be 0.3 ng / ml-2.0 ng / ml.

[0101] Steps for isolating pluripotent stem cell-derived three-dimensional intestinal organoids into single cells or small cell aggregates

[0102] In the present disclosure, in the step of separating pluripotent stem cell-derived three-dimensional intestinal organoids into single cells or small cell aggregates, the intestinal organoids can be separated by treating EDTA, trypsin, or both EDTA and trypsin.

[0103] More specifically, it is preferred to perform treatment with EDTA, trypsin, or both EDTA and trypsin in a state where the extracellular matrix remaining in the three-dimensional intestinal organoid is removed as much as possible to purify the isolated single cells or small cell aggregates.

[0104] During this process, the three-dimensional intestinal organoids can be carefully separated by pipetting as needed, and more preferably, the separation can be close to single cells.

[0105] The isolated single cells or small cell aggregates can be briefly cultured or washed in a basal medium as needed, and can be cultured in the intestinal stem cell culture medium according to the present invention.

[0106] In a study involving WNT / R-floorplate responsive protein activators, activators of prostaglandin signaling pathways, and receptor tyrosine Steps for two-dimensional culture in culture medium containing acid kinase ligands

[0107] Provided is a culture medium for producing an intestinal stem cell population used in this article, which is used to culture the intestinal stem cell population in two dimensions, and the culture medium mainly contains a WNT / R-floor plate response protein activator, an activator of a prostaglandin signaling pathway, and a receptor tyrosine kinase ligand.

[0108] The WNT / R-floor response protein activator is used to regulate the stem cell characteristics and proliferation of two-dimensional intestinal stem cells by activating the WNT signal transduction system, and can be any one or more selected from the group consisting of R-floor response protein 1, R-floor response protein 2, R-floor response protein 3, R-floor response protein 4 and R-floor response protein mimics. Preferably, the WNT / R-floor response protein activator can be R-floor response protein 1.

[0109] The culture medium may include a WNT / R-floorplate activator at a concentration of 50 ng / ml, 100 ng / ml, 200 ng / ml, 300 ng / ml, 500 ng / ml, 600 ng / ml, 700 ng / ml, 800 ng / ml, 900 ng / ml, 1 μg / ml, 1.5 μg / ml or 2 μg / ml or more. Preferably, the culture medium may include R-floorplate activator at a concentration of about 50 ng / ml to about 800 ng / ml.

[0110] The activator of the prostaglandin signaling pathway is used to regulate the proliferation of the intestinal stem cell population by activation of the prostaglandin signaling system, and can 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, the activator of the prostaglandin signaling pathway can be prostaglandin E2 (PGE2).

[0111] As an activator of the prostaglandin signaling pathway, it is preferred that prostaglandin E2 is treated 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.

[0112] The receptor tyrosine kinase ligand is used to prevent apoptosis and regulate proliferation of intestinal stem cell populations, and can be any one selected from the group consisting of epidermal growth factor (EGF), transforming growth factor-α (TGF-α), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF). Preferably, the receptor tyrosine kinase ligand can be epidermal growth factor (EGF).

[0113] Receptor tyrosine kinase ligands can be treated at concentrations such as 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.

[0114] The combination of the WNT / R-floorplate activator, the prostaglandin signaling pathway activator and the receptor tyrosine kinase ligand is preferably R-floorplate 1, prostaglandin E2 (PGE2) and epidermal growth factor (EGF).

[0115] The combination of WNT / R-floor plate responsive protein activators, activators of the prostaglandin signaling pathway, and receptor tyrosine kinase ligands is essential for the maintenance of stem cell properties, cell growth and proliferation, and has the efficacy of stem cell enrichment and long-term culture.

[0116] Although the present disclosure is not limited in this regard, WNT / R-floor plate response protein activators can show improvements in stem cell characteristics and / or cell proliferation, activators of the prostaglandin signaling pathway can show improvements in stem cell characteristics and / or cell proliferation, and receptor tyrosine kinase ligands can show improvements in cell survival and / or cell proliferation.

[0117] In addition to the above-mentioned essential components, the culture medium of the present invention may also include any one or more selected from the group consisting of B27, N-acetyl-L-cysteine ​​(NAC), nicotinamide, gastrin, TGF-β inhibitors, Wnt signaling pathway activators, BMP inhibitors and p38 inhibitors for stable long-term subculture.

[0118] B27, N-acetyl-L-cysteine ​​(NAC) and nicotinamide can be added as components of the basal medium, and in particular, B27 can be replaced by a universal preparation comprising one or more of the ingredients selected from the following list: biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-α-tocopherol (vitamin E), albumin, insulin and transferrin.

[0119] The TGF-β 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, preferably A-83-01.

[0120] The Wnt signaling pathway activator can 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, preferably Wnt3a.

[0121] The BMP inhibitor may be Noggin, Dorsomorphin, DMH1 or LDN-193189, preferably Noggin.

[0122] 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 preferably SB202190.

[0123] The concentration of each of these additional components can be appropriately adjusted within the general range commonly used in the culture medium.

[0124] Furthermore, to prevent cell loss during subculture, the culture medium may further include a ROCK inhibitor, a Notch activator, or both a ROCK inhibitor and a Notch activator in the initial stage of culture, for example, immediately after the start of culture, on day 1, day 2, day 3, day 4, or day 5.

[0125] More specifically, in the initial stage of two-dimensional culture of single cells or small cell clusters in a culture medium including a WNT / R-floor plate response protein activator, an activator of the prostaglandin signaling pathway, and a receptor tyrosine kinase ligand, the culture medium may also include a ROCK inhibitor, a Notch activator, or all ROCK inhibitors and Notch activators. This may be 1 day, 2 days, 3 days, 4 days, or 5 days after the start of culture.

[0126] Rho-associated protein kinase (ROCK) inhibitors are used to inhibit the activity of serine / threonine kinases that serve as target proteins of Rho (Rho A, Rho B, and Rho C), and may 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-isoquinolyl)sulfonyl]-hexahydro-1H-1,4-diazepan dihydrochloride (H-1 152). More preferably, the ROCK inhibitor may be R-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632).

[0127] Y-27632 can be treated in an amount of, for example, about 0.1 μM to about 100 μM or about 0.1 μM to about 50 μM.

[0128] The Notch activator refers to a protein or a small molecule compound that activates the Notch pathway function, and preferably may be Jagged-1 (JAG1).

[0129] Jagged-1 (JAG1) can be treated 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.

[0130] The intestinal stem cell population according to the present disclosure can be cultured on feeder cells or plates coated with an extracellular matrix as required. The extracellular matrix is ​​the same as described above.

[0131] The intestinal stem cell population according to the present disclosure can be cultured for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days, as well as 2 weeks or 3 weeks or longer, but the present disclosure is not limited thereto, and subculture can be performed as needed.

[0132] The term "intestinal stem cells" herein refers to differentiated cells derived from intestinal epithelial tissue that have the ability to self-replicate and differentiate into various specific cells present in the intestinal epithelium. Intestinal stem cells can differentiate into, for example, intestinal stem cells, intestinal progenitor cells, intestinal epithelial cells, intestinal goblet cells, enteroendocrine cells, Paneth cells, etc.

[0133] The term "intestinal stem cell population" herein refers to the state of intestinal stem cells aggregation, and may be in the form of a tissue, a cell cluster, or a single cell.

[0134] The intestinal stem cell population disclosed herein can show increased expression levels of any one or more markers selected from the group consisting of LGR5, CD44, SOX9, LRIG1, LYZ, AXIN2, CTNNB and MKI67. In addition, any one or more markers selected from the group consisting of LDHB, EIF3E, SOX9 and SHH can be expressed.

[0135] More specifically, the intestinal stem cell population can show increased expression levels of LGR5, CD44, SOX9, LRIG1, LYZ, AXIN2, CTNNB or MKI67 compared to a control group cultured in a medium not including a WNT / R-floor plate response protein activator. LGR5, CD44, SOX9, LRIG1, LYZ, AXIN2, CTNNB or MKI67 are intestinal stem cell markers, which means that when an increased expression level is shown, the stem cell properties and self-renewal functions of the stem cells are maintained.

[0136] Alternatively, by analyzing gene expression patterns through single-cell RNA sequencing, intestinal stem cells may express LDHB, EIF3E, SOX9, or SHH, which are specific markers for intestinal stem and progenitor cells.

[0137] In the intestinal stem cell population of the present disclosure, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 88%, 89% or 90% or more of the cells constituting the population may be intestinal stem cells or progenitor cells. More specifically, based on the total number of cells in the population, the intestinal stem cell population may have 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 88%, 89% or 90% or more of the cells, including S phase cells, LGR5+ stem cells and early intestinal cells.

[0138] The S phase cell refers to a cell that is in the S phase, which is a step of DNA replication, among cells undergoing proliferation according to the cell cycle.

[0139] LGR5+ stem cells refer to stem cells expressing LGR5, which is an indicator indicating adult stem cell activity, and LGR5 refers to a target gene of Wnt signaling and a factor of R-floorplate receptor that amplifies Wnt signaling.

[0140] Early enterocytes are progenitor cells of epithelial cells that surround the small and large intestines and include early enterocyte 1 and early enterocyte 2.

[0141] In addition, early intestinal cells can be used by freezing or thawing cells under freezing conditions as needed. Even under such long-term subculture conditions and / or freezing and thawing conditions, the characteristics of the cells are maintained and the culture can be continued.

[0142] Therefore, the present disclosure provides a method for cryopreserving an intestinal stem cell population, the method comprising the following steps: (a) isolating pluripotent stem cell-derived three-dimensional intestinal organoids into single cells or small cell aggregates;

[0143] (b) culturing the single cell or the small cell aggregate in two dimensions in a culture medium comprising a WNT / R-floorplate response protein activator, an activator of a prostaglandin signaling pathway, and a receptor tyrosine kinase ligand to generate an intestinal stem cell population; and

[0144] (c) Cryopreservation of the resulting intestinal stem cell population.

[0145] If necessary, after centrifuging the cells, the intestinal stem cell population produced by step (b) can be resuspended at a predetermined concentration (cells / mL).

[0146] The freezing medium may also include, for example, any one selected from the group consisting of: dimethyl sulfoxide (DMSO); glucose; 1,2-propylene glycol; ethylene glycol; glycerol; formamide; ethylene glycol or butane-2,3 diol; hydroxyethyl starch (HES), dextran, sucrose, trehalose, lactose, raffinose, ribitol, mannitol and polyvinyl pyrrolidone (PVP).

[0147] Preferably, the cells are commercially available or suspended in a freezing medium suitable for freezing and thawing cells. The freezing medium may be Recovery TM Cell Freezing Medium (Gibco) culture medium.

[0148] In some cases, 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 (Bio Life Solutions, Inc., Bothell, Washington) is a cell culture medium containing 10% (v / v) DMSO and substantially free of animal components or serum. CS5 (Bio Life Solutions, Inc.) medium is a cell culture medium containing 5% (v / v) DMSO and substantially free of animal components or serum. Cells can be placed in vials and cryogenically frozen. The temperature sufficient to freeze the composition is about -80 ° C to about -190 ° C.

[0149] If necessary, the cryopreserved cells can be thawed by a commonly known thawing method and used as an intestinal stem cell population. In addition, if necessary, the number of cells can be increased by subculture.

[0150] The present disclosure also provides an intestinal stem cell population produced according to the culture method.

[0151] Methods for producing intestinal epithelial cells

[0152] The present invention provides a method for producing intestinal epithelial cells by culturing a population of intestinal stem cells in a chemically defined differentiation medium via air-liquid interface culture.

[0153] The present invention provides a method for producing intestinal epithelial cells, which comprises the step of culturing an intestinal stem cell population in a differentiation medium containing an activator of a prostaglandin signaling pathway, a receptor tyrosine kinase ligand, a p38 inhibitor, a WNT / R-floorplate response protein activator and nicotinamide by air-liquid interface culture.

[0154] More specifically, a method for producing intestinal epithelial cells is provided, the method comprising the following steps: (a) isolating pluripotent stem cell-derived three-dimensional intestinal organoids into single cells or small cell aggregates;

[0155] (b) culturing the single cell or the small cell aggregate in two dimensions in a culture medium comprising a WNT / R-floorplate response protein activator, an activator of a prostaglandin signaling pathway, and a receptor tyrosine kinase ligand to generate an intestinal stem cell population; and

[0156] (c) Intestinal stem cell populations were cultured in a differentiation medium containing an activator of the prostaglandin signaling pathway, a receptor tyrosine kinase ligand, a p38 inhibitor, a WNT / R-floor planarian activator, and nicotinamide via air-liquid interface culture.

[0157] The contents of the above-mentioned pluripotent stem cells, organoids and intestinal stem cells include the above descriptions, which will not be repeated here to avoid excessive complexity of this specification.

[0158] Furthermore, since the culture medium of step (b) contains the above-mentioned WNT / R-floorplate response protein activator, activator of prostaglandin signaling pathway and receptor tyrosine kinase ligand, redundant description thereof will be omitted to avoid excessive complexity of this specification.

[0159] The term "differentiation medium" herein refers to a cell growth medium that allows undifferentiated stem cells to be generated into cells having some or all of the characteristics of differentiated cells when cultured in the medium, and includes a basal medium.

[0160] Differentiation media herein optimized for intestinal epithelial cell production contain an activator of the prostaglandin signaling pathway, a receptor tyrosine kinase ligand, a p38 inhibitor, a WNT / R-floorplanin activator, and nicotinamide.

[0161] The term "gas-liquid interface culture" herein may refer to culture in a partially open culture container or a culture container partially filled with culture medium, but the present disclosure is not limited thereto. For example, the surface of the cell or organoid may be exposed to air. The gas may be air and is not limited to a mixture of gases and components found in the surrounding environment. Specifically, the present disclosure contemplates and encompasses gas mixtures having components different from the surrounding environment, for example, mixtures containing enriched specific components or mixtures depleted or removed specific components.

[0162] When cell is cultivated at air-liquid interface, cell can contact with the air on the upper side of porous matrix and contact with the cell culture medium on bottom side, to cultivate cells on porous matrix.For example, culture medium of sufficient volume can be added to the bottom of the culture vessel containing porous matrix (for example, filter element), so that culture medium contacts with the bottom surface of the cell present on porous matrix, but cell is not sealed or immersed.Can use any material that will not adversely affect cell growth and differentiation to form suitable porous matrix.Exemplary porous matrix can be formed by the polymer of for example polyethylene terephthalate (PET), polyester or polycarbonate.Suitable porous matrix can be coated or not coated. Examples of commercially available extracellular matrices include extracellular matrix proteins from Engelbreth-Holm-Swarm (EHS) murine sarcoma cells (Invitrogen) and basement membrane preparations (e.g., Cultrex (registered trademark) basement membrane extract (Trevigen, Inc.), type I collagen (Invitrogen), or Vitrogel (registered trademark) (The Well Bioscience Inc.). Alternatively, the porous matrix can be coated with Matrigel (trademark) (BD Biosciences).

[0163] That is, intestinal stem cells can be cultured in the presence of a transport well plate coated with an extracellular matrix. The extracellular matrix can be any of the extracellular matrices described above.

[0164] Preferably, the extracellular matrix can be Matrigel. The porosity of the matrix needs to be sufficient to maintain cell viability and promote cell differentiation.

[0165] Suitable matrices include those having a pore size 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, and preferably about 0.4 μm, and about 50 million to about 120 million pores / cm 2 , about 60 million to about 110 million pores / cm 2 , about 70 million to about 100 million pores / cm 2 , preferably about 80 million to about 100 million pores / cm 2 , about 90 million to about 100 million pores / cm 2 , more preferably about 100 million pores / cm 2 Filter element with a pore density of

[0166] It may be advantageous to replace or regenerate the culture medium every day or every other day. Cells grown on top of a porous matrix are usually not single cells, but cells are in sheets or in aggregated clusters. Cells cultured at the air-liquid interface can experience much higher oxygen tension than cells immersed in the culture medium.

[0167] The intestinal epithelial cells of the present disclosure may show an increased expression level of at least one marker selected from the group consisting of VIL1, ECAD, FABP1, KRT20, LCT, LYZ and MUC2.

[0168] More specifically, intestinal epithelial cells may have the following characteristics: the expression of at least one marker gene of LGR5, CD44, MKI67, SOX9, ASCL2, OLFM4, AXIN2 or CTNNB, which are intestinal stem cell markers, is reduced, while the expression of VIL1, ECAD, FABP1, KRT20, LCT, LYZ or MUC2, which are intestinal epithelial cell markers, is increased.

[0169] Furthermore, intestinal epithelial cells can exhibit increased expression levels of AKR1B15, DHRS11, GALNT4, GALNT5, DHRS3, RDH10, AADAC, NR1I2, SULTE1, DOUX2, FABP1, SLC6A20, SLC43A1 and / or CLDN3 compared to intestinal stem cells.

[0170] Intestinal epithelial cells can include enterocytes, mucus-secreting cells, hormone-secreting cells, and Paneth cells.

[0171] The production of intestinal epithelial cells according to the present disclosure is not limited thereto, but may be performed for 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days, as well as 2 weeks, 3 weeks or longer.

[0172] The present invention provides intestinal epithelial cells produced according to the method for producing intestinal epithelial cells.

[0173] use

[0174] 1) Medicinal use

[0175] The present invention provides a method for treating patients suffering from intestinal diseases or at risk of developing intestinal diseases by using intestinal stem cells or intestinal epithelial cells as cell therapeutic agents.

[0176] The present disclosure provides a tissue therapeutic agent comprising an intestinal stem cell population and / or intestinal epithelial cells.

[0177] The present disclosure provides a pharmaceutical composition comprising an intestinal stem cell population and / or intestinal epithelial cells.

[0178] The present disclosure provides a pharmaceutical composition for preventing or treating intestinal diseases, comprising an intestinal stem cell population and / or intestinal epithelial cells.

[0179] The present disclosure provides a pharmaceutical composition for assisting intestinal transplantation, comprising an intestinal stem cell population and / or intestinal epithelial cells.

[0180] The present disclosure provides a pharmaceutical composition for preventing or treating intestinal diseases, comprising: an intestinal stem cell population and / or intestinal epithelial cells; and intestinal organoids.

[0181] Intestinal stem cell populations and / or intestinal epithelial cells can be used for therapeutic purposes as a biotechnological technique for restoring cellular or tissue function.

[0182] 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. In particular, it is contemplated that the pharmaceutical composition can be used as a material for regenerating and rebuilding damaged (including dysfunctional) intestinal tissue.

[0183] Thus, the pharmaceutical composition according to the present disclosure may be a tissue therapeutic agent.

[0184] Preferably, the cells may be an intestinal stem cell population.

[0185] In the present disclosure, the intestinal disease may be any one or more intestinal diseases selected from the group consisting of leaky gut syndrome, short bowel syndrome, irritable bowel syndrome, Crohn's disease, ulcerative colitis, intestinal Behcet's disease, infectious enteritis, ischemic bowel disease and radiation enteritis.

[0186] According to the present disclosure, the intestinal stem cell population can be used as a pharmaceutical composition to assist intestinal transplantation of small intestinal organoids.

[0187] A prerequisite for transplantation efficiency and regenerative therapy efficiency is that blood is supplied to the transplanted cells or tissues through angiogenesis by rapid transplantation at the transplantation site. Conventional small intestinal organoids or intestinal stem cells have low implantation rates and insufficient vascularization during intestinal transplantation, resulting in very low initial transplantation efficiency. The intestinal stem cell population described in the present disclosure can be used as an adjuvant to improve the efficacy of intestinal transplantation by improving the implantation rate and vascularization efficiency during intestinal transplantation.

[0188] Furthermore, the intestinal stem cell population disclosed herein can be used as a composition for treating intestinal diseases, wherein the composition comprises an intestinal stem cell population and intestinal organoids.

[0189] In this transplantation, the intestinal stem cell population can be used for transplantation together with at least one biodegradable support selected from the group consisting of fibrin, laminin, collagen, gelatin, chitosan, alginate, hyaluronic acid, dextran, polylactic acid, poly(glycolic acid) (PGA), lactic acid-glycolic acid copolymer (poly(PLGA)), poly-ε-(caprolactone), polyanhydrides, polyorthoesters, polyvinyl alcohol, polyethylene glycol, polyurethane, polyacrylic acid, poly-N-isopropylacrylamide, poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) copolymers, their copolymers and their mixtures.

[0190] That is, the cells used as a target of a gastric transplant material can be used for transplantation as they are or embedded in the above-mentioned support.

[0191] In addition, dimethyl sulfoxide (DMSO) and the like may be added for the purpose of protecting cells; antibiotic substances and the like may be added to block bacterial incorporation; and various components (vitamins, cytokines, growth factors, steroids, etc.) may be added to the transplant material of the present disclosure for the purpose of activating cells, proliferation or inducing differentiation.

[0192] The graft material disclosed herein can also be used to construct an in vivo test system. For example, the above-mentioned graft material can be transplanted into experimental animals, such as mice, rats, guinea pigs, hamsters, pigs, cynomolgus monkeys, rhesus monkeys, chimpanzees, etc., to produce humanized animals (human intestinal models). Such humanized animals are particularly suitable for experiments such as pharmacokinetics and toxicity tests, and are expected to contribute to studies such as the initial passage effect of oral drugs or drug-induced enteritis.

[0193] The term "prevention" herein refers to all actions to inhibit or delay the onset of intestinal diseases by administering the composition, and the term "treatment" herein refers to all actions to improve or benefit the symptoms of intestinal diseases by administering the composition.

[0194] The composition of the present disclosure may include 1.0×10 5 to 1.0×10 10 cells / 1ml, preferably 1.0×10 6 to 1.0 to 10 9 cells / 1ml.

[0195] The pharmaceutical composition of the present disclosure can be formulated into various dosage forms, such as liquid preparations, suspensions, etc., according to conventional methods.

[0196] The pharmaceutical composition of the present invention can be formulated into a unit dosage form of a pharmaceutical preparation suitable for administration to a patient according to the conventional methods of the pharmaceutical field, and the preparation includes an effective dose administered once or multiple times. Preferred preparations suitable for this purpose include parenteral preparations, such as injections, infusions and transplants. In addition, the pharmaceutical composition may include pharmaceutically acceptable common inert carriers and diluents. Pharmaceutically acceptable carriers and diluents can be biologically and physiologically friendly to intestinal stem cell groups and recipients to be transplanted with intestinal stem cell groups. Diluents may include saline, water-soluble buffers, solvents and / or dispersion media, but the disclosure is not limited thereto. In addition, for example, injections may also include preservatives, analgesics, solubilizers, stabilizers, etc., and topical preparations may also include substrates, excipients, lubricants, preservatives, etc.

[0197] The compositions of the present disclosure may be uncooked or frozen for subsequent use. When the compositions are to be frozen, standard cryopreservatives (e.g., DMSO, glycerol, and Cell freezing medium (Cascade Biologics) was added to the cell population.

[0198] In addition, the composition can be transplanted and administered using a commonly used administration method in the art, preferably directly transplanted or transplanted into the diseased site of a patient in need of treatment, but the present disclosure is not limited thereto. In addition, administration can be performed by non-surgical administration using a catheter and surgical administration (e.g., injection or transplantation after an incision in the diseased site). The dose can be 5×10 5 Up to 10 8 / 60kg adult or 5×10 5 Up to 10 8 However, it should be understood that the actual dosage of the active ingredient should be determined according to various relevant factors, such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age and gender, etc., and therefore the dosage does not limit the scope of the present disclosure in any way.

[0199] The present disclosure also provides a pharmaceutical composition comprising an intestinal stem cell population for preventing or treating intestinal diseases.

[0200] The present disclosure also provides a use of an intestinal stem cell population in producing a drug for preventing or treating intestinal diseases.

[0201] The present disclosure provides a method for treating an intestinal disease, comprising the step of administering an intestinal stem cell population to a subject in need thereof.

[0202] The term "subject" refers to any animal that has or may develop an intestinal disease, including humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quail, cats, dogs, mice, rats, rabbits, or guinea pigs.

[0203] The present disclosure also provides a pharmaceutical composition comprising an intestinal stem cell population for assisting intestinal transplantation.

[0204] The present disclosure also provides a use of an intestinal stem cell population in producing a drug for assisting intestinal transplantation.

[0205] A method of assisting intestinal transplantation is provided, comprising the step of administering a population of intestinal stem cells to a subject in need thereof.

[0206] The present disclosure also provides a pharmaceutical composition comprising an intestinal stem cell population for assisting intestinal transplantation.

[0207] The present disclosure also provides a use of an intestinal stem cell population in producing a drug for assisting intestinal transplantation.

[0208] A method of assisting intestinal transplantation is provided, comprising the step of administering a population of intestinal stem cells to a subject in need thereof.

[0209] The present disclosure also provides a pharmaceutical composition for preventing or treating intestinal diseases, comprising: an intestinal stem cell population; and small intestinal organoids.

[0210] The present disclosure also provides a use of an intestinal stem cell population and a small intestinal organoid in producing a drug for preventing and treating intestinal diseases.

[0211] Provided is a method for treating intestinal diseases, comprising the step of administering an intestinal stem cell population and a small intestinal organoid to a subject in need thereof.

[0212] 2) Model

[0213] The present disclosure also provides an intestinal stem cell and / or intestinal epithelial cell model.

[0214] Specifically, the model can be used as a model by directly using intestinal stem cells or by using intestinal epithelial cells differentiated from intestinal stem cells.

[0215] Intestinal stem cells according to the present disclosure can be used as cell therapeutic agents for regenerative treatment of damaged intestinal tissues, and can help provide better results for research and clinical application of tissue regeneration. More specifically, since the intestinal stem cells according to the present disclosure are formed by a stem cell population that is transplanted into tissues and plays a key role in tissue regeneration, the probability of implantation in damaged tissues is high, and damaged tissues can be quickly repaired by direct regeneration.

[0216] In addition, this model can be used for various evaluations, such as screening to discover therapeutic agents important for regulating stem cell regeneration and proliferation and drug toxicity evaluation.

[0217] If desired, the intestinal stem cells of the present disclosure can also be used as a system model for developing intestinal stem cell lines, such as a system model for stem cell lines through gene editing.

[0218] The term "stem cell line" refers to cells that can differentiate into other cell types, either fully differentiated to have a specific specialized function, or can be maintained in an undifferentiated state.

[0219] Intestinal stem cells according to the present disclosure can produce transformed cell lines by applying gene editing technology such as using lentivirus or CRISPR-Cas9, and the transformed cell lines provide various research methods, such as large-capacity drug screening, efficacy evaluation, and real-time tracking of cell function.

[0220] Furthermore, intestinal stem cells can be labeled with any one fluorescent protein selected from the group consisting of green fluorescent protein (GFP), blue fluorescent protein (CFP), yellow fluorescent protein (YFP), and red fluorescent protein (DsRed), or can be used to analyze the characteristics of target cells by converting fluorescent proteins.

[0221] In particular, new cell lines can also be provided which can be monitored in real time by conversion to fluorescent proteins, etc.

[0222] The present disclosure provides an intestinal epithelial cell model, comprising intestinal epithelial cells. Preferably, the intestinal epithelial model can be a small intestinal epithelial model.

[0223] In an intestinal epithelial cell model comprising intestinal epithelial cells differentiated from the intestinal stem cell population disclosed herein, 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.

[0224] The intestinal epithelial cell model can help provide more accurate results in research and clinical applications on bacterial or viral infection and intestinal drug metabolism. More specifically, the intestinal epithelial cell model not only has the shape and functional characteristics of living tissue, but also has high stability and reproducibility, making it likely to be used to evaluate drug metabolism, bacterial or viral infection, microbial infection, etc.

[0225] In other words, the intestinal epithelial cell model can be used as a platform for bacterial or viral infection, microbial infection, drug metabolism, etc., in order to provide accurate predictions for symptom studies, drug side effects, safety, and interactions regarding bacterial or viral infection and microbial infection.

[0226] The intestinal epithelial cells produced according to the production method of the present disclosure or an intestinal epithelial cell model comprising the same can be used as an infectious disease model.

[0227] For example, the intestinal epithelial cells produced according to the production method of the present disclosure or the intestinal epithelial cell model comprising the same can be used as a bacterial or viral infectious disease model or a microbial infectious disease model. For these bacteria, viruses or microorganisms, any known bacteria, viruses or microorganisms can be used. According to one embodiment of the present disclosure, the intestinal epithelial cells produced according to the production method of the present disclosure or the intestinal epithelial cell model comprising the same can be used as a SARS-CoV-2 viral infectious disease model.

[0228] The present disclosure provides a method for screening therapeutic agents using the intestinal epithelial cell model according to the present disclosure.

[0229] Accordingly, the present disclosure provides a drug screening method comprising the steps of: (a) infecting an intestinal epithelial cell model with bacteria or viruses; (b) treating the infected intestinal epithelial cell model with a drug; and (c) confirming a response according to the drug treatment.

[0230] In addition, a drug evaluation method is provided, comprising the following steps: (a) treating the intestinal epithelial cell model with a drug; and (b) evaluating the absorption rate or bioavailability of the drug in the intestinal epithelial cell model in (a).

[0231] Furthermore, a method for providing an intestinal epithelial cell model for disease modeling is provided, the method comprising the step of infecting the intestinal epithelial cell model with bacteria or viruses.

[0232] In the present disclosure, a step of treating a test substance on an intestinal epithelial cell model may be performed. In the present disclosure, a test substance is a substance predicted to prevent, alleviate or treat an intestinal-related disease, and examples of drug candidate substances, test compounds or test compositions may include small molecule drugs, antibodies, antisense nucleotides, short interfering RNAs, short hairpin RNAs, nucleic acids, proteins, peptides, other extracts or natural substances, but the present disclosure is not limited thereto. In the present disclosure, when the expression of a biomarker protein of a disease or an mRNA encoding the biomarker protein increases or decreases after treating the test substance compared to before treatment, a step of selecting the test substance as a therapeutic agent may be performed.

[0233] Examples of such diseases include bacterial or viral infectious diseases, microbial infectious diseases or enteric-related diseases.

[0234] The term "bacterial or viral infectious disease or microbial infectious disease" refers to any disease that can be caused by infection with pathogenic bacteria or viruses or microorganisms.

[0235] In the present disclosure, intestinal related diseases include inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), Crohn's disease, ulcerative colitis (UC), short bowel syndrome, enterocolitis, Hirschsprung's disease (which is a genetic intestinal disease), and celiac disease, but the present disclosure is not limited thereto.

[0236] Unless otherwise defined, all technical terms used in the present disclosure are used with the same meanings as those generally understood by those skilled in the art in the relevant field of the present disclosure. In addition, preferred methods or samples are described in this specification, but methods or samples similar or equivalent thereto are also included in the scope of the present disclosure. The contents of all publications cited herein are incorporated into the present disclosure.

[0237] Beneficial Effects

[0238] By the intestinal stem cell culture method according to the present disclosure, homogeneous intestinal stem cells can be easily and quickly cultured. In addition, through long-term subculture and freezing / thawing processes, stable large-scale culture can be performed, and culture can be performed while maintaining cell characteristics, thereby constructing a highly reproducible intestinal stem cell culture system.

[0239] Intestinal stem cell models produced by culture systems can be used as new research models for human intestinal stem cell research. In particular, intestinal stem cells derived from intestinal organoids differentiated from patient-specific induced pluripotent stem cells are patient-specific cell sources and can therefore be used as cell sources and research models for developing regenerative therapeutics for intestinal diseases and producing artificial organs.

[0240] Furthermore, by differentiating intestinal stem cells derived from intestinal organoids into intestinal epithelial cells according to the intestinal epithelial cell production method disclosed herein, a 2.5-dimensional intestinal epithelial cell model having intestinal cell diversity and a structure similar to intestinal tissue can be obtained.

[0241] The intestinal epithelial cell model is a model with increased nutrient and material metabolism enzymes, which can evaluate the absorption and metabolism of various substances and can act as a barrier to divide the outside and inside of the body. In addition, the intestinal epithelial cell model can be used to model various diseases (including infectious diseases), and can also be used in various fields such as screening of therapeutic agents for treating diseases and evaluating the efficacy and toxicity of new drug candidate materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0242] Figure 1 Schematic representation of the method used to isolate intestinal stem cell populations from pluripotent stem cell-derived three-dimensional intestinal organoids to culture intestinal stem cell populations in two dimensions.

[0243] Figure 2The images show the cell morphology during the culture of two-dimensional intestinal stem cell populations on feeders and Matrigel.

[0244] Figure 3 Figure 3 is a graph showing the morphology of an intestinal stem cell population derived from embryonic stem cell-derived three-dimensional intestinal organoids (hESC-hIOs) during two-dimensional culture on feeders and Matrigel.

[0245] Figure 4 is a diagram showing the results of screening various types of extracellular matrix coating conditions in order to maximize the engraftment of a two-dimensional intestinal stem cell population.

[0246] Figure 4 (a) is a diagram showing the cell morphology of a two-dimensional intestinal stem cell population when cultured in culture vessels coated with 0.2% gelatin, 10 μg / ml Col I, 50 μg / ml Col I, 1% Matrigel, and 5% Matrigel, respectively.

[0247] Figure 4 (b) is a display of the Figure 4 (a) is a graph showing the results of calculating the surface area by using the area of ​​the confirmed cell colonies.

[0248] Figure 5 This figure shows the results of screening for optimal medium components for culturing two-dimensional intestinal stem cell populations.

[0249] Figure 5 (a) is a graph showing the colony size of the intestinal stem cell population when a single factor was removed from the culture medium components by crystal violet (CV) staining.

[0250] Figure 5 (b) is a graph showing the result of calculating the surface area based on the colony size confirmed by crystal violet (CV) staining in (a) by using the Image J program.

[0251] Figure 6 The diagram shows the morphology of an intestinal stem cell population in a two-dimensional culture medium when the intestinal stem cell population is cultured in a medium from which essential factors (R-floorplate reactant protein 1, EGF, and PGE2) are respectively removed.

[0252] Figure 7 The results showed that WNT3A and R-floor response protein 1, two components of the culture medium, play an important role in maintaining the stem cell properties and self-renewal of the two-dimensional intestinal stem cell population by activating the WNT signaling system.

[0253] Figure 7(a) is a graph demonstrating by qPCR that the expression of marker genes associated with stem cell properties and self-renewal of a two-dimensional intestinal stem cell population is reduced when the WNT signaling system is inhibited by depletion of WNT3A and R-floor response protein 1.

[0254] Figure 7 (b) The results of immunofluorescence staining of EdU and KI67-labeled proteins confirmed that the growth capacity of the 2D intestinal stem cell population was reduced when the WNT signaling system was inhibited by depleting WNT3A and R-floor plate response protein 1.

[0255] Figure 8 are morphological images showing growth-arrested two-dimensional intestinal stem cell populations when the WNT signaling system is inhibited by removal of WNT ligands (WNT3A or R-floorplate responder 1) or treatment with WNT inhibitors (WNT-C59 or XAV939).

[0256] Fig. 9 It is a morphological diagram showing a two-dimensional intestinal stem cell population in which cell proliferation is inhibited when the EGFR signaling system is inhibited due to removal of the EGF ligand or treatment with an EGF inhibitor (PD0325901), as well as the results of cell survival / death (Calcein-AM (survival) / Etidium homodimer (death)) assays.

[0257] Fig.10 The results show that when the PGE2 signal transduction system is inhibited by removing the PGE2 ligand or treating with a PGE2 inhibitor (EP2i or EP4i), the growth of the two-dimensional intestinal stem cell population is inhibited.

[0258] Fig.10 (a) is a morphological image showing that the growth of a two-dimensional intestinal stem cell population is inhibited when the PGE2 signaling system is inhibited by removal of the PGE2 ligand or treatment with a PGE2 inhibitor (EP2i or EP4i).

[0259] Fig.10 (b) is a diagram showing that among PTGER2 receptors (PTGER1 to PTGER4) and PGE2 synthase receptors (PTGES), PGE2 and PTGER4 are specifically expressed in two-dimensional intestinal stem cells.

[0260] Fig.11 The results show that stable long-term culture is possible through subculture of two-dimensional intestinal stem cell populations.

[0261] Fig.11 (a) is a morphological diagram showing the two-dimensional intestinal stem cell population at P0, P1, P3, P5, P10, P20 and P30.

[0262] Fig.11 (b) is a graph showing the increase in the total number of cells during subculture.

[0263] Fig.12 When removed from the culture medium Figure 6 When other factors except the essential factors of the culture medium (R-floor plate response protein 1, EGF and PGE2) were added, these factors did not affect the growth of two-dimensional intestinal stem cells at P0, but the cell migration and proliferation capabilities were reduced during subculture.

[0264] Fig.13 It is a graph showing that cell engraftment is increased by treatment with a NOTCH activator (Jagged-1 or valproic acid) or a ROCK inhibitor (Y-27632) during subculture of two-dimensional intestinal stem cells.

[0265] Fig.13 (a) is a graph showing the morphology of transplanted two-dimensional intestinal stem cell populations when treated with NOTCH activators and ROCK inhibitors alone or simultaneously.

[0266] Fig.13 (b) shows the results of cell counting using the Countess III cell counter. Fig.13 (a) Graph showing the results of measuring the cell number of the transplanted two-dimensional intestinal stem cell population.

[0267] Fig.14 A diagram showing the morphological characteristics of cells when a two-dimensional intestinal stem cell population is cultured under optimized culture conditions.

[0268] Fig.14 (a) is a diagram showing that a two-dimensional intestinal stem cell population cultured under optimized culture conditions is formed by a monolayer.

[0269] Fig.14 (b) is a graph showing 100% survival rate of a two-dimensional intestinal stem cell population cultured under optimized culture conditions.

[0270] Fig.15 A graph showing that under optimized culture conditions, two-dimensional intestinal stem cells can be cryopreserved and thawed and can be cultured highly reproducibly with little variability between batches.

[0271] Fig.15 (a) is a diagram showing the cell morphology of a two-dimensional intestinal stem cell population thawed after cryopreservation after culturing for 2 days, 4 days, and 8 days.

[0272] Fig.15 (b) is a graph showing that two-dimensional intestinal stem cell populations can be cultured for a long time with little variability between batches.

[0273] Fig.16Schematic diagram of the analytical process for analyzing the cellular composition and characteristics of a two-dimensional intestinal stem cell population by single-cell RNA sequencing (scRNA-seq).

[0274] Fig.17 The results of the analysis of marker gene expression of epithelial cells and mesenchymal stromal cells show that the two-dimensional intestinal stem cell population includes a large number of cells that exhibit epithelial cell properties compared with mesenchymal stromal cells.

[0275] Fig.18 This is a diagram showing that the genes of a two-dimensional intestinal stem cell population were analyzed and showed similar characteristics to intestinal epithelial cells of a 6- to 8-week-old fetus.

[0276] Fig.18 (a) is a diagram showing the results of comparing the expression of marker genes of two-dimensional intestinal stem cell populations from fetuses to adults and human intestinal epithelial cells in a heat map.

[0277] Fig.18 (b) shows the similarity of marker gene expression patterns in a dendrogram. Fig.18 (a) is a graph of the results of hierarchical clustering.

[0278] Fig.19 A graph showing the types and composition of cells that make up a two-dimensional intestinal stem cell population based on single-cell transcriptome analysis results.

[0279] Fig.19 (a) A diagram showing the types and distribution of cells that make up the two-dimensional intestinal stem cell population on UMAP based on the analysis results of single-cell transcriptomes.

[0280] Fig.19 (b) is based on Fig.19 (a) Results Table showing the percentage of cells that make up the two-dimensional intestinal stem cell population.

[0281] Fig. 20 The figure shows the results of comparing the analysis results of single-cell transcriptomes of a two-dimensional intestinal stem cell population with the analysis results of single-cell transcriptomes of human intestinal epithelium disclosed in a reference.

[0282] Fig. 20 (a) A diagram showing the types and distribution of cells that make up the human intestinal epithelium on UMAP by analyzing single-cell transcriptomes reported in references.

[0283] Fig. 20 (b) is a diagram showing that the cell composition of the two-dimensional intestinal stem cell population is mainly composed of intestinal epithelial stem cells and progenitor cells, wherein the analysis results of single-cell transcriptomes of the two-dimensional intestinal stem cell population are similar to those of Fig. 20 The results of (a) are shown together on the same UMAP.

[0284] Fig. 20 (c) is a diagram showing the types and expression patterns of marker genes specifically expressed in each type of cells constituting the human intestinal epithelium by analyzing single-cell transcripts.

[0285] Fig.21 The two-dimensional intestinal stem cell population is shown to include multiple cells that exhibit characteristics of stem cells or progenitor cells by analyzing the expression patterns of marker genes that specifically express stem cells.

[0286] Fig. 22 is a diagram showing the results of validation by immunofluorescence staining to confirm that the two-dimensional intestinal stem cell population is mainly composed of cells exhibiting stem cell and progenitor cell properties.

[0287] Fig. 22 (a) is a diagram showing the results of confirming that most cells of the two-dimensional intestinal stem cell population are stem cells or progenitor cells by immunofluorescence staining of stem cell-specific marker proteins LDHB, EIF3E, SOX9, and KI67.

[0288] Fig. 22 (b) is a diagram showing the results of confirming that the two-dimensional stem cell population was not differentiated into secretory cells, as shown by the fact that the expression of secretory cell marker proteins MUC2 and CHGA was not observed at all in the differentiated cells constituting the intestinal epithelium.

[0289] Fig.23 Schematic diagram of a method for differentiating a two-dimensional intestinal stem cell population into 2.5-dimensional intestinal epithelial cells via air-liquid interface culture.

[0290] Fig.24 It is a diagram showing the cell morphology during differentiation of a two-dimensional intestinal stem cell population after removing a single factor from the culture medium to search for essential factors important for differentiation into 2.5-dimensional intestinal epithelial cells through air-liquid interface culture.

[0291] Fig.25 A diagram showing that a 2D intestinal stem cell population can normally differentiate into 2.5-dimensional intestinal epithelial cells by air-liquid interface culture in a minimal medium consisting of essential factors.

[0292] Fig.25 (a) is a graph showing that when a two-dimensional intestinal stem cell population is differentiated into 2.5-dimensional intestinal epithelial cells through air-liquid interface culture in a culture medium for culturing a two-dimensional intestinal stem cell population (complete medium (Full M)) and a minimal medium (minimal M) composed of essential factors, the entire population is normally differentiated.

[0293] Fig.25(b) is a diagram showing that various types of pluripotent stem cell-derived two-dimensional intestinal stem cell populations were differentiated into 2.5-dimensional intestinal epithelial cells by air-liquid interface culture in minimal medium.

[0294] Fig.25 (c) is a graph showing that 2.5-dimensional intestinal epithelial cells differentiated by air-liquid interface culture can be cultured with high reproducibility with little variation between batches.

[0295] Fig.26 A graph showing that when a two-dimensional intestinal stem cell population is differentiated into 2.5-dimensional intestinal epithelial cells by air-liquid interface culture, the expression of differentiated cell marker genes gradually increases as the differentiation proceeds.

[0296] Fig. 27 The graph shows that when a two-dimensional intestinal stem cell population is differentiated into 2.5-dimensional intestinal epithelial cells by air-liquid interface culture, villus-like structures are formed and the expression of marker proteins of differentiated cells increases.

[0297] Fig. 27 (a) is a graph showing the cell cross-sections confirmed by H&E staining and the expression levels of marker proteins confirmed by immunofluorescence staining on days 4, 8, and 12 after a two-dimensional intestinal stem cell population was differentiated into 2.5-dimensional intestinal epithelial cells by air-liquid interface culture.

[0298] Fig. 27 (b) shows the measurement of Fig. 27 (a) is a graph showing the thickness of cell cross sections confirmed by H&E staining.

[0299] Fig. 27 (c) is a graph showing that after a two-dimensional intestinal stem cell population was differentiated into 2.5-dimensional intestinal epithelial cells by air-liquid interface culture, the barrier function of intestinal epithelial cells on days 4, 8, and 12 was confirmed by measuring transepithelial electrical resistance (TEER).

[0300] Fig.28 The figure shows the results of confirming a two-dimensional intestinal stem cell population and 2.5-dimensional intestinal epithelial cells differentiated by air-liquid interface culture by using principal component analysis (PCA) of transcript expression patterns for comparative analysis with pluripotent stem cells (hPSCs), immature three-dimensional intestinal organoids (control hIOs), mature three-dimensional intestinal organoids (mature hIOs), functional intestinal epithelial cells (hIECs), and human intestinal epithelial tissue (hSI).

[0301] Fig.29The diagram shows the differences in the expression patterns of gene clusters and representative genes when differentiation into intestinal epithelial cells is performed by transcript analysis of a two-dimensional intestinal stem cell population and 2.5-dimensional intestinal epithelial cells differentiated by air-liquid interface culture.

[0302] Fig.29 (a) is a table showing the types of gene clusters whose expression patterns increased or decreased when a two-dimensional intestinal stem cell population was differentiated into 2.5-dimensional intestinal epithelial cells by air-liquid interface culture.

[0303] Fig.29 (b) is verified by qPCR experiment Fig.29 (a) Graph showing expression pattern results of representative genes of the gene clusters found.

[0304] Fig.30 is a schematic diagram of the process of producing an intestinal stem cell line expressing a fluorescent protein by gene delivery using lentivirus.

[0305] Fig.31 The diagrams show the morphology of each production step of an intestinal stem cell line expressing a fluorescent protein and the expression pattern of the fluorescent protein.

[0306] Fig.31 (a) is a diagram showing the morphology and fluorescent protein expression pattern of an intestinal stem cell line, wherein, immediately after using lentivirus to deliver a fluorescent gene (after spin infection), clones are selected using antibiotics (after selection and expansion), after selection, they are separated into single cells using trypsin (trypsin-EDTA) to produce a single-cell-derived cell line, and immediately after subculturing (after cell seeding), and after culturing, the intestinal stem cell line is made to have a specific size or larger (after expansion).

[0307] Fig.31 (b) is the date shown by separation by treatment with collagenase type IV + neutral protease Fig.31 (a) Diagram of the morphology and fluorescent protein expression pattern of cells in which single-cell-derived fluorescent protein-expressing clones were produced and then only individual clones were isolated for culture.

[0308] Fig.32 Is displayed using Fig.31 Figure 3. Cell morphology and fluorescent protein expression patterns of intestinal stem cell lines produced in the experiment after differentiating three-dimensional intestinal organoids into 2.5-dimensional intestinal epithelial cells through air-liquid interface culture.

[0309] Fig.32 (a) is a graph showing the cell morphology and fluorescent protein expression pattern of an intestinal stem cell line expressing fluorescent protein on days 6, 14, and 20 after differentiation into three-dimensional intestinal organoids in a Matrigel dome.

[0310] Fig.32 (b) is a diagram showing the intestinal epithelial cell morphology and fluorescent protein expression pattern on day 8 after the intestinal stem cell line expressing fluorescent protein was differentiated into 2.5-dimensional intestinal epithelial cells by air-liquid interface culture.

[0311] Fig.33 This is a schematic diagram illustrating the process used to model intestinal diseases and transplant intestinal stem cells. This figure demonstrates the confirmation of the regenerative therapeutic effect using a two-dimensional intestinal stem cell population and its applicability as a cell therapy agent.

[0312] Fig.34 A diagram depicting the process of using mouse colonoscopy to transplant a two-dimensional intestinal stem cell population and the condition of the mice after transplantation.

[0313] Fig.34 (a) is a diagram showing a photograph of a mouse colonoscope device capable of injecting cells captured using a mouse colonoscope (left) and an image of a transplanted two-dimensional intestinal stem cell population (right).

[0314] Fig.34 (b) is a diagram showing the condition of mice immediately after transplantation of a two-dimensional intestinal stem cell population.

[0315] Fig.35 is a graph illustrating changes in body weight over time after transplantation of Matrigel or 2D intestinal stem cells into mice.

[0316] Fig.36 Included are tables and graphs illustrating post-transplant survival of mice transplanted with either Matrigel or 2D intestinal stem cells.

[0317] Fig.37 The figure shows the results of confirming the regenerative effect of intestinal epithelium using mouse colonoscopy on days 3 and 14 after transplantation of Matrigel or two-dimensional intestinal stem cells into mice with intestinal damage. This evaluation was performed using Hot-EDTA before and after modeling intestinal epithelial damage.

[0318] Fig.38 is a diagram showing engraftment of a two-dimensional intestinal stem cell population in damaged intestinal epithelium.

[0319] Fig.38 (a) is a diagram showing the engraftment of a two-dimensional intestinal stem cell population labeled with DiR fluorescent dye in the damaged intestinal site observed on day 14 after transplantation using the IVIS device.

[0320] Fig.38 (b) is a diagram confirming the normal engraftment of an intestinal stem cell line expressing green fluorescent protein (GFP) in damaged intestinal epithelium. This was confirmed by transplanting an intestinal stem cell line, isolating the intestine 14 days later, and verifying the expression of fluorescent protein.

[0321] Fig.39 is a diagram demonstrating the regenerative capacity of a two-dimensional intestinal stem cell population, showing normal regeneration of damaged intestinal epithelium after transplantation of the population into the damaged site.

[0322] Fig.39 (a) is a diagram illustrating that intestinal epithelial tissue regeneration occurs only when a 2D intestinal stem cell population is transplanted. This is shown by transplanting Matrigel or a 2D intestinal stem cell population into the damaged site, followed by isolating the intestinal tissue 14 days later and analyzing the intestinal epithelial tissue morphology by H&E and AB-PAS staining.

[0323] Fig.39 (b) is a diagram showing the results of confirming mouse intestinal regeneration by human intestinal stem cells indicated by human-specific antibody expression throughout the crypt-villus structure. This was verified by transplanting intestinal stem cells into damaged intestinal sites and isolating the intestine 14 days later to confirm human-specific cytokeratin expression.

[0324] Fig.40 Schematic diagram of the overall process of modeling the disease by infecting SARS-CoV-2 virus into 2.5-dimensional intestinal epithelial cells differentiated from immature or mature 2D intestinal stem cell populations through air-liquid interface culture.

[0325] Fig.41 The graph shows the results of observing the morphology of an immature or mature two-dimensional intestinal stem cell population differentiated into 2.5-dimensional intestinal epithelial cells by air-liquid interface culture on the 2nd, 4th, 6th, 8th, and 10th day after the start of differentiation.

[0326] Fig.42 A graph showing that intestinal epithelial cells differentiated from a mature two-dimensional intestinal stem cell population have higher expression of marker genes of intestinal maturity than intestinal epithelial cells differentiated from an immature two-dimensional intestinal stem cell population.

[0327] Fig.43 The figure shows the results confirming that intestinal epithelial cells differentiated from a mature two-dimensional intestinal stem cell population have higher expression of receptors important for SARS-CoV-2 infection than the intestinal epithelial cells differentiated from an immature two-dimensional intestinal stem cell population.

[0328] Fig.43 (a) is a graph showing the results of analyzing only the expression of receptors important for SARS-CoV-2 infection among the analysis results of transcripts of intestinal epithelial cells differentiated from mature or immature two-dimensional intestinal stem cell populations.

[0329] Fig.43(b) is a graph showing the results of confirming the expression level of ACE2 protein in intestinal epithelial cells differentiated from mature or immature two-dimensional intestinal stem cell populations using immunofluorescence staining. The expression level of ACE2 protein confirmed that Fig.43 (a) Increased expression of ACE2 protein, a receptor important for SARS-CoV-2 infection.

[0330] Fig.44 is a graph showing the results of confirming viral transcripts using qPCR in intestinal epithelial cells, wherein intestinal epithelial cells differentiated from a mature two-dimensional intestinal stem cell population experienced higher levels of SARS-CoV-2 infection than intestinal epithelial cells differentiated from an immature two-dimensional intestinal stem cell population. DETAILED DESCRIPTION

[0331] Hereinafter, the present disclosure will be described in more detail with reference to Examples. The Examples are intended to describe the present disclosure in more detail, and the scope of the present disclosure is not limited to these embodiments.

[0332] Experimental Example 1. Cell Culture and iPSC Production

[0333] hESC (human embryonic stem cells) and hPSC (human induced pluripotent stem cells) including hiPSC (human induced pluripotent stem cells) were cultured by known methods (Molecular carcinogenesis 55, 387-396 (2016), Proteomics 15, 2220-2229 (2015)). Non-insertion-hiPSCs were transfected by electroporation according to known methods and reprogrammed using EpisomaliPSC reprogramming vectors (Catalog No. A14703. Invitrogen, Carlsbad, CA, USA).

[0334] Five days after electroporation, fibroblasts were plated at 1 × 10 5 100 cells / well were seeded on 6-well plates coated with Matrigel (BD Biosciences, San Diego, CA, USA) and cultured in E8 medium (Stem Cell Technologies, Vancouver, Canada). After 3 weeks, hiPSC colonies were selected and the cell number was increased for subculture and subsequent characterization.

[0335] Experimental Example 2. Differentiation of hPSCs into intestinal organoids (HIOs) for production of 3D intestinal organoids

[0336] Human intestinal organoids (hIO) were prepared using known methods (Nature 470, 105-109 (2011)). To induce definitive endoderm, hPSCs were plated on Matrigel or ECMatrix-coated TM-511 culture dish, and in RPMI 1640 medium containing 0%, 0.2% and 2% purified fetal bovine serum (dFBS, HyClone, Thermo Fisher Scientific Inc., Waltham, MA, USA) with 100ng / ml activin A (R&D Systems, Minneapolis, MN, USA) treatment for 3 days. In addition, in order to differentiate hPSC into 3D hindgut spheres, 500ng / ml FGF4 (R&D Systems) and 3μM CHIR99021 (TOCRIS) were treated with RPMI 1640 medium containing 2% dFBS for 4-6 days. Starting from the fourth day, the spheroids induced into the hindgut were inserted into Matrigel (BD Biosciences) and cultured in hIO medium (2mM L-glutamine, 1% penicillin-streptomycin and 15mM HEPES buffer in advanced DMEM F12) containing 1X B27 (Invitrogen), 200 to 250ng / ml R-floor plate reaction protein 1 (R&D Systems), 100ng / ml EGF (R&D Systems) and 40 to 50ng / ml head protein (R&D Systems), and subcultured every 10 to 14 days. In order to mature the intestinal organoids, 1ng / ml interleukin 2 (IL-2, R&D Systems) was added to the hIO medium for about 2 passages and cultures.

[0337] Experimental Example 3. Method for isolating and culturing intestinal stem cells from three-dimensional intestinal organoids

[0338] The three-dimensional intestinal organoids were separated from the matrigel dome, and the remaining three-dimensional intestinal organoids were removed as much as possible by pipetting. The separated organoids were placed in 1ml 0.25% trypsin-EDTA (TE, Invitrogen) and incubated in a 37°C water bath for about 5 minutes. Then, less than five pipettings were performed to allow the organoids to separate into single cells and small clumps, and basal medium was added to make its total volume 10ml. The cells fed into the centrifuge were placed on a culture dish coated with feeder cells or 1% matrigel (Corning). Thereafter, 200 ng / ml of R-floor plate reaction protein 1 (R&D Systems), 100 ng / ml of EGF (R&D Systems) and 2.5 μM prostaglandin E2 (Sigma-aldrich) were used as main components, and an intestinal stem cell culture medium (2 mM L-glutamine, 1% penicillin-streptomycin and 15 mM HEPES buffer in advanced DMEM) containing one or more selected from the group consisting of 1X B27 (Invitrogen), 80 ng / ml of noggin (R&D Systems), 10 nM [Leucine 15]-gastrin I (Sigma-aldrich), 100 ng / ml of 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 auxiliary components was added. F12 medium) and subcultured every 7 to 10 days. 1 μM Jagged-1 (Anaspac) and / or 2.5 μM Y-27632 (Tocirs) were added to the intestinal stem cell culture medium 2 days before subculture.

[0339] Experimental Example 4. Freezing and Thawing of Intestinal Stem Cells

[0340] To freeze intestinal stem cells, after 3 to 5 days of subculture, the intestinal stem cells were washed once or twice with PBS and treated with TE for 5 minutes at 37°C in the same manner as the subculture to separate the intestinal stem cells into single cells or small cell clumps, and then washed once with hIO medium (2 mM L-glutamine, 1% penicillin-streptomycin and 15 mM HEPES buffer in advanced DMEM F12). Thereafter, freezing medium (Recovery Medium) was added. TM Cell culture freezing medium, Gibco) was used to fully suspend the cells, and the resulting suspension was then frozen and stored in a LN2 tank for a long time.

[0341] Prepare warm (37°C) culture medium in advance for thawing frozen intestinal stem cells. Thaw the frozen cells quickly, wash once with intestinal stem cell culture medium, and culture in intestinal stem cell culture medium containing WNT / R-floor plate response protein activator, prostaglandin signaling pathway activator and receptor tyrosine kinase ligand in a culture dish coated with matrigel. Replace the culture medium of intestinal stem cells every 2 days, and after 3-7 days of culture, subculture in the same way according to the state of the cells at the time of initial thawing.

[0342] Experimental Example 5. Method for culturing and differentiating intestinal epithelial cells using an air-liquid interface

[0343] The intestinal stem cells grown to a density of 70% to 80% were washed 1-2 times with PBS and then treated with TE for 5 to 7 minutes in an incubator at 37°C. The intestinal stem cells separated into single cells were collected and then diluted with hIO medium. The cells were collected by centrifugation, the supernatant was removed, the intestinal stem cell medium was added thereto and mixed well, and then the cell number was measured using a CountessIII cell counter (Thermo Scientific, Inc.). 2.5-3.5×10 5 Cells were placed in the insert of a 12-transport well plate (Corning) coated with 1% matrigel and cultured in an incubator. After the cell density reached 100%, the culture medium of the upper layer was completely removed and the culture medium of the lower layer was replaced with a differentiation medium (containing 2mM L-glutamine, 1% penicillin-streptomycin and 15mM HEPES buffer in advanced DMEM F12), wherein the differentiation medium contained 200ng / ml of R-floor plate reaction protein 1 (R&D Systems), 100ng / ml of EGF (R&D Systems), 2.5μM prostaglandin E2 (Sigma-aldrich), 10μM SB202190 (Sigma-aldrich) and 10mM nicotinamide (Sigma-aldrich). Thereafter, the upper surface was washed once with PBS or hIO culture medium every 2 days, and the lower layer was replaced with a new differentiation culture medium and cultured for 8-12 days.

[0344] Experimental Example 6. Method for measuring cell viability

[0345] To measure the survival rate of two-dimensional intestinal stem cell populations cultured in culture dishes coated with 1% matrigel, a kit capable of distinguishing and staining viable and dead cells (live / dead viability / cytotoxicity kit, Invitrogen) was used. Viable cells were stained with calcein-AM and dead cells were stained with ethidium homodimer-1. Stained cells were observed by fluorescence microscopy (Olympus).

[0346] Experimental Example 7. Method for measuring cell growth rate

[0347] The two-dimensional intestinal stem cell population was seeded on a culture dish coated with 1% matrigel and then cultured for 2 to 7 days. Thereafter, the culture medium was removed, washed once or twice with PBS (Sigma-aldrich), and TE (Invitrogen) was added thereto to separate the cells. After centrifugation, culture medium was added to separate the cells into single cells, and then the cell number was measured using a Countess III cell counter (Thermo Scientific, Inc.).

[0348] Experimental Example 8. Crystal violet (CV) staining

[0349] The intestinal stem cell population was fixed with 4% paraformaldehyde (PFA) and stained with 0.02% crystal violet solution (Sigma-aldrich) for 10 minutes at room temperature. Thereafter, images were acquired after washing three times with sterile water. The colony size of the intestinal stem cell population was analyzed using Image J software (National Institute of Health).

[0350] Experimental Example 9. Quantitative Real-Time RT-PCR (qRT-PCR)

[0351] 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 by known methods (Cho et al. Oncotarget 6, 23837-23844, 2015) in a 7500 fast real-time PCR system (Applied Biosystems, Foster City, CA, USA). All experiments were repeated three times, and the CT value of each target gene was calculated using the software provided by the manufacturer. The base sequences of the primers used are shown in Table 1.

[0352] [Table 1]

[0353]

[0354]

[0355]

[0356] Experimental Example 10. Immunofluorescence test of cells and tissues

[0357] Immunofluorescence test was performed according to a known method (Kwak et al., Biochemical and biophysical research communications, 457, 554-560, 2015). Specifically, two-dimensional intestinal stem cell populations and differentiated intestinal epithelial cells or intestinal tissues were fixed with 4% paraformaldehyde (PFA) and permeabilized with PBS containing 0.1% Triton X-100.

[0358] Differentiated intestinal epithelial cells or intestinal tissues were cryoprotected with sucrose, and then the membranes inserted into the wells were lysed and placed vertically into optimal lysis temperature (OCT) compound (Sakura Finettek, Tokyo, Japan) and then frozen. The frozen sections were then cut into 10 μm sections using a cryostat at -20°C and permeabilized with PBS containing 0.1% Triton X-100 for immunofluorescence testing.

[0359] After blocking with 4% BSA, the cells were incubated with primary antibodies at 4°C overnight. Thereafter, the mixture was incubated with secondary antibodies for 1 hour at room temperature. The primary antibodies used are shown in Table 2. DAPI was added to visualize the nuclei. Slides were observed using EVOS FL Auto2 (Thermo Fisher), Axiovert 200M microscope (Carl Zeiss, Gottingen, Germany) or fluorescence microscope (IX51, Olympus, Japan).

[0360] [Table 2]

[0361]

[0362] Experimental Example 11. Single-cell RNA sequencing

[0363] The two-dimensional intestinal stem cell population was washed 2 to 3 times with PBS (Sigma-aldrich), then 0.25% TE (Invitrogen) was added thereto, and the cells were separated for a sufficient time of 10 minutes or longer, and then separated into single cells using a 40 μm cell strainer (BD Bioscience). The cells were diluted with PBS containing 0.04% BSA, and then the cell number and viability were measured using a Countess III cell counter (Thermo Scientific, Inc.). In order to construct a transcript library, the Chromium Next GEM single cell 3' kit v3.1 (10X Genomics) was used. In brief, approximately 5,000 single cell transcript libraries were generated by diluting cells in Chromium NextGEM Chip G, and then approximately 60,000 base sequences of each cell were analyzed using a Novasek 6000 sequencer (Illumina).

[0364] Experimental Example 12. RNA Sequencing and RNA Quantification

[0365] For RNA sequencing and quantification, first, RNA samples with RNA integrity number (RIN) values ​​of 7.5 or more were prepared by Agilent 2100 Bioanalyzer system (Agilent Biotechnologies, Palo Alto, USA), and mRNA libraries were prepared by Illumina TruSeq kit. Sequencing was performed using Illumina HiSeq2500 machine (Illumina, San Diego, CA, USA). Sequencing quality was determined by FastQC package, and trimmed read lengths of 50 bases or less were excluded. Thereafter, mapping was performed by HISAT2 (v2.0.5), and hg19 was used for human genome information. Differentially expressed genes between samples were analyzed by Cuffquant and Cuffnorm (Cufflinks v2.2.1).

[0366] Experimental Example 13. Bioinformatics Analysis

[0367] In order to analyze the results of single-cell transcript sequencing, the 10X Genomics software CellRanger (version 3.1) was used to process the initial data and construct a gene expression matrix. In addition, the analysis results of single-cell transcripts of fetal and adult intestinal epithelial tissue reported in the reference (Elementalite et al., 2020) were used for transcript comparative analysis. The integrated data were normalized and feature selected using the Scanpy package v1.8, and clustering and cell type annotation were performed using the main processed data. Thereafter, Spearman correlation was used for data integration, and the composition ratio of each cell was calculated.

[0368] Bioinformatics analysis was performed using IPA analysis software (Ingeneity systems, Redwood City, CA, USA), protein analysis by evolution (PANTHER, http: / / www.pantherdb.org) database, and DAVID bioinformatics resource 6.7 (http: / / david.abc.ncifcrf.gov). Gene ontology (GO) / pathways of functional grouping were analyzed using the Cytoscape software platform (Cytoscape software platform, version 3.3.0, http: / / apps.cytoscape.org / apps / cluego) combined with the ClugGO plugin (Version 2.2.5, http: / / www.cytoscape.org / what_is_cytoscape.html).

[0369] Experimental Example 14. Production of a cell line expressing fluorescent protein by lentiviral infection

[0370] To generate an intestinal stem cell line expressing fluorescent protein (green fluorescent protein; eGFP), lentivirus expressing EF-1α-Gene X-IRES2-EGFP-IRES-Puro was purchased from GeneCopoeia (MD, USA). 5 The 2D intestinal stem cells were centrifuged at 2,500 rpm for 90 minutes in a medium containing lentivirus and 8 μg / ml polybrene, and then additional medium was added and cultured for 48 hours. For clonal selection, the cells were cultured in a medium containing 1 μg / ml puromycin until only colonies expressing fluorescent protein remained.

[0371] In order to obtain clones derived from single cells, cells are separated into single cells by TE treatment, and then cells are plated and cultured at low density until the size of the colony reaches a certain size or larger. Type IV collagenase and dispase (Dispase) are mixed for 5 minutes to harvest colonies as a whole, and single colonies are separated by a pipette. The separated single colonies are transferred to a new plate and cultured to grow to a sufficient size, and differentiated into 3-dimensional organoids in a matrix gel dome to confirm the differentiation ability of the intestinal stem cell line expressing fluorescent protein, or differentiated into 2.5-dimensional intestinal epithelial cells using air-liquid interface culture.

[0372] Experimental Example 15. Two-dimensional intestinal stem cell transplantation experiment using colonoscopy

[0373] To confirm the tissue regeneration capacity of 2D intestinal stem cell populations, an intestinal epithelial injury model was prepared using hot EDTA in male NIG mice (NOD / SCID IL2Rg gene deletion, 6-12 weeks old; GHBio, Daejon, South Korea). 1.5 × 10 SCs were transplanted per mouse using a colon endoscopy syringe (Image1 Hub HD H3-Z; D-Light C; Rigid HOPKINS telescope; Karl Storz, Tuttlinen, Germany; and optimized syringe; Vetcom, Gwacheon, South Korea). 6 Two-dimensional intestinal stem cells (Matrigel transplantation group, n = 3; intestinal stem cell population transplantation group, n = 5). After transplantation, the cells were sealed with Vetbond tissue adhesive (3M, MN, USA) for 6 to 12 hours. Thereafter, the transplantation site was monitored using colonoscopy on days 0, 3, and 14, and intestinal tissue was isolated from euthanized mice to confirm the regenerative capacity on day 14.

[0374] Experimental Example 16. Tissue Analysis Using a Fluorescence Stereomicroscope

[0375] To confirm the presence of intestinal stem cells growing in the damaged intestinal epithelium, bright-field and fluorescent images of the mouse intestine on day 14 after transplantation were captured using a stereomicroscope (SZX16, Olympus, Japan).

[0376] Experimental Example 17. Histological (Hematoxylin & Eosin (H&E)) staining experiment

[0377] For histopathological analysis, intestinal tissue or intestinal epithelial cells were cryoprotected with sucrose, and then the membrane inserted into the well was lysed and placed vertically in an optimal lysis temperature (OCT) compound (Sakura Finettek, Tokyo, Japan) and then frozen. Then, the frozen sections were cut into 10 μm sections at -20 ° C using a cryostat microtome, adhered to a slide glass, and H&E stained according to published methods. The slides were observed using an optical microscope (BX53F, Olympus, Japan).

[0378] Experimental Example 18. Experiment for measuring epithelial transepithelial electrical resistance (TEER)

[0379] To confirm the barrier function of differentiated intestinal epithelial cells, epithelial cells and resistance were measured using an epithelial tissue volt / ohmmeter (EVOM, WPI, FL, USA). All upper and lower layers of the transwell plate (transwell) in which intestinal epithelial cells were cultured were washed with PBS, and then new culture medium was added thereto, and electrodes were immersed in the upper and lower layers one by one, and then the TEER value was measured.

[0380] Experimental Example 19. SARS-CoV-2 Virus Infection Experiment

[0381] Differentiated intestinal epithelial cells were infected with SARS-CoV-2 virus at 0.01 and 0.001 multiplicity of infection (MOI) produced in Vero cells for 1 hour. After that, the medium containing the virus was carefully removed, and then new medium was added and further cultured for 72 hours. After that, the cells were harvested for RNA isolation and purification to detect viruses that infect intestinal epithelial cells.

[0382] Experimental Example 20. Statistical Analysis

[0383] All results are expressed as mean ± standard error (sem), and all experiments were repeated at least three times. P values ​​were determined using a two-tailed t-test or one-tailed ANOVA. Unless otherwise stated, all statistically significant analyses were calculated by comparison with controls.

[0384] Example 1. Isolation and culture of intestinal stem cell populations from three-dimensional intestinal organoids

[0385] In order to easily and quickly culture high-purity intestinal stem cells on a large scale, a new method for isolating and concentrating intestinal stem cells from three-dimensional intestinal organoids was constructed ( Figure 1 Newly developed techniques for culturing intestinal stem cells allow for the two-dimensional culture of intestinal stem cell populations on feeder cells or plates coated with 1% Matrigel, and for the generation of three-dimensional intestinal organoids derived from various types of pluripotent stem cell lines ( Figure 2 and Figure 3 ) to isolate and culture only intestinal stem cells.

[0386] As a result of coating tests using various coating materials to maximize the engraftment rate of the intestinal stem cell population, it was confirmed that the engraftment rate was the highest when 1% Matrigel was coated ( Figure 4 ). However, it was confirmed that even when gelatin, collagen, etc. were used, an excellent transplantation rate was shown, so that the intestinal stem cell population can be easily cultured without the need for a xenobiotic component.

[0387] Example 2. Development of a culture medium with optimized composition for culturing two-dimensional intestinal stem cell populations

[0388] To minimize performance variability between batches of 2D intestinal stem cell populations, screens were performed to exclude the use of chemically undefined factors and to newly discover media compositions containing factors with defined composition and capacity.

[0389] The results confirmed that WNT / R-floorplate responsive protein activators, activators of the prostaglandin signaling pathway, and receptor tyrosine kinase ligands play an important role in the survival of intestinal stem cell populations. In particular, with regard to these signaling systems, it was confirmed that the combination of R-floorplate responsive protein 1, PGE2, and EGF corresponds to the most suitable factor combination for the survival of two-dimensional intestinal stem cell populations ( Figure 5 and 6 ).

[0390] In order to confirm the effects of the above essential factors in detail, the difference in effect depending on the presence or absence of each factor was examined in more detail.

[0391] Among the essential factors, R-floorplate responder 1 was found to activate the WNT signaling pathway ( Figure 7 and Figure 8 ) regulate the stem cell properties and proliferation of 2D intestinal stem cells.

[0392] Furthermore, EGF was found to prevent the death of intestinal stem cell populations and regulate proliferation by activating the EGF-EGFR signaling system ( Fig. 9 ).

[0393] Finally, it was found that PGE2 regulates the proliferation of intestinal stem cell populations by activating the PGE2-EP2 / 4 signaling pathway ( Fig.10 ).

[0394] Example 3. Construction of a method for subculturing and cryopreservation of a two-dimensional intestinal stem cell population

[0395] In order to increase the utilization of the two-dimensional intestinal stem cell population, stable long-term culture, large-scale culture, cryopreservation and thawing were tested. First, it was confirmed that more than 30 subcultures could be stably performed in the optimized culture medium and that a large number of cells could be proliferated without cell loss ( Fig.11 ).

[0396] On the other hand, factors other than the essential factors in the culture medium composition factors did not affect the transplantation and initial proliferation of the two-dimensional intestinal stem cell population. However, it was confirmed that for long-term subculture, factors such as B27, noggin, gastrin, WNT3A, A-83-01, SB202190, N-acetylcysteine ​​and nicotinamide were required for more effective long-term culture ( Fig.12 ).

[0397] Furthermore, in order to prevent cell loss during subculture, other factors important for subculture were discovered. As a result, it was confirmed that when Jagged-1 or valproic acid was added to activate the Notch signaling system, or Y-27632 was added to inhibit ROCK activity, the subculture efficiency was improved, and it was confirmed that when both signals were controlled at the same time, the subculture efficiency was maximized ( Fig.13 ). Under optimized conditions, it was confirmed that the two-dimensional intestinal stem cell population was formed by a monolayer, showed a 100% survival rate and could be stably cultured ( Fig.14 ), and confirmed that it can be frozen and thawed ( Fig.15 (a)), and also confirmed that long-term subculture without batch-to-batch variability ( Fig.15 (b)).

[0398] Example 4. Methods for analyzing single cell transcriptomes (scRNA-seq) to characterize two-dimensional intestinal stem cell populations method

[0399] Single-cell transcriptome profiling was performed to characterize the 2D intestinal stem cell population isolated and cultured from 3D intestinal organoids, and a new analysis method was designed for transcript expression pattern analysis and comparison with previous research results reported in references ( Fig.16 ).

[0400] Example 5. Verification of the Cellular Properties and Composition of the Two-Dimensional Intestinal Stem Cell Population Using Single-cell Transcriptome Analysis Results

[0401] As a result of analyzing the characteristics of the two-dimensional intestinal stem cell population based on the results of single-cell transcriptome analysis, it was confirmed that almost all of the two-dimensional intestinal stem cell populations showed characteristics of intestinal epithelial cells based on marker gene expression analysis, and a few cells showed characteristics of intestinal mesenchymal stromal cells ( Fig.17 ). When compared with the single-cell transcriptome analysis of human intestinal epithelial tissue at developmental stages reported in reference ( Fig.18 ), the two-dimensional intestinal stem cell population exhibiting the described properties showed characteristics most similar to those of the human fetal intestinal epithelium.

[0402] Furthermore, it was confirmed that the cells constituting the two-dimensional intestinal stem cell population were mainly composed of stem cells and progenitor cells among cells showing characteristics of intestinal epithelial cells, and that 90% or more of the total cells were stem cells and progenitor cells ( Fig.19 When the results of the two-dimensional intestinal stem cell population analysis were compared with the results of the single-cell transcriptome analysis of fetal intestinal epithelial tissue published in the reference, it was confirmed that the majority of cells were distributed in the group distributed by intestinal stem cells and progenitor cells ( Fig. 20 ).

[0403] Furthermore, as a result of confirming the expression distribution of marker genes (LDHB, EIF3E, SOX9, and SHH) known as markers for intestinal stem and progenitor cells, it was confirmed that all marker genes were expressed in almost all cells, thereby confirming that most of the two-dimensional intestinal stem cell populations exhibited the characteristics of intestinal stem cells or progenitor cells ( Fig.21 ).

[0404] Example 6. Verification of the Cellular Composition of the Two-Dimensional Intestinal Stem Cell Population Using Immunofluorescence Staining

[0405] To confirm the characteristics of the two-dimensional intestinal stem cell population, as a result of confirming the cell composition using immunofluorescence staining, it was confirmed that marker proteins of intestinal stem and progenitor cells (LDHB, EIF3E, and SOX9) were expressed in most cells, and that some cells were actively proliferating (KI67+ cells) ( Fig. 22 (a)). On the other hand, it was confirmed that the marker protein of absorptive cells (FABP1) was expressed in some cells among the differentiated cells, but the marker proteins of secretory cells (MUC2 and CHGA) were not observed ( Fig. 22 (a) and (b)).

[0406] The results confirmed that the two-dimensional intestinal stem cell population was mainly composed of multiple stem cells and progenitor cells, with very few differentiated cells.

[0407] Example 7. Differentiation of a two-dimensional intestinal stem cell population into intestinal epithelial cells using air-liquid interface culture

[0408] In order to differentiate a two-dimensional intestinal stem cell population into highly functional intestinal epithelial cells, a new differentiation method using air-liquid interface culture was established ( Fig.23 In this case, a culture medium factor screening was performed to construct an optimal culture medium for optimizing the differentiation of two-dimensional intestinal stem cell populations into intestinal epithelial cells, and by discovering five essential components ( Fig.24 ) can find the minimum culture medium composition.

[0409] Specifically, it was confirmed that activators of the prostaglandin signaling pathway, receptor tyrosine kinase ligands, p38 inhibitors, WNT / R-floorplate activators and nicotinamide are essential factors for differentiation into intestinal epithelial cells. In particular, treatment with R-floorplate 1, EGF, PGE2, SB202190 and nicotinamide was confirmed as essential factors showing excellent effects on differentiation into intestinal epithelial cells, which were used as the minimum medium composition.

[0410] It was confirmed that when the two-dimensional intestinal stem cell population was differentiated into intestinal epithelial cells in a defined minimal medium, the differentiation that occurred was similar to the differentiation that occurred when the two-dimensional intestinal stem cell population was differentiated in a two-dimensional intestinal stem cell population culture medium. Specifically, it was confirmed that all two-dimensional intestinal stem cell populations derived from various types of pluripotent cell lines were successfully differentiated into intestinal epithelial cells ( Fig.25).

[0411] Example 8. Analysis of intestinal epithelial cell characteristics by marker gene expression analysis

[0412] In order to analyze the characteristics of intestinal epithelial cells differentiated by air-liquid interface culture, the expression patterns of marker genes specifically expressed in intestinal stem cells and epithelial cells were detected. When gene expression in intestinal epithelial cells was detected by qPCR, it was confirmed that the expression of some stem cell marker genes was decreased, and the expression levels of most differentiated cell marker genes were increased ( Fig.26 ).

[0413] Furthermore, as a result of confirming the cross-sectional shape and expression pattern of marker proteins by collecting cells on days 4, 5, and 12 after differentiation into intestinal epithelial cells, it was confirmed that over time, the cross-section of intestinal epithelial cells produced a crypt-like structure and the height of the intestinal epithelium increased ( Fig. 27 (a) and (b)). At the same time, as intestinal epithelial cells gradually develop over time, it was confirmed that the expression levels of marker proteins of differentiated cells gradually increased in proportion ( Fig. 27 (a)). As mentioned above, the TEER value was measured to confirm that the degree of differentiation of intestinal epithelial cells increased over time, and the barrier function of intestinal epithelial cells also continued to increase ( Fig. 27 (c)). Therefore, it was confirmed that the intestinal stem cell population was successfully differentiated into intestinal epithelial cells by air-liquid interface culture.

[0414] Example 9. Comparative analysis of transcripts of various intestinal epithelial cells to identify intestinal stem cell populations and intestinal epithelial cells Characteristics of cells

[0415] When the analysis was performed by comparing the expression patterns of transcripts from pluripotent stem cells, 3D intestinal organoids derived from them, functional intestinal epithelial cells, and actual human intestinal tissue, it was confirmed that the 2D intestinal stem cell population segregated into cells that displayed characteristics distinct from those of other cells ( Fig.28 However, when the two-dimensional intestinal stem cell population was differentiated into intestinal epithelial cells using air-liquid interface culture, it was confirmed that the changes in transcript expression patterns were similar to those of other intestinal epithelial cells, and in particular, the cells were grouped most closely resembling functional intestinal epithelial cells ( Fig.28 In this case, when the gene sets with the largest differences in expression levels between the two-dimensional intestinal stem cell population and the intestinal epithelial cells were identified, it was confirmed that the two-dimensional intestinal stem cell population showed high expression of genes related to cell proliferation, and the intestinal epithelial cells showed high expression of metabolic genes ( Fig.29 ). The results confirmed that the two-dimensional intestinal stem cell population exhibited the characteristics of stem cells that activated cell proliferation, and intestinal epithelial cells could function as differentiated cells with the function of metabolizing various nutrients or substances.

[0416] Example 10. Development of an intestinal stem cell line expressing fluorescent protein using lentivirus

[0417] To confirm the various uses of 2D intestinal stem cell populations, we investigated whether gene-edited cell lines could be generated by introducing external genes ( Fig.30 ). First, in order to introduce external genes, a lentivirus containing a fluorescent protein is infected into the two-dimensional intestinal stem cell population, and the fluorescent protein is injected into the two-dimensional intestinal stem cell population. Thereafter, in order to select only cells expressing the fluorescent protein, positive selection is performed using antibiotics, and the selected cells are used to produce monoclones derived from single cells ( Fig.31 To verify the functionality of the constructed single clones, it was confirmed that when the single clones were differentiated into three-dimensional organoids in Matrigel domes or into intestinal epithelial cells using air-liquid interface culture, the differentiation pattern was the same as that of normal cells ( Fig.32 ). The results confirmed that gene-edited cell lines can be produced using two-dimensional intestinal stem cell populations.

[0418] Example 11. Verification of the regenerative capacity of two-dimensional intestinal stem cell populations using a mouse model of intestinal epithelial tissue damage experiment

[0419] To verify the applicability of the 2D intestinal stem cell population as a cell therapy agent, experiments were performed by transplanting the population into a mouse model with damaged intestinal epithelial tissue induced by hot EDTA ( Fig.33 ). Transplantation of a two-dimensional intestinal stem cell population into lesions using murine colonoscopy ( Fig.34 ). It was observed that mice transplanted with 2D intestinal stem cell populations showed a faster rate of weight recovery and a higher survival rate compared with the Matrigel-transplanted control group ( Fig.35 and Fig.36 Colonoscopic observations before and after transplantation confirmed that when the two-dimensional intestinal stem cell population ( Fig.37 ), lesions recovered faster and with lower immune response. This effect was attributed to the successful engraftment of the two-dimensional intestinal stem cell population in the lesions ( Fig.38 ), which promotes the regeneration of damaged intestinal tissue ( Fig.39 ). Thus, it was demonstrated that when the 2D intestinal stem cell population was transplanted into damaged intestinal epithelial tissue, it effectively regenerated the injury, demonstrating its potential as a biomaterial for cell therapy in the regeneration of intestinal epithelial tissue.

[0420] Example 12. Modeling of SARS-CoV-2 infectious disease using a 2.5-dimensional intestinal epithelial cell model

[0421] To validate the utility of 2.5-dimensional intestinal epithelial cells derived from 2D intestinal stem cell populations, infectious disease modeling using SARS-CoV-2 virus ( Fig.40). In order to confirm whether there is a difference in sensitivity to SARS-CoV-2 virus infection according to the maturity of intestinal epithelial cells, two-dimensional intestinal stem cell populations were isolated from immature / mature three-dimensional intestinal organoids, and immature / mature intestinal epithelial cells were produced using air-liquid interface culture. As a result of monitoring cell morphology after 10 days of air-liquid interface culture, it was confirmed that there was no difference between immature and mature intestinal epithelial cells ( Fig.41 ), but there were differences in the expression of maturation-related marker genes ( Fig.42 ).

[0422] In particular, it was confirmed that among the receptors important for SARS-CoV-2 virus infection, the expression level of ACE2 is higher in mature intestinal epithelial cells ( Fig.43 ), and thus confirmed that SARS-CoV-2 infection is more susceptible in mature intestinal epithelial cells ( Fig.44 ).

[0423] Based on these results, it was confirmed that 2.5-D intestinal epithelial cells derived from 2D intestinal stem cell populations can be used as a cell model to simulate various diseases including infectious diseases.

Claims

1. A method for culturing an intestinal stem cell population, the method comprising: (a) Dissociation of pluripotent stem cell-derived 3D intestinal organoids into single cells or small cell aggregates; as well as (b) culturing the single cell or the small cell aggregate in two dimensions in a culture medium comprising a WNT / R-floorplate response protein activator, an activator of a prostaglandin signaling pathway, and a receptor tyrosine kinase ligand.

2. The method according to claim 1, wherein: The WNT / R-floor response protein activator is any one or more selected from the group consisting of R-floor response protein 1, R-floor response protein 2, R-floor response protein 3, R-floor response protein 4 and R-floor response protein mimetic substances.

3. The method according to claim 1, wherein: The activator of the prostaglandin signaling pathway is 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).

4. The method 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-α (TGF-α), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF).

5. The method according to claim 1, wherein: The culture medium in (b) further comprises any one or more selected from the group consisting of B27, N-acetyl-L-cysteine ​​(NAC), nicotinamide, gastrin, a TGF-β inhibitor, a Wnt signaling pathway activator, a BMP inhibitor and a p38 inhibitor.

6. The method according to claim 5, wherein: The TGF-β inhibitor is any one selected from the group consisting of A-83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-36494 and SJN-251.

7. The method according to claim 5, wherein: The Wnt signaling pathway activator is 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.

8. The method according to claim 5, wherein: The BMP inhibitor is noggin, doxorubicin, DMH1 or LDN-193189.

9. The method according to claim 5, 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.

10. The method according to claim 1, wherein: The culture medium in (b) further comprises a ROCK inhibitor, a Notch activator, or both a ROCK inhibitor and a Notch activator in the initial stage of culture.

11. The method according to claim 1, wherein: The culture medium in (b) is selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Ham's F12, DMEM / F12, DMEM / F12 and Advanced DMEM / F12.

12. The method according to claim 1, wherein: The intestinal stem cell population shows increased expression levels of any one or more markers selected from the group consisting of LGR5, CD44, SOX9, LRIG1, LYZ, AXIN2, CTNNB and MKI67.

13. The method according to claim 1, wherein: The intestinal stem cell population expresses any one or more markers selected from the group consisting of LDHB, EIF3E, SOX9 and SHH.

14. The method according to claim 1, wherein: The intestinal stem cell population comprises 80% or more of the cells relative to the total cells of the intestinal stem cell population, and the 80% or more of the cells include S phase cells, LGR5+ stem cells and early intestinal epithelial cells.

15. The method according to claim 1, wherein: The pluripotent stem cell-derived three-dimensional intestinal organoids are produced by the following steps: (a) culturing pluripotent stem cells in a medium comprising any one or more selected from the group consisting of Nodal, activin A, activin B, BMP4, Wnt3a, CHIR99021 and bFGF to differentiate the stem cells into definitive endoderm; (b) culturing the definitive endoderm in a medium comprising 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-GKEAPPQSpP-NH2) and CHIR99021; and fibroblast growth factor (FGF) to differentiate the definitive endoderm into three-dimensional hindgut spheroids; and (c) culturing the three-dimensional hindgut spheroids in a culture medium comprising a BMP inhibitor; a WNT / R-floor plate response protein activator; a receptor tyrosine kinase ligand; and any 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 colivirin to produce three-dimensional intestinal organoids.

16. The method according to claim 15, wherein: In (c), the culture medium further comprises one or more selected from the group consisting of ROCK inhibitor, B27, N-acetyl-L-cysteine ​​(NAC), N2, nicotinamide, gastrin, IGF-1, modulin-1β, bFGF, A-83-01 and SB202190.

17. The method according to claim 1, wherein: When separating the pluripotent stem cell-derived three-dimensional intestinal organoids into the single cells or the small cell aggregates, the intestinal organoids are treated with EDTA, trypsin, or both the EDTA and the trypsin, and dissociated into the single cells or the small cell aggregates.

18. An intestinal stem cell population produced by the culture method according to any one of claims 1 to 17.

19. A method for producing intestinal epithelial cells, the method comprising: (a) Dissociation of pluripotent stem cell-derived 3D intestinal organoids into single cells or small cell aggregates; (b) culturing the single cell or the small cell aggregate in two dimensions in a culture medium comprising a WNT / R-floor plate response protein activator, an activator of a prostaglandin signaling pathway, and a receptor tyrosine kinase ligand to produce an intestinal stem cell population; as well as (c) Intestinal stem cell populations were cultured in a differentiation medium containing an activator of the prostaglandin signaling pathway, a receptor tyrosine kinase ligand, a p38 inhibitor, a WNT / R-floor planarian activator, and nicotinamide via air-liquid interface culture.

20. The method according to claim 19, wherein: The air-liquid interface culture is to culture the intestinal stem cells under a transport well plate coated with an extracellular matrix.

21. The method according to claim 19, wherein: The WNT / R-floor response protein activator is any one or more selected from the group consisting of R-floor response protein 1, R-floor response protein 2, R-floor response protein 3, R-floor response protein 4 and R-floor response protein mimetic substances.

22. The method according to claim 19, wherein: The activator of the prostaglandin signaling pathway is 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).

23. The method according to claim 19, wherein: The receptor tyrosine kinase ligand is any one selected from the group consisting of epidermal growth factor (EGF), transforming growth factor-α (TGF-α), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF).

24. The method according to claim 19, 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.

25. The method of claim 19, wherein: The intestinal epithelial cells show increased expression levels of any one or more markers selected from the group consisting of VIL1, ECAD, FABP1, KRT20, LCT, AXIN2, LYZ and MUC2.

26. The method of claim 19, wherein: The intestinal epithelial cells show increased expression levels of any 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.

27. The method of claim 19, wherein: The intestinal epithelial cells include intestinal cells, mucus-secreting cells, hormone-secreting cells and Paneth cells.

28. An intestinal epithelial cell produced by the production method according to any one of claims 19 to 27. An intestinal epithelial cell model, comprising the intestinal epithelial cell according to claim 28.

30. The intestinal epithelial cell model according to claim 29, wherein The intestinal epithelial cell model has a crypt-villus structure.

31. A drug screening method comprising: (a) infecting the intestinal epithelial cell model according to claim 29 with bacteria or viruses; (b) drug-treated infected intestinal epithelial cell model; and (c) Confirm the response to drug treatment.

32. A drug evaluation method comprising: (a) treating the intestinal epithelial cell model according to claim 29 with a drug; as well as (b) evaluating the absorption rate or bioavailability of the drug in the intestinal epithelial cell model described in (a).

33. A method for providing an intestinal epithelial cell model for disease modeling, the method comprising infecting the intestinal epithelial cell model according to claim 29 with bacteria or viruses.

34. A tissue therapeutic agent comprising the intestinal stem cell population according to claim 18.

35. A pharmaceutical composition for assisting intestinal transplantation, comprising the intestinal stem cell population according to claim 18.

Images