Method for producing intestinal epithelial cells

Abstract

The present invention addresses the problem of providing a means for producing intestinal epithelial cells having a function closer to that of living cells. According to the present invention, a method for producing intestinal epithelial cells from intestinal progenitor cells is provided, the method comprising subjecting the intestinal progenitor cells to (a-1) culturing in the presence of a DNA methyltransferase inhibitor and subsequently to (a-2) culturing in the absence of a DNA methyltransferase inhibitor, wherein the mode of culture is changed from liquid culture to air-liquid interface culture in the middle of a culture period including culture (a-1) and culture (a-2).

Description

Method for producing intestinal epithelial cells

[0001] The present invention relates to a method for producing intestinal epithelial cells.

[0002] Many pharmaceuticals are orally administered, and accurate prediction of intestinal drug absorption and metabolism is crucial for new drug development. Currently, Caco-2 cells derived from human colon carcinoma are widely used as a model system for the small intestine. However, the expression pattern of drug transporters in Caco-2 cells differs from that of the human small intestine. Furthermore, Caco-2 cells show little expression of CYP3A4, a major drug-metabolizing enzyme in the small intestine, and little induction of CYP3A4 by PXR ligands. This makes it difficult to accurately evaluate pharmacokinetics in the small intestine.

[0003] Additionally, there are evaluation systems that use animal models as an evaluation system that is relatively close to the in vivo state in humans, but due to species differences with humans and ethical issues, there is a trend to replace them with alternative methods that do not use animals and to reduce the number of animals used.

[0004] Therefore, to comprehensively evaluate drug metabolism and membrane permeability in the small intestine, it is desirable to use primary small intestinal epithelial cells, but these are difficult to obtain. Therefore, we are conducting research on the induction of differentiation of stem cells and progenitor cells into intestinal cells, aiming to apply this evaluation system.

[0005] Patent Document 1 describes a method for producing intestinal cells, which includes culturing intestinal cells produced from pluripotent stem cells in a medium that does not contain 5-aza-2'-deoxycytidine. Patent Document 1 also describes a method for producing intestinal cells in a medium that does not contain 5-aza-2'-deoxycytidine, in which at least a portion of the culture is performed as an air-liquid phase culture.

[0006] International Publication No. 2022 / 168908

[0007] As described in Patent Document 1, methods for inducing differentiation of stem cells or progenitor cells into intestinal epithelial cell-like cells are known, but there remains a high need for in vitro production of mature intestinal epithelial cells that can be used in pharmacokinetic assays, toxicity tests, etc. Therefore, an objective of the present invention is to provide a means for producing intestinal epithelial cells that have functions more similar to those of living cells.

[0008] In order to solve the above-mentioned problems, the present inventors have improved the culture method of the invention described in Patent Document 1. As a result, the present inventors have found that a method for producing intestinal epithelial cells from intestinal progenitor cells, which comprises culturing intestinal progenitor cells (a-1) in the presence of a DNA methyltransferase inhibitor, followed by culturing intestinal progenitor cells (a-2) in the absence of a DNA methyltransferase inhibitor, and switching from liquid culture to air-liquid interface culture midway through the culture period including the culture of (a-1) and the culture of (a-2), makes it possible to produce mature intestinal epithelial cells with functions more similar to those of in vivo intestinal epithelial cells, thereby completing the present invention.

[0009] Specifically, the present invention relates to the following: [1] A method for preparing intestinal epithelial cells from intestinal progenitor cells, comprising culturing intestinal progenitor cells (a-1) in the presence of a DNA methyltransferase inhibitor, followed by culturing intestinal progenitor cells (a-2) in the absence of a DNA methyltransferase inhibitor, and switching from liquid culture to air-liquid interface culture midway through a culture period including the cultures (a-1) and (a-2). [2] The method according to [1], wherein the culture period for (a-1) is 1 to 12 days, the culture period for (a-2) is 1 to 12 days, the liquid culture period is 1 to 12 days, and the air-liquid interface culture period is 1 to 12 days. [3] The method according to [1] or [2], wherein the culture for (a-1) is liquid culture, and the culture for (a-2) is air-liquid interface culture. [4] The method according to any one of [1] to [3], wherein the DNA methyltransferase inhibitor is 5-aza-2'-deoxycytidine. [5] The method according to any one of [1] to [4], wherein (a-1) and (a-2) comprise culturing in the presence of an epidermal growth factor, a cAMP signal activator, a MEK1 / 2 inhibitor, and a TGFβ receptor inhibitor. [6] The method according to [5], wherein the cAMP signal activator is forskolin, the MEK1 / 2 inhibitor is PD98059, and the TGFβ receptor inhibitor is A8301. [7] The method according to any one of [1] to [6], wherein the intestinal progenitor cells are cells obtained by the following steps: (3) culturing intestinal stem cells in the presence of an epidermal growth factor and a cAMP signal activator. [8] The method according to [7], wherein the culture period in step (3) is 2 to 12 days. [9] The method according to [7] or [8], wherein the intestinal stem cells are cells obtained by the following steps: (1) differentiating pluripotent stem cells into endoderm cells, and (2) differentiating the endoderm cells obtained in step (1) into intestinal stem cells.

[10] The method according to [9], wherein step (1) comprises culturing pluripotent stem cells in the presence of activin A, and step (2) comprises culturing endodermal cells in the presence of epidermal growth factor.

[11] The method according to any one of [1] to

[10] , wherein the intestinal progenitor cells are derived from pluripotent stem cells.

[12] The method according to any one of [1] to

[11] , wherein the pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells.

[13] The method according to any one of [1] to

[11] , wherein the pluripotent stem cells are human induced pluripotent stem cells.

[14] A culture medium for use in the method according to any one of [1] to

[13] , for preparing intestinal epithelial cells, comprising an epidermal growth factor, a cAMP signal activator, a MEK1 / 2 inhibitor, a TGFβ receptor inhibitor, and a DNA methyltransferase inhibitor.

[15] A culture medium for use in the method according to any one of [1] to

[13] , for preparing intestinal epithelial cells, comprising an epidermal growth factor, a cAMP signal activator, a MEK1 / 2 inhibitor, and a TGFβ receptor inhibitor, but not a DNA methyltransferase inhibitor.

[16] A kit comprising the culture medium for preparing intestinal epithelial cells according to

[14] and intestinal progenitor cells.

[17] A kit comprising the culture medium for preparing intestinal epithelial cells according to

[15] and intestinal progenitor cells.

[0010] The present invention also relates to the following:

[18] Intestinal epithelial cells obtained by the method according to any one of [1] to

[13] .

[19] A method for evaluating the pharmacokinetics or toxicity of a test substance using the intestinal epithelial cells according to

[18] .

[20] The method according to

[19] , wherein the pharmacokinetics is metabolism, absorption, excretion, drug interactions, induction of drug-metabolizing enzymes, or induction of drug transporters.

[21] The method according to

[19] or

[20] , comprising the following steps (i) to (iii): (i) preparing a cell layer composed of the intestinal epithelial cells according to

[18] ; (ii) contacting the cell layer with a test substance; and (iii) quantifying the test substance that has permeated the cell layer, and evaluating the absorbability or membrane permeability, drug interactions, induction of drug-metabolizing enzymes, induction of drug transporters, or toxicity of the test substance.

[22] The method according to

[19] or

[20] , comprising the following steps (I) and (II): (I) contacting the intestinal epithelial cells according to

[18] with a test substance; (II) measuring and evaluating the metabolism or absorption, drug interaction, induction of drug-metabolizing enzymes, induction of drug transporters, or toxicity of the test substance.

[23] A cell preparation comprising the intestinal epithelial cells according to

[18] .

[0011] According to the present invention, it is possible to prepare mature intestinal epithelial cells that have functions more similar to those of intestinal epithelial cells in vivo.

[0012] Figure 1 shows the effect of medium and ALI culture on the expression of each gene (SI (Figure 1A), CYP3A4 (Figure 1B), and CES2 (Figure 1C)) (F-hiSIEC®). Mean ± SD (n = 3), human adult small intestine (ASI) = 100, *P < 0.05, **P < 0.01 vs. LL & conv. Medium. LL: liquid culture; ALI: ALI culture; Conv. Medium: medium containing 5-aza-2'-deoxycytidine; New Medium: medium without 5-aza-2'-deoxycytidine. Figure 2 is a heat map of RNA-seq data (F-hiSIEC®), showing the expression of intestinal and hepatic markers. LL: Liquid-liquid culture; ALI: ALI culture; Conv. Medium: Medium containing 5-aza-2'-deoxycytidine; New Medium: Medium without 5-aza-2'-deoxycytidine. Figure 3 shows a heat map of RNA-seq data (F-hiSIEC®) based on transcriptome data comparing small intestinal and large intestinal epithelia (Wang et al., J Exp Med, 217, e20191130, 2020). LL: Liquid-liquid culture; ALI: ALI culture; Conv. Medium: Medium containing 5-aza-2'-deoxycytidine; New Medium: Medium without 5-aza-2'-deoxycytidine. Figure 4 shows the results of GO enrichment analysis of RNA-seq data and gene expression analysis by RT-qPCR. Genes with FDR < 0.05 and 1 < Log2 [TPM of ALI & New medium group / TPM of LL & Conv. medium group] were analyzed. RT-qPCR shows the mean value of n = 4. Figure 5 shows the results of drug-metabolizing enzyme activity evaluation (F-hiSIEC (registered trademark)). Mean ± SD (n = 3-4), **P < 0.01. LL: liquid culture, ALI: ALI culture, Conv. Medium: medium containing 5-aza-2'-deoxycytidine, New Medium: medium without 5-aza-2'-deoxycytidine. Figure 6 shows the F of midazolam. gThe figures show estimated values ​​of . Mean ± SD (n = 3). LL: liquid culture, ALI: ALI culture, Conv. Medium: medium containing 5-aza-2'-deoxycytidine, New Medium: medium without 5-aza-2'-deoxycytidine. Figure 7 shows the effect of the duration of 5-aza-2'-deoxycytidine addition on gene expression (Windy strain). Mean ± SD (n = 3). The following groups were tested: a group without 5-aza-2'-deoxycytidine from days 14 to 26 after the initiation of differentiation (0day5aza); a group with 4 days of 5-aza-2'-deoxycytidine treatment from days 14 to 18 after the initiation of differentiation (4days5aza); a group with 8 days of 5-aza-2'-deoxycytidine treatment from days 14 to 22 after the initiation of differentiation (8days5aza); and a group with 12 days of 5-aza-2'-deoxycytidine treatment from days 14 to 26 after the initiation of differentiation (12days5aza, conventional protocol). After examining these conditions, we determined that the optimal differentiation conditions were 4 days of 5-aza-2'-deoxycytidine treatment from days 14 to 18 after the initiation of differentiation and 8 days of ALI culture from days 18 to 26 after the initiation of differentiation. Figure 8 shows the effect of ALI culture duration on gene expression (Windy strain). Mean ± SD (n = 4). To examine the duration of ALI culture, we prepared three groups: a group without ALI culture (LL), a group with ALI culture from days 22 to 26 after the start of differentiation (4-day ALI), a group with ALI culture from days 18 to 26 after the start of differentiation (8-day ALI), and a group with ALI culture for 12 days from days 14 to 26 after the start of differentiation (12-day ALI). When examining ALI culture, 5 μM 5-aza-2'-deoxycytidine was added for 4 days from days 14 to 18 after the start of differentiation. Figure 9 shows gene expression in cells differentiated under optimized conditions (Windy strain). Mean ± SD (n = 4), human adult small intestine (ASI) = 100, **P < 0.01. Conventional protocol: Conventional protocol. New protocol: 5-aza-2'-deoxycytidine was added for 4 days from day 14 to day 18 after the start of differentiation, and the ALI culture period was 8 days from day 18 to day 26 after the start of differentiation.Figure 10 shows the drug-metabolizing enzyme activity of cells differentiated under optimized conditions (Windy strain). Mean ± SD (n = 4), *P < 0.05, **P < 0.01. Conventional protocol: New protocol. 5-Aza-2'-deoxycytidine was added for 4 days from day 14 to day 18 after the start of differentiation, and ALI culture was performed for 8 days from day 18 to day 26 after the start of differentiation.

[0013] [Method for Producing Intestinal Epithelial Cells from Intestinal Progenitor Cells] In one aspect, the present invention relates to a method for producing intestinal epithelial cells from intestinal progenitor cells (hereinafter, sometimes referred to as the production method of the present invention). The production method of the present invention allows for the production of intestinal epithelial cells having properties similar to those of living cells.

[0014] <Production Method> In one embodiment, the production method of the present invention comprises culturing intestinal progenitor cells (a-1) in the presence of a DNA methyltransferase inhibitor, followed by (a-2) culturing in the absence of a DNA methyltransferase inhibitor, and switching from liquid culture to air-liquid interface culture midway through the culture period including the culture of (a-1) and the culture of (a-2).

[0015] (Culturing in the Presence or Absence of a DNA Methyltransferase Inhibitor) (a-1) "In the presence of a DNA methyltransferase inhibitor" is synonymous with a culture system containing a DNA methyltransferase inhibitor. As used herein, the term "culture system" refers to a system that may include a culture medium, a culture medium, and the like. The culture medium may be any medium suitable for culturing intestinal cells, and may be, for example, Dulbecco's Modified Eagle's Medium (DMEM). In one embodiment, "culturing in the presence of a DNA methyltransferase inhibitor" refers to culturing in a medium supplemented with a DNA methyltransferase inhibitor.

[0016] Examples of DNA methyltransferase inhibitors include 5-aza-2'-deoxycytidine (5aza), 5-azacytidine, RG108, and zebularine, with 5aza being preferred. The concentration of the DNA methyltransferase inhibitor added to the culture system in (a-1) (for 5aza) is typically 1 μM to 25 μM, preferably 2.5 μM to 10 μM. When using a compound other than 5aza, a person skilled in the art can determine the concentration based on the above concentration range, taking into account the characteristics of the compound used and the differences in the properties (particularly differences in activity) of the exemplified compounds. Furthermore, the appropriateness of the determined concentration range can be confirmed by preliminary experiments similar to those described in the Examples below.

[0017] The lower limit of the period (a-1) (culture period), i.e., the period during which intestinal progenitor cells are cultured in the presence of a DNA methyltransferase inhibitor, may be, for example, 1 day or more, preferably 2 days or more, more preferably 3 days or more, and even more preferably 4 days or more, while the upper limit is not particularly limited and may be, for example, 30 days or less, 27 days or less, 24 days or less, 18 days or less, 15 days or less, 12 days or less, 11 days or less, 10 days or less, 9 days or less, or 8 days or less, preferably 7 days or less, more preferably 6 days or less, and even more preferably 5 days or less. Any of the above upper and lower limits of the number of days may be combined to represent the period (range).

[0018] (a-2) is culture in the absence of a DNA methyltransferase inhibitor following the culture of (a-1). "Subsequently" means that the culture of (a-2) is carried out after the culture of (a-1), and an operation such as medium change may be included between (a-1) and (a-2).

[0019] The lower limit of the period (culture period) of (a-2) may be, for example, 1 day or more, 2 days or more, 3 days or more, or 4 days or more, preferably 5 days or more, more preferably 6 days or more, even more preferably 7 days or more, and particularly preferably 8 days or more, while the upper limit is not particularly limited, and may be, for example, 30 days or less, 27 days or less, 24 days or less, 18 days or less, 15 days or less, or 12 days or less, preferably 11 days or less, more preferably 10 days or less, and even more preferably 9 days or less. Any of the above upper and lower limits of the number of days may be combined to represent the period (range).

[0020] (Liquid Culture and Air-Liquid Interface Culture) "Liquid culture" (sometimes called liquid-liquid culture) refers to culturing cells in a liquid culture system without contact with gas. Examples of liquid culture methods include seeding cells in a culture vessel equipped with a cell-adhesive culture surface and culturing them in liquid medium after adhesion, but this is not limiting as long as the method cultures cells in a liquid culture system without contact with gas. The liquid medium may be any suitable liquid medium for culturing intestinal cells, such as DMEM.

[0021] The lower limit of the liquid culture period may be, for example, 1 day or more, preferably 2 days or more, more preferably 3 days or more, and even more preferably 4 days or more, while the upper limit is not particularly limited and may be, for example, 30 days or less, 27 days or less, 24 days or less, 21 days or less, 18 days or less, 15 days or less, 12 days or less, 11 days or less, 10 days or less, or 9 days or less, preferably 8 days or less, more preferably 7 days or less, even more preferably 6 days or less, and particularly preferably 5 days or less. Any of the above upper and lower limits of the number of days may be combined to represent the period (range).

[0022] "Changing from liquid culture to air-liquid interface culture" means that air-liquid interface culture is performed after liquid culture, but operations such as medium exchange and cell adhesion to the culture surface may be included between liquid culture and air-liquid interface culture.

[0023] "Air-Liquid Interface Culture" (ALI, also referred to as "Air-Liquid Interface Culture" or "Air-Liquid Interface Culture") refers to culture in which only a portion of the cell surface is in contact with the gas phase, rather than the entire cell surface (excluding the portion attached to the culture surface and the portion in contact with or attached to other cells) being in contact with the culture medium. In this specification, the term "contact with the gas phase" refers to a state in which the medium remains between the cell surface and the gas phase after the medium is removed, or the medium permeates from the liquid phase to the gas phase, i.e., an unintended small amount of medium is present between the cell surface and the gas phase. The method of the present invention can be used with any culture vessel equipped with a cell-adhesive culture surface, although this is not particularly limited. The cell-adhesive culture surface is preferably coated with a basement membrane component (e.g., laminin, type IV collagen, entactin, heparan sulfate proteoglycan, vitronectin, fibronectin) or a fragment thereof. In particular, a culture surface coated with laminin-511 or its E8 fragment is recommended. A highly purified recombinant form of the E8 fragment of laminin-511 (recombinant human laminin-511-E8 protein) is commercially available (product name: iMatrix-511, manufactured by Nippi Corporation and sold by Matrixome Co., Ltd.). The culture vessel is not particularly limited, and cell culture inserts, dishes, flasks, multiwell plates, etc. can be used. Culture can also be performed on an organ-on-a-chip (Microphysiological System, MPS: biomimetic system).

[0024] In one embodiment, cells are cultured on a semi-permeable membrane (porous membrane) to form a cell layer. Specifically, for example, a culture vessel equipped with an insert (e.g., Transwell (registered trademark) from Corning) is used, and cells are seeded and cultured in the insert to obtain a cell layer. According to this embodiment, a cell layer suitable for use in an evaluation system in which evaluation is performed by quantifying a test substance that has permeated the cell layer is obtained.

[0025] Air-liquid interface culture can be initiated, for example, by using a culture vessel equipped with an insert (typically consisting of an insert and a well plate that serves as the insert), seeding cells into the insert, and removing the culture medium from the insert after the cells have adhered (preferably after a cell layer has formed), resulting in a state in which an air phase exists above the cells and a liquid phase (culture medium) exists below the cells (i.e., the side that is adhered to the insert). Alternatively, instead of removing the culture medium from the insert, air-liquid interface culture can be performed by removing the culture medium from the well, resulting in a state in which a liquid phase exists above the cells and an air phase exists below the cells.

[0026] The lower limit of the period of air-liquid interface culture may be, for example, 1 day or more, 2 days or more, or 3 days or more, preferably 4 days or more, more preferably 5 days or more, even more preferably 6 days or more, particularly preferably 7 days or more, and most preferably 8 days or more, while the upper limit is not particularly limited, and may be, for example, 30 days or less, 27 days or less, 24 days or less, 21 days or less, 18 days or less, 15 days or less, 12 days or less, preferably 11 days or less, more preferably 10 days or less, and even more preferably 9 days or less. Any of the above upper and lower limits of the number of days may be combined to represent the period (range).

[0027] Other culture conditions for the culture of (a-1) and the culture of (a-2) may be conditions commonly employed in the culture of animal cells, such as 37°C and 5% CO2. A preferred example of the basal medium is DMEM / F12 (a mixture of DMEM and Ham's F12 medium). The culture system may contain appropriate additives, such as antibiotics, non-essential amino acids (NEAA), and L-glutamine.

[0028] The period of (a-1) and (a-2) (the sum of the period of (a-1) and the period of (a-2)) may be, for example, 7 to 60 days, preferably 8 to 45 days, more preferably 9 to 30 days, and even more preferably 10 to 15 days. In one preferred embodiment, the culture of (a-1) is liquid culture, and the culture of (a-2) is air-liquid interface culture.

[0029] (Culturing in the presence of epidermal growth factor, cAMP signal activator, MEK1 / 2 inhibitor, and TGFβ receptor inhibitor) The culture (a-1) and the culture (a-2) may comprise culturing in the presence of one or more selected from the group consisting of epidermal growth factor, cAMP signal activator, MEK1 / 2 inhibitor, and TGFβ receptor inhibitor. In one embodiment, "culturing in the presence of one or more selected from the group consisting of epidermal growth factor, cAMP signal activator, MEK1 / 2 inhibitor, and TGFβ receptor inhibitor" refers to culturing in a medium supplemented with one or more selected from the group consisting of epidermal growth factor, cAMP signal activator, MEK1 / 2 inhibitor, and TGFβ receptor inhibitor.

[0030] The concentration of epidermal growth factor (EGF, also known as epidermal growth factor, epidermal cell growth factor, epidermal cell growth factor, etc.) added to the culture system may be, for example, 1 to 500 ng / mL, preferably 3 to 200 ng / mL, more preferably 5 to 100 ng / mL, even more preferably 10 to 50 ng / mL, and particularly preferably 15 to 30 ng / mL. In some embodiments, a peptide compound having activity equivalent to or greater than that of epidermal growth factor may be used in place of epidermal growth factor. Furthermore, peptide compounds having activity equivalent to or greater than that of not only epidermal growth factor but also each of the factors described herein (including factors, inhibitors, ligands, cytokines, etc.) can also be used. Such peptide compounds, also referred to as alternative peptide compounds, are expected to be developed in the future, and it is naturally anticipated that they will be used by those skilled in the art.

[0031] The cAMP signal activator may be one or more selected from the group consisting of cAMP derivatives, cAMP degrading enzyme inhibitors and cAMP activators, and is preferably a cAMP activator.

[0032] As cAMP derivatives, for example, PKA activators (e.g., 8-Br-cAMP (8-Bromoadenosine-3′,5′-cyclic monophosphate sodium salt, CAS Number : 76939-46-3), 6-Bnz-cAMP (N6-Benzoyladenosine-3',5'-cyclic monophosphate sodium salt salt, CAS Number : 1135306-29-4), cAMPS-Rp ((R)-Adenosine, cyclic 3',5'-(hydrogenphosphorothioate)triethylammonium salt, CAS Number : 151837-09-1), cAMPS-Sp ((S)-Adenosine, cyclic 3',5'-(hydrogenphosphorothioate)triethylammonium salt, CAS Number : 93602-66-5), Dibutyryl-cAMP (N6,O2'-Dibutyryl adenosine 3',5'-cyclic monophosphate sodium salt salt, CAS Number : 16980-89-5), 8-Cl-cAMP (8-Chloroadenosine- 3', 5'- cyclic monophosphate salt, CAS Number : 124705-03-9)), Epac activators (Rp-8-Br-cAMPS (8-Bromoadenosine 3',5'-cyclic Monophosphothioate, Rp-Isomer).Examples of cAMP derivatives that can be used include 8-CPT-cAMP (8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate sodium salt, CAS Number: 129735-00-8), 8-CPT-cAMP (8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate monosodium, CAS Number: 93882-12-3), and 8-pCPT-2'-O-Me-cAMP (8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium, CAS Number: 634207-53-7). The concentration of the cAMP derivative added (in the case of 8-Br-cAMP) is, for example, 0.1 mM to 10 mM, preferably 0.2 mM to 5 mM, and more preferably 0.5 mM to 2 mM. When using the exemplified compounds, i.e., compounds other than 8-Br-cAMP, the concentration of the compound added can be determined by a person skilled in the art based on the above concentration range, taking into account the differences (particularly the activity) between the properties of the compound used and the exemplified compound (8-Br-cAMP). Furthermore, whether the selected concentration range is appropriate can be confirmed by preliminary experiments.

[0033] cAMP degrading enzyme inhibitors include IBMX (3-isobutyl-1-methylxanthine) (MIX), Theophylline, Papaverine, Pentoxifylline (Trental), KS-505 , 8-Methoxymethyl-IBMX, Vinpocetine (TCV-3B), EHNA, Trequinsin (HL-725), Lixazinone (RS-82856), (LY-186126), Cilostamide (OPC3689), Bemoradan (RWJ-22867), Anergrelide (BL4162A), Indolidan (LY195115), Cilostazol (OPC-13013), Milrinone (WIN47203), Siguazodan (SKF-94836), 5-Methyl-imazodan (CI 930), SKF-95654, Pirilobendan (UD-CG Examples of cAMP inhibitors include AR-L 115 BS), Enoximone (MDL 17043), Imazodan (CL 914), SKF-94120, Vesnarinone (OPC 8212), Rolipram (Ro-20-1724), (ZK-62711), Denbufylline, Zaprinast (M&B-22,948), Dipyridamole, Zaprinast (M&B-22,948), Dipyridamole, Zardaverine, AH-21-132, and Sulmazol (AR-L 115 BS). The concentration of the cAMP inhibitor (in the case of IBMX) is typically 0.05 mM to 5 mM, preferably 0.1 mM to 3 mM, and more preferably 0.2 mM to 1 mM. When using an exemplified compound, i.e., a compound other than IBMX, the concentration of the compound to be added can be determined by a person skilled in the art in accordance with the above concentration range, taking into consideration the differences (particularly the differences in activity) between the properties of the compound to be used and the properties of the exemplified compound (IBMX). Whether the set concentration range is appropriate can be confirmed by a preliminary experiment.

[0034] Examples of cAMP activators that can be used include forskolin, indomethacin, NKH477 (colforsin daropate), cell-derived toxic proteins (pertussis toxin, cholera toxin), PACAP-27, PACAP-38, and SKF83822. The concentration of a cAMP activator added to a culture system (forskolin) can range from 1 μM to 200 μM, preferably from 5 μM to 100 μM, more preferably from 10 μM to 50 μM, even more preferably from 20 μM to 40 μM, and particularly preferably from 30 μM. When using an exemplified compound, i.e., a compound other than forskolin, the concentration can be determined within the above concentration range by one skilled in the art, taking into account the differences in the properties (particularly activity) between the compound used and the exemplified compound (forskolin). The appropriateness of the selected concentration range can be confirmed by preliminary experiments similar to those described in the Examples below.

[0035] Examples of MEK1 / 2 inhibitors include PD98059, PD184352, PD184161, PD0325901, U0126, MEK inhibitor I, MEK inhibitor II, MEK1 / 2 inhibitor II, and SL327, with PD98059 being preferred. The concentration of the MEK1 / 2 inhibitor added to the culture system (in the case of PD98059) may be, for example, 4 μM to 100 μM, preferably 10 μM to 40 μM. When using a compound other than PD98059, the concentration can be determined by a person skilled in the art based on the above concentration range, taking into account the characteristics of the compound used and the differences in the properties of the exemplified compounds (particularly differences in activity). Whether the set concentration range is appropriate can be confirmed by a preliminary experiment based on the examples described below.

[0036] The TGFβ receptor inhibitor preferably exhibits inhibitory activity against one or more of the TGF-β receptors ALK4, ALK5, and ALK7. Examples include A-83-01, SB431542, SB-505124, SB525334, D4476, ALK5 inhibitor, LY2157299, LY364947, GW788388, and RepSox, with A-83-01 being particularly preferred. Alternatively, a peptide compound with TGFβ inhibitory activity may be used instead of the TGFβ receptor inhibitor. The peptide compound with TGFβ inhibitory activity may be a TGFβ inhibitory peptide, i.e., one that binds to TGFβ and inhibits the binding of TGFβ to the TGFβ receptor, or a TGFβ receptor inhibitory peptide, i.e., one that binds to the TGFβ receptor and inhibits the binding of TGFβ to the TGFβ receptor. Examples of TGFβ inhibitory peptides include the TGFβ1 inhibitor peptide PG-002 (PeptiGrowth). The concentration of the TGFβ receptor inhibitor added to the culture system (in the case of A-83-01) may be, for example, 0.1 μM to 2.5 μM, preferably 0.2 μM to 1 μM. When using a compound other than A-83-01, the concentration can be determined by a person skilled in the art based on the above concentration range, taking into account the characteristics of the compound used and the differences in the characteristics (particularly differences in activity) of the exemplified compounds. Furthermore, whether the set concentration range is appropriate can be confirmed by a preliminary experiment based on the examples described below.

[0037] In one preferred embodiment, the culture of (a-1) and the culture of (a-2) comprise culture in the presence of epidermal growth factor, a cAMP signal activator, a MEK1 / 2 inhibitor, and a TGFβ receptor inhibitor. In another preferred embodiment, the culture of (a-1) and the culture of (a-2) are culture in the presence of epidermal growth factor, a cAMP signal activator, a MEK1 / 2 inhibitor, and a TGFβ receptor inhibitor, wherein the cAMP signal activator is forskolin, the MEK1 inhibitor is PD98059, and the TGFβ receptor inhibitor is A8301.

[0038] <Intestinal progenitor cells> In the production method of the present invention, intestinal progenitor cells can be used. As used herein, "intestinal progenitor cells" refers to cells that partially function as intestinal epithelial cells but are more immature than intestinal epithelial cells, and that have limited differentiation and self-renewal capabilities compared to stem cells. Self-renewal capability refers to the ability to produce daughter cells with the same differentiation capabilities as the cell itself through cell division. The intestinal progenitor cells used in the production method of the present invention may be obtained by any method known to those skilled in the art, and may be, for example, intestinal progenitor cells derived from a living organism, mesenchymal stem cells, or pluripotent stem cells.

[0039] Intestinal progenitor cells can be identified by methods known to those skilled in the art. Cell markers may be used as indicators for identification, for example, in the method using RNA sequence analysis shown in the Examples section, when the Z-score of one or more, two or more, three or more, four or more, or five or more of the intestinal epithelial cell markers and one or more, two or more, three or more, or four or more of the intestinal stem cell markers is greater than 0; when the Z-score of the intestinal epithelial cell marker is smaller than that of the intestinal epithelial cells and the Z-score of the intestinal stem cell marker is larger than that of the intestinal stem cells; when the Z-score of the intestinal epithelial cell marker is larger than that of the intestinal stem cells and the Z-score of the intestinal stem cell marker is smaller than that of the intestinal stem cells, and the Z-score of the intestinal stem cell marker is smaller.

[0040] F-hiSIEC (registered trademark) may be used as the intestinal progenitor cells. F-hiSIEC (registered trademark) can be purchased from Fujifilm Corporation. F-hiSIEC (registered trademark) is preferably thawed and seeded in a culture vessel or the like. When seeding F-hiSIEC (registered trademark), F-hiSIEC (registered trademark) Seeding medium can be used, and for subsequent culture, F-hiSIEC (registered trademark) Culture medium can be used. F-hiSIEC (registered trademark) Seeding medium and F-hiSIEC (registered trademark) Culture medium can be purchased from Fujifilm Corporation.

[0041] (Step (3): Differentiation into intestinal progenitor cells) The intestinal progenitor cells may be obtained by any method known to those skilled in the art. In one embodiment, the intestinal progenitor cells may be, for example, cells obtained by the following step (3): (3) A step of culturing intestinal stem cells in the presence of epidermal growth factor and a cAMP signal activator.

[0042] Step (3) can be understood as "a step of inducing differentiation into intestinal epithelial cells while maintaining stemness." "In the presence of epidermal growth factor and cAMP signal activator" is synonymous with a culture system containing these. In one embodiment, "culturing in the presence of epidermal growth factor and cAMP signal activator" refers to culturing in a medium supplemented with epidermal growth factor and cAMP signal activator.

[0043] The types and concentrations of the epidermal growth factor and cAMP signal activator can be appropriately selected in the same manner as in (a-1) and (a-2).

[0044] The period (culture period) for step (3) may be, for example, 2 to 12 days, preferably 3 to 11 days, more preferably 4 to 10 days, and even more preferably 5 to 9 days. If this period is too short, the differentiation of intestinal stem cells into intestinal progenitor cells will not progress, and the method of the present invention will not be suitable for producing mature intestinal epithelial cells. Note that this period can be set longer by repeating the passaging procedure.

[0045] In a preferred embodiment, a passaging procedure is performed in step (3). That is, subculture is performed during step (3). The passaging procedure is considered to be particularly effective for purifying intestinal epithelial stem cells (improving purity and uniformity) and for more selectively inducing differentiation into intestinal epithelial cells.

[0046] Passaging may be performed, for example, once every 24 hours to once every three days. Too many passaging procedures can result in significant damage to the cells, leading to a decrease in production efficiency. Passaging procedures can be performed according to standard procedures. For example, when the cells reach confluence or subconfluence, a portion of the cells can be harvested and transferred to another culture vessel, and the culture can be continued. Cells can be recovered using a cell dissociation solution. Examples of cell dissociation solutions include proteolytic enzymes such as trypsin-EDTA, collagenase IV, and metalloproteases, which can be used alone or in combination. Those with minimal cytotoxicity are preferred. Commercially available cell dissociation solutions include Dispase (Eidea), TrypLE (Invitrogen), and Accutase (MILLIPORE).

[0047] <Intestinal stem cells> Intestinal stem cells obtained by any method known to those skilled in the art can be used. Intestinal stem cells may be, for example, intestinal stem cells derived from a living body, mesenchymal stem cells, or pluripotent stem cells. As used herein, "intestinal stem cells" refer to cells that have the ability to differentiate into intestinal epithelial cells such as absorptive epithelial cells, goblet cells, and Paneth cells, and also have the ability to self-replicate.

[0048] Intestinal stem cells can be determined or evaluated, for example, using the expression of intestinal stem cell markers as an indicator. For example, in the method using RNA sequencing analysis described in the Examples section, intestinal stem cells can be determined or evaluated when the Z-score of the intestinal stem cell marker is higher than that of other cells, including intestinal epithelial cells, or is greater than 0. Examples of intestinal stem cell markers include leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), ephrin B2 receptor (EphB2), OLFM4, ASCL2, BMI1, MSI1, etc.

[0049] In one embodiment, the intestinal stem cells may be cells obtained by the following steps: (1) differentiating pluripotent stem cells into endoderm cells, and (2) differentiating the endoderm cells obtained in step (1) into intestinal stem cells.

[0050] (Step (1): Differentiation into Endodermal Cells) In this step, pluripotent stem cells are cultured and differentiated into endoderm cells. In other words, pluripotent stem cells are cultured under conditions that induce differentiation into endoderm cells. The culture conditions are not particularly limited, as long as the pluripotent stem cells differentiate into endoderm cells. For example, the cells may be cultured in a medium supplemented with activin A according to a conventional method. In this case, the concentration of activin A in the medium can be, for example, 10 ng / mL to 200 ng / mL, preferably 20 ng / mL to 150 ng / mL, and more preferably 30 ng / mL to 120 ng / mL. Alternatively, a peptide compound having activity equivalent to or greater than that of activin A may be used instead of activin A. From the viewpoint of cell proliferation rate and maintenance, it is preferable to further add serum or a serum substitute (e.g., knockout serum replacement (KSR)) to the medium. The serum is not limited to fetal bovine serum (FBS), and human serum, sheep serum, etc. can also be used. The amount of serum or serum substitute added may be, for example, 0.1% (v / v) to 10% (v / v).

[0051] Wnt ligands (e.g., Wnt-3a, peptide compounds with activity equivalent to or greater than that of Wnt-3a, etc.) or GSK-3 inhibitors (e.g., CHIR99021, CHIR98014, BIO, SB415286, SB216763, TWS119, A1070722, etc.) may be added to the culture medium to promote differentiation into endoderm.

[0052] Step (1) may involve two or more stages of culture. The first stage of culture is carried out in a serum-free medium or a medium containing a relatively low concentration of serum (e.g., 0.1% (v / v) to 1% (v / v)), and the subsequent second and subsequent stages of culture are carried out in a medium containing a higher serum concentration than that of the first stage of culture (e.g., 1% (v / v) to 10% (v / v)). Using such two or more stages of culture is preferable because the first stage of culture suppresses the proliferation of undifferentiated cells, and the subsequent second and subsequent stages allow the proliferation of differentiated cells.

[0053] The period (culture period) of step (1) is, for example, 1 to 10 days, preferably 2 to 7 days. When two-stage culture is employed in step (1), the culture period of the first stage can be, for example, 1 to 7 days, preferably 2 to 5 days, and the culture period of the second stage can be, for example, 1 to 6 days, preferably 1 to 4 days.

[0054] (Step (2): Differentiation into Intestinal Stem Cells) In this step, the endoderm cells obtained in step (1) are cultured and differentiated into intestinal stem cells. In other words, the endoderm cells are cultured under conditions that induce differentiation into intestinal stem cells. The culture conditions are not particularly limited as long as the endoderm cells differentiate into intestinal stem cells. In step (2), the culture is preferably performed in the presence of FGF2 (fibroblast growth factor 2). As FGF2, for example, human FGF2 (e.g., human recombinant FGF2) or a peptide compound having activity equivalent to or greater than that of FGF2 can be used. The culture can also be performed in the presence of FGF4 (fibroblast growth factor 4) or a peptide compound having activity equivalent to or greater than that of FGF2. Step (2) may also include culture in a culture system further containing a GSK-3 inhibitor (e.g., CHIR99021, CHIR98014, BIO, SB415286, SB216763, TWS119, A1070722, etc.).

[0055] Typically, the cell population obtained through step (1) or a portion thereof is subjected to step (2) without selection. Alternatively, endoderm cells may be selected from the cell population obtained through step (1) before step (2) is performed. The selection of endoderm cells may be performed, for example, using a flow cytometer (cell sorter) using cell surface markers as indicators.

[0056] "In the presence of FGF2" is synonymous with a culture system containing FGF2. In one embodiment, "culturing in the presence of FGF2" refers to culturing in a medium supplemented with FGF2. An example of the concentration of FGF2 added to a culture system is 50 ng / mL to 2.5 μg / mL, preferably 150 ng / mL to 500 ng / mL.

[0057] An example of the concentration of FGF4 added to the culture system is 50 ng / mL to 2.5 μg / mL, preferably 150 ng / mL to 500 ng / mL. An example of the concentration of a GSK-3 inhibitor added to the culture system (in the case of CHIR99021) is 600 nM to 60 μM, preferably 1 μM to 20 μM. When using an exemplified compound, i.e., a compound different from CHIR99021, the concentration to be added can be determined by one skilled in the art based on the above concentration range, taking into account the characteristics of the compound used and the differences in the characteristics of the exemplified compound (particularly differences in activity). Whether the set concentration range is appropriate can be confirmed by a preliminary experiment based on the examples described below.

[0058] The period of step (2) (culture period) may be, for example, 1 to 10 days, preferably 2 to 8 days, and more preferably 3 to 6 days. If the culture period is too short, the expected effects (increased differentiation efficiency, promotion of acquisition of intestinal stem cell functions) will not be fully achieved. On the other hand, if the culture period is too long, differentiation efficiency will decrease.

[0059] Subculture may be performed during steps (1) and (2). For example, when the cells reach confluence or subconfluence, a portion of the cells may be harvested and transferred to another culture vessel, and the culture may continue. Cells may be recovered using a cell dissociation solution or the like. Examples of cell dissociation solutions include proteolytic enzymes such as trypsin-EDTA, collagenase IV, and metalloproteases, which may be used alone or in combination. Preferably, these solutions are less cytotoxic. Commercially available examples of such cell dissociation solutions include Dispase (Eidia), TrypLE (Invitrogen), and Accutase (MILLIPORE). The recovered cells may be treated with a cell strainer or the like to obtain a dispersed (dispersed) state before being subcultured. Meanwhile, in each step of the present invention, medium changes may be performed as needed. For example, medium changes may be performed every 24 hours to every three days.

[0060] <Intestinal epithelial cells> As used herein, the term "intestinal epithelial cells" refers to intestinal epithelial cells and cells having similar properties. Whether intestinal epithelial cells have been obtained by the production method of the present invention can be determined or evaluated, for example, by using as an index the expression of intestinal epithelial cell markers, peptide uptake, or the induction of vitamin D receptor-mediated expression of drug-metabolizing enzymes in the obtained cells. Examples of intestinal epithelial cell markers include ATP-binding cassette transporter B1 / multidrug resistance protein 1 (ABCB1 / MDR1), ATP-binding cassette transporter G2 / breast cancer resistance protein (ABCG2 / BCRP), cytochrome P450 3A4 (CYP3A4), cytochrome P450 2C9 (CYP2C9), cytochrome P450 2C19 (CYP2C19), cytochrome P450 2D6 (CYP2D6), fatty acid binding protein 2 (FABP), pregnane X receptor (PXR), solute carrier (SLC) family member 5A1 / sodium-coupled glucose transporter 1 (SLC5A1 / SGLT1), solute carrier (SLC) family member 15A1 / peptide transporter 1 (SLC15A1 / PEPT1), and solute carrier (SLC) family member 20A1 / sodium-coupled glucose transporter 1 (SLC20A1 / SGLT2). These include sucrase-isomaltase (SI), uridine diphosphate-glucuronosyltransferase 1A1 (UGT1A1), uridine diphosphate-glucuronosyltransferase 1A4 (UGT1A4), villin 1, and carboxylesterase 2A1 (CES2A1). Among these, the following are particularly effective markers: sucrase-isomaltase, which is highly specific to the intestinal epithelium; CYP3A4, a major drug-metabolizing enzyme in the small intestine; SLC15A1 / PEPT1, which is involved in peptide absorption in the small intestine; SLC5A1 / SGLT1, a glucose transporter expressed on the apical membrane of the small intestine; SLCO2B1 / OATP2B1, which is involved in organic anion absorption in the small intestine; and CES2A1, a hydrolase highly expressed in the small intestine.

[0061] To obtain a cell population consisting only of target cells (intestinal epithelial cells) or a cell population containing a high proportion (high purity) of target cells, the cell population after culture can be selected and separated using cell surface markers characteristic of the target cells as an indicator.

[0062] Furthermore, if the expression of genes that serve as intestinal epithelial cell markers is increased compared to a control, it can be determined or evaluated that more mature intestinal epithelial cells have been obtained. Furthermore, if the activity of drug-metabolizing enzymes in the small intestine is increased compared to a control, it can also be determined or evaluated that mature intestinal epithelial cells have been obtained. Examples of drug-metabolizing enzymes in the small intestine include CYP3A4 / 5, CYP2C9, CYP2C19, and CYP2D6. Substrates for each enzyme are known to those skilled in the art, and they can be used to measure the activity of drug-metabolizing enzymes.

[0063] In one embodiment, absorptive epithelial cells are obtained by the production method of the present invention. Absorptive epithelial cells are intestinal epithelial cells that are responsible for absorbing nutrients and water from the intestinal lumen. As used herein, "absorptive epithelial cells" refers to absorptive epithelial cells and cells with similar properties. The fact that absorptive epithelial cells have been obtained by the production method of the present invention can be determined or evaluated, for example, by using the induction of expression of genes related to nutrient absorption in the obtained cells as an indicator. Examples of genes related to nutrient absorption include CD36, ENPP7, APOA4, APOA1, ABCG8, KCNQ1, and SLC5A1.

[0064] In one embodiment, the present invention relates to intestinal epithelial cells obtained by the production method of the present invention. The cells obtained by the production method of the present invention are more mature intestinal epithelial cells than those obtained without culture in the absence of a DNA methyltransferase inhibitor or without air-liquid interface culture. The intestinal epithelial cells obtained by the production method of the present invention exhibit increased expression levels of one or more mRNAs selected from the group consisting of SI, CYP3A4, CYP2C9, CYP2C19, CES2, SGLT1, BCRP, and MDR1, preferably by 3% or more, 5% or more, 10% or more, or 15% or more, more preferably 20% or more, and even more preferably 30% or more, compared to intestinal epithelial cells produced by a culture method that does not include air-liquid interface culture. Those skilled in the art can measure mRNA expression levels using known methods, such as real-time RT-PCR.

[0065] The intestinal epithelial cells obtained by the method of the present invention have an Fg value similar to that of living humans. The Fg value represents the proportion of a drug that avoids metabolism in the small intestine, and is sometimes referred to as small intestinal availability. For example, under certain conditions, the Fg value is 1.0 × 10 5 The test compound is permeated from the apical to the basal side of intestinal epithelial cells at a density of 100-150 cells, and the amounts of the test compound (substrate) and its metabolite (metabolite) measured before and after permeation are calculated using the following formula:

[0066]

[0067] The predetermined conditions may be, for example, seeding cells into the insert, pre-incubating, then replacing the apical medium with medium containing the test compound, and measuring the amounts of the test compound and its metabolites on the apical and basal sides 60 minutes later. Measurement of the compound and metabolite amounts may be performed, for example, by LC-MS / MS. The test compound may be, for example, midazolam, which is metabolized by CYP3A4. In this case, the concentration of midazolam contained in the apical medium may be, for example, 5 μM. The metabolite of midazolam may be 1'-hydroxymidazolam. In one preferred embodiment, the intestinal epithelial cells obtained by the production method of the present invention have an Fg value for midazolam of 0.8 or less, preferably 0.7 or less, and more preferably 0.67 or less, which may be close to the Fg value of 0.55 in the human body.

[0068] <Pluripotent stem cells> "Pluripotent stem cells" refer to cells that have both the ability to differentiate into all cells that make up a living organism (pluripotency) and the ability to produce daughter cells with the same differentiation potential through cell division (self-renewal ability). Pluripotency can be evaluated by transplanting the cells to be evaluated into nude mice and testing for the presence or absence of teratoma formation containing cells from each of the three germ layers (ectoderm, mesoderm, and endoderm).

[0069] Examples of pluripotent stem cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), and induced pluripotent stem cells (iPS cells), but are not limited to these as long as they are cells that have both pluripotency and self-renewal capabilities. ES cells or iPS cells are preferably used. iPS cells are more preferably used. Pluripotent stem cells are preferably mammalian (e.g., primates such as humans, chimpanzees, and cynomolgus monkeys, and rodents such as mice and rats), and particularly preferably human cells.

[0070] ES cells can be established, for example, by culturing preimplantation early embryos, the inner cell mass that constitutes the early embryo, single blastomeres, etc. (Manipulating the Mouse Embryo: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994); Thomson, J.A. et al., Science, 282, 1145-1147 (1998)). As the early embryo, an early embryo produced by nuclear transfer of a nucleus from a somatic cell may be used (Wilmut et al. (Nature, 385, 810 (1997)), Cibelli et al. (Science, 280, 1256 (1998)), Iritani A. et al. (Protein, Nucleic Acid, Enzyme, 44, 892 (1999)), Baguisi et al. (Nature Biotechnology, 17, 456 (1999)), Wakayama et al. (Nature, 394, 369 (1998); Nature Genetics, 22, 127 (1999); Proc. Natl. Acad. Sci. USA, 96, 14984 (1999)), Rideout III et al. (Nature Genetics, 24, 109 (2000), Tachibana et al. (Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer, Cell, 153, 1228-1238 (2013)). Parthenogenetic embryos may also be used as initial loci (Kim et al. (Science, 315, 482-486 (2007)), Nakajima et al. (Stem Cells, 25, 983-985 (2007)), Kim et al. (Cell Stem Cell, 1, 346-352 (2007)), Revazova et al. (Cloning Stem Cells, 9, 432-449 (2007)), Revazova et al. (Cloning Stem Cells, 10, 11-24 (2008))).In addition to the above-mentioned papers, for the production of ES cells, references that can be made include Strelchenko N., et al. Reprod Biomed Online. 9: 623-629, 2004; Klimanskaya I., et al. Nature 444: 481-485, 2006; Chung Y., et al. Cell Stem Cell 2: 113-117, 2008; Zhang X., et al. Stem Cells 24: 2669-2676, 2006; Wassarman PM et al. Methods in Enzymology, Vol. 365, 2003. Note that fused ES cells obtained by cell fusion of ES cells and somatic cells are also included in the embryonic stem cells used in the production method of the present invention.

[0071] Some ES cells are available from repositories or commercially available, such as human ES cells from the Institute for Frontier Medical Sciences, Kyoto University (e.g., KhES-1, KhES-2, and KhES-3), WiCell Research Institute, and ESI BIO.

[0072] ES cells can be established by culturing primordial germ cells in the presence of LIF, bFGF, and SCF (Matsui et al., Cell, 70, 841-847 (1992); Shamblott et al., Proc. Natl. Acad. Sci. USA, 95(23), 13726-13731 (1998); Turnpenny et al., Stem Cells, 21(5), 598-609 (2003)).

[0073] "Induced pluripotent stem cells (iPS cells)" are cells with pluripotency (multipotency) and proliferation capacity that are produced by reprogramming somatic cells, for example, by introducing reprogramming factors. Induced pluripotent stem cells exhibit properties similar to ES cells. The somatic cells used to produce iPS cells are not particularly limited, and may be differentiated somatic cells or undifferentiated stem cells. Their origin is also not particularly limited, but somatic cells from mammals (e.g., primates such as humans, chimpanzees, and cynomolgus monkeys, and rodents such as mice and rats), with human somatic cells being particularly preferred. iPS cells can be produced by various methods reported to date. Furthermore, it is naturally anticipated that future iPS cell production methods will also be applied.

[0074] Using iPS cells derived from patients with intestinal diseases (iPS cells prepared from the patient's somatic cells) makes it possible to generate disease-specific intestinal epithelial cells. These iPS cells are prepared from somatic cells (e.g., skin, blood, mononuclear cells, etc.) collected from the patient. Examples of intestinal diseases include intractable inflammatory bowel diseases (Crohn's disease, ulcerative colitis), polyps, colon cancer, and drug-induced enteritis. Disease-specific intestinal epithelial cells are useful as models of intestinal pathology and are expected to be used in drug evaluation systems and contribute to elucidating disease mechanisms (e.g., molecular mechanisms related to onset, pathogenesis, and progression).

[0075] The most basic method for generating iPS cells is to introduce four transcription factors, Oct3 / 4, Sox2, Klf4, and c-Myc, into cells using a virus (Takahashi K, Yamanaka S: Cell 126(4), 663-676, 2006; Takahashi, K, et al: Cell 131(5), 861-72, 2007). Human iPS cells have been reported to be established by introducing four factors: Oct4, Sox2, Lin28, and onog (Yu J, et al: Science 318(5858), 1917-1920, 2007). iPS cells have also been established by introducing three factors other than c-Myc (Nakagawa M, et al.: Nat. Biotechnol. 26(1), 101-106, 2008), two factors Oct3 / 4 and Klf4 (Kim JB, et al.: Nature 454(7204), 646-650, 2008), or only Oct3 / 4 (Kim JB, et al.: Cell 136(3), 411-419, 2009). In addition, methods for introducing proteins, the product of gene expression, into cells have also been reported (Zhou H, Wu S, Joo JY, et al.: Cell Stem Cell 4, 381-384, 2009; Kim D, Kim CH, Moon JI, et al.: Cell Stem Cell 4, 472-476, 2009). On the other hand, it has been reported that the use of histone methyltransferase G9a inhibitor BIX-01294, or histone deacetylase inhibitors such as valproic acid (VPA) or BayK8644 can improve production efficiency and reduce the amount of factors introduced (Huangfu D, et al.: Nat. Biotechnol. 26(7), 795-797, 2008; Huangfu D, et al.: Nat. Biotechnol. 26(11), 1269-1275, 2008; Silva J, et al.: PLoS. Biol. 6(10), e 253, 2008).Gene transfer methods have also been investigated, and in addition to retroviruses, lentiviruses (Yu J, et al: Science 318 (5858), 1917-1920, 2007), adenoviruses (Stadtfeld M, et al: Science 322 (5903), 945-949, 2008), plasmids (Okita K, et al: Science 322 (5903), 949-953, 2008), transposon vectors (Woltjen K, Michael IP, Mohseni P, et al: Nature 458, 766-770, 2009; Kaji K, Norrby K, Pac a A, et al: Nature 458, 771-775, 2009; Yusa K, Rad R, Takeda J, et al: Nat Methods 6, 363-369, 2009) or episomal vectors (Yu J, Hu K, Smuga-Otto K, Tian S, et al: Science 324, 797-801, 2009) have been developed for gene transfer.

[0076] Cells that have undergone transformation into iPS cells, i.e., reprogramming, can be selected using the expression of pluripotent stem cell markers (undifferentiated markers) such as Fbxo15, Nanog, Oct / 4, Fgf-4, Esg-1, and Cript as indicators. The selected cells are then recovered as iPS cells.

[0077] iPS cells can also be obtained from, for example, Kyoto University or the RIKEN BioResource Center.

[0078] As used herein, the terms "to differentiate" and "to induce (differentiation)" mean to induce differentiation along a specific cell lineage.

[0079] [Culture Medium for Preparing Intestinal Epithelial Cells] In one aspect, the present invention relates to a culture medium for preparing intestinal epithelial cells (hereinafter, sometimes referred to as the culture medium of the present invention) to be used in the preparation method of the present invention.

[0080] In one embodiment, the medium of the present invention contains an epidermal growth factor, a cAMP signal activator, a MEK1 / 2 inhibitor, a TGFβ receptor inhibitor, and a DNA methyltransferase inhibitor (hereinafter, this medium will be referred to as medium a-1). In another embodiment, the medium of the present invention contains an epidermal growth factor, a cAMP signal activator, a MEK1 / 2 inhibitor, and a TGFβ receptor inhibitor, but does not contain a DNA methyltransferase inhibitor (hereinafter, this medium will be referred to as medium a-2). In this specification, the term "medium of the present invention" refers to medium a-1 and medium a-2, unless otherwise specified.

[0081] The concentration of epidermal growth factor in the medium of the present invention may be, for example, 1 to 500 ng / mL, preferably 3 to 200 ng / mL, more preferably 5 to 100 ng / mL, even more preferably 10 to 50 ng / mL, and particularly preferably 15 to 30 ng / mL.

[0082] The cAMP signal activator in the medium of the present invention may be one or more selected from the group consisting of cAMP derivatives, cAMP degrading enzyme inhibitors, and cAMP activators, and is preferably a cAMP activator. The cAMP signal activator in the medium of the present invention may be selected as described in the section on the production method of the present invention in this specification.

[0083] The concentration of the cAMP signal activator in the medium of the present invention, for example, in the case of a cAMP derivative (8-Br-cAMP), is 0.1 mM to 10 mM, preferably 0.2 mM to 5 mM, and more preferably 0.5 mM to 2 mM. When using an exemplified compound, i.e., a compound other than 8-Br-cAMP, the concentration can be determined by one skilled in the art within the above concentration range, taking into account the differences (particularly activity) between the properties of the compound used and the properties of the exemplified compound (8-Br-cAMP). Furthermore, whether the determined concentration range is appropriate can be confirmed by preliminary experiments.

[0084] The concentration of the cAMP signal activator in the medium of the present invention, for example, a cAMP degrading enzyme inhibitor (IBMX), is 0.05 mM to 5 mM, preferably 0.1 mM to 3 mM, and more preferably 0.2 mM to 1 mM. When using an exemplified compound, i.e., a compound other than IBMX, a person skilled in the art can set the concentration according to the above concentration range, taking into consideration the differences in the properties of the compound used and the properties of the exemplified compound (IBMX) (particularly differences in activity). Furthermore, whether the set concentration range is appropriate can be confirmed by preliminary experiments.

[0085] The concentration of the cAMP signal activator in the medium of the present invention, for example, for the cAMP activator (forskolin), may be, for example, 1 μM to 200 μM, preferably 5 μM to 100 μM, more preferably 10 μM to 50 μM, even more preferably 20 μM to 40 μM, and particularly preferably 30 μM. When using an exemplified compound, i.e., a compound other than forskolin, the concentration can be determined by one skilled in the art based on the above concentration range, taking into account the differences in the properties of the compound used and the properties of the exemplified compound (forskolin) (particularly differences in activity). Furthermore, whether the set concentration range is appropriate can be confirmed by preliminary experiments similar to those described in the Examples below.

[0086] The MEK1 / 2 inhibitor in the medium of the present invention may be, for example, PD98059, PD184352, PD184161, PD0325901, U0126, MEK inhibitor I, MEK inhibitor II, MEK1 / 2 inhibitor II, or SL327, with PD98059 being preferred. The concentration of the MEK1 / 2 inhibitor in the medium of the present invention may be, for example, 4 μM to 100 μM in the case of PD98059, and is preferably 10 μM to 40 μM. When using a compound other than PD98059, the concentration can be determined in accordance with the above concentration range by a person skilled in the art, taking into account the characteristics of the compound used and the differences in the properties of the exemplified compounds (particularly differences in activity). Furthermore, whether the set concentration range is appropriate can be confirmed by a preliminary experiment similar to the Examples described below.

[0087] The TGFβ receptor inhibitor in the medium of the present invention preferably exhibits inhibitory activity against one or more of the TGF-β receptors ALK4, ALK5, and ALK7. Examples include A-83-01, SB431542, SB-505124, SB525334, D4476, ALK5 inhibitor, LY2157299, LY364947, GW788388, and RepSox, with A-83-01 being particularly preferred. Alternatively, a peptide compound with TGFβ inhibitory activity may be used instead of the TGFβ receptor inhibitor. The peptide compound with TGFβ inhibitory activity may be a TGFβ inhibitory peptide or a TGFβ receptor inhibitory peptide. An example of a TGFβ inhibitory peptide is the TGFβ1 inhibitor peptide PG-002 (PeptiGrowth). The concentration of the TGFβ receptor inhibitor in the medium of the present invention (in the case of A-83-01) may be, for example, 0.1 μM to 2.5 μM, preferably 0.2 μM to 1 μM. When using a compound other than A-83-01, a person skilled in the art can set the concentration according to the above concentration range, taking into account the characteristics of the compound used and the differences in the characteristics (particularly differences in activity) of the exemplified compounds. Furthermore, whether the set concentration range is appropriate can be confirmed by a preliminary experiment similar to the examples described below.

[0088] The DNA methyltransferase inhibitor in Medium A-1 is, for example, 5-aza-2'-deoxycytidine (5aza), 5-azacytidine, RG108, Zebularine, etc., with 5aza being preferred. The concentration of the DNA methyltransferase inhibitor in Medium A-1 (for 5aza) is, for example, 1 μM to 25 μM, preferably 2.5 μM to 10 μM. When using a compound other than 5aza, a person skilled in the art can determine the concentration based on the above concentration range, taking into account the characteristics of the compound used and the differences in the properties (particularly differences in activity) of the exemplified compounds. Furthermore, whether the determined concentration range is appropriate can be confirmed by a preliminary experiment based on the examples described below.

[0089] The medium of the present invention may further contain additives suitable for the production method of the present invention. Examples of additives include serum, amino acids, insulin, transferrin, progesterone, selenium, and putrescine. The serum may be, for example, mammalian serum, preferably fetal bovine serum (FBS). The serum concentration in the medium of the present invention may be, for example, 1-5%, and preferably 2-4%. The amino acids are preferably essential amino acids such as L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-glycine, L-proline, and L-serine, or L-alanyl-L-glutamine. The amino acid concentration in the medium of the present invention may be, for example, 0.1-10%, and preferably 0.5-5%.

[0090] The culture medium of the present invention is preferably a liquid culture medium. In one preferred embodiment, medium a-1 is a culture medium for use in liquid culture. In another preferred embodiment, medium a-2 is a culture medium for use in air-liquid interface culture.

[0091] [Kit] In one aspect, the present invention relates to a kit comprising the medium of the present invention and intestinal progenitor cells (hereinafter, sometimes referred to as the kit of the present invention). The intestinal progenitor cells contained in the kit of the present invention may be obtained by any method. The number of intestinal progenitor cells contained in the kit of the present invention is not particularly limited, and may be, for example, 1 x 10 4 ~1×10 8 cells, 1 × 10 5 ~1×10 7 In one embodiment, the intestinal progenitor cells contained in the kit of the present invention are frozen.

[0092] The kit of the present invention may include a container containing the culture medium of the present invention and a container containing intestinal progenitor cells. These containers are not particularly limited as long as they are suitable for storing the culture medium or cells. The kit of the present invention may also include an instruction manual describing operating instructions such as the culture method.

[0093] [Uses of Intestinal Epithelial Cells] (First Use) As a first use of the intestinal epithelial cells obtained by the production method of the present invention, various assays are provided. The intestinal epithelial cells obtained by the production method of the present invention can be used in a model system of the intestinal tract, particularly the small intestine, and are useful for evaluating pharmacokinetics (absorption, metabolism, etc.) and toxicity in the intestinal tract, particularly the small intestine. In one embodiment, a method for evaluating the pharmacokinetics or toxicity of a test substance using intestinal epithelial cells obtained by the production method of the present invention is provided.

[0094] Specifically, intestinal epithelial cells obtained by the production method of the present invention can be used to test the absorbability or membrane permeability of a test substance, drug interactions, induction of drug-metabolizing enzymes, induction of drug transporters, toxicity, etc. That is, in one aspect, the present invention provides a method for evaluating the absorbability or membrane permeability of a test substance, drug interactions, pharmacokinetics such as induction of drug-metabolizing enzymes and drug transporters, toxicity, etc., as one use of intestinal epithelial cells. This method includes the steps of: (i) preparing a cell layer composed of intestinal epithelial cells obtained by the production method of the present invention; (ii) contacting the cell layer with a test substance; and (iii) quantifying the test substance that has permeated the cell layer and evaluating the absorbability or membrane permeability, drug interactions, induction of drug-metabolizing enzymes, induction of drug transporters, or toxicity of the test substance. Note that the absorbability of a test substance can also be evaluated by a method according to another aspect including steps (I) and (II) described below.

[0095] In step (i), intestinal epithelial cells are typically cultured on a semipermeable membrane (porous membrane) to form a cell layer. Specifically, for example, a culture vessel equipped with a culture insert (e.g., Transwell (registered trademark) provided by Corning Incorporated) is used, and cells are seeded and cultured in the culture insert to obtain a cell layer composed of intestinal epithelial cells.

[0096] The "contact" in step (ii) is typically carried out by adding the test substance to the medium. The timing of adding the test substance is not particularly limited. Therefore, the test substance may be added at a certain time point after starting culture in a medium not containing the test substance, or the culture may be started in a medium containing the test substance in advance.

[0097] Organic or inorganic compounds of various molecular sizes can be used as test substances. Examples of organic compounds include nucleic acids, peptides, proteins, lipids (simple lipids, complex lipids (phosphoglycerides, sphingolipids, glycosylglycerides, cerebrosides, etc.)), prostaglandins, isoprenoids, terpenes, steroids, polyphenols, catechins, and vitamins (B1, B2, B3, B5, B6, B7, B9, B12, C, A, D, E, etc.). Existing or potential components of pharmaceuticals, nutritional foods, food additives, pesticides, and cosmetics are also suitable test substances. Plant extracts, cell extracts, culture supernatants, etc. can also be used as test substances. Two or more test substances can be added simultaneously to examine interactions, synergies, and other effects between the test substances. Test substances can be natural or synthetic. In the latter case, efficient assay systems can be constructed using, for example, combinatorial synthesis techniques.

[0098] The period for contacting the test substance can be set arbitrarily. The contact period is, for example, 10 minutes to 3 days, preferably 1 hour to 1 day. The contact may be performed in multiple batches.

[0099] In step (iii), the test substance that has permeated the cell layer is quantified. For example, when a culture vessel equipped with a culture insert such as Transwell (registered trademark) is used, the test substance that has permeated the culture insert, i.e., the test substance that has migrated through the cell layer into the upper or lower vessel, is quantified using a measurement method such as mass spectrometry, liquid chromatography, or immunological techniques (e.g., fluorescence immunoassay (FIA) or enzyme immunoassay (EIA)), depending on the test substance. Based on the quantification results (the amount of test substance that has permeated the cell layer) and the amount of test substance used (typically the amount added to the culture medium), the test substance's absorbability or membrane permeability, drug interactions, induction of drug-metabolizing enzymes, induction of drug transporters, or toxicity are determined and evaluated.

[0100] In another aspect, the present invention also provides a method for evaluating the metabolism or absorption of a test substance, which comprises the steps of (I) contacting the test substance with intestinal epithelial cells obtained by the production method of the present invention, and (II) measuring and evaluating the metabolism or absorption, drug interaction, induction of drug-metabolizing enzymes, induction of drug transporters, or toxicity of the test substance.

[0101] Step (I), i.e., contacting the intestinal epithelial cells with the test substance, can be carried out in the same manner as step (ii) above, except that it is not essential to form a cell layer in advance.

[0102] After step (I), the metabolism or absorption of the test substance, drug interactions, induction of drug-metabolizing enzymes, induction of drug transporters, or toxicity are measured and evaluated (step (II)). The metabolism and other properties may be measured and evaluated immediately after step (I), i.e., immediately after contact with the test substance, without any substantial time interval, or after a certain period of time (e.g., 10 minutes to 5 hours) has elapsed. Metabolism can be measured, for example, by detecting metabolites. In this case, the culture medium after step (I) is usually used as a sample, and predicted metabolites are measured qualitatively or quantitatively. An appropriate measurement method may be selected depending on the metabolite; for example, mass spectrometry, liquid chromatography, immunological techniques (e.g., fluorescence immunoassay (FIA) and enzyme immunoassay (EIA)), etc., can be used.

[0103] Typically, when a metabolic product of the test substance is detected, it is determined or evaluated that "the test substance has been metabolized." Furthermore, the metabolic amount of the test substance can be evaluated according to the amount of metabolic product. The metabolic efficiency of the test substance may be calculated based on the detection result of the metabolic product and the amount of the test substance used (typically the amount added to the medium).

[0104] It is also possible to measure the metabolism of a test substance using the expression of drug-metabolizing enzymes (cytochrome P450 (particularly CYP3A4), uridine diphosphate-glucuronosyltransferase (particularly UGT1A8 and UGT1A10), sulfotransferase (particularly SULT1A3, etc.)) in intestinal epithelial cells as an indicator. The expression of drug-metabolizing enzymes can be evaluated at the mRNA level or protein level. For example, an increase in the mRNA level of a drug-metabolizing enzyme can be determined to indicate that the test substance has been metabolized. Similarly, an increase in the activity of a drug-metabolizing enzyme can be determined to indicate that the test substance has been metabolized. As with the case of using metabolites as indicators, quantitative determination and evaluation can also be performed based on the expression level of the drug-metabolizing enzyme.

[0105] To evaluate the absorption of a test substance, for example, the amount of the test substance remaining in the culture medium is measured. Typically, the test substance is quantified using the culture medium after step (I) as a sample. An appropriate measurement method can be selected depending on the test substance. For example, mass spectrometry, liquid chromatography, immunological techniques (e.g., fluorescence immunoassay (FIA) and enzyme immunoassay (EIA)), etc. can be used. Typically, when a decrease in the content of the test substance in the culture medium is observed, it is determined and evaluated that the test substance has been absorbed. Furthermore, the amount or absorption efficiency of the test substance can be determined and evaluated depending on the degree of decrease. Absorption can also be evaluated by measuring the amount of test substance taken up into the cells.

[0106] The measurement and evaluation of metabolism and the measurement and evaluation of absorption may be carried out simultaneously or in parallel.

[0107] (Second Use) A second use of the intestinal epithelial cells obtained by the production method of the present invention is to provide a cell preparation containing intestinal epithelial cells. The cell preparation of the present invention is applicable to the treatment of various intestinal diseases. In particular, it is expected to be used as a material for regenerating and rebuilding damaged (including dysfunctional) intestinal epithelial tissue. In other words, it is expected to contribute to regenerative medicine. The cell preparation of the present invention can be prepared, for example, by suspending the intestinal epithelial cells obtained by the method of the present invention in physiological saline or a buffer solution (e.g., phosphate buffer), or by producing a three-dimensional tissue (organoid or spheroid) using the cells. To enable the administration of a therapeutically effective amount of cells, a single dose of, for example, 1 x 10 5 pieces ~ 1×10 10 The amount of cells contained can be adjusted appropriately taking into consideration the purpose of use, the target disease, the sex, age, weight, condition of the affected area, the condition of the cells, etc. of the recipient.

[0108] The cell preparation of the present invention may contain dimethyl sulfoxide (DMSO), serum albumin, etc. for the purpose of protecting the cells, antibiotics, etc. for the purpose of preventing bacterial contamination, and various components (vitamins, cytokines, growth factors, steroids, etc.) for the purpose of activating, proliferating, or inducing differentiation of the cells, etc. Furthermore, the cell preparation of the present invention may contain other components acceptable for formulation (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, physiological saline, etc.).

[0109] (Third Use) A third use of the intestinal epithelial cells obtained by the production method of the present invention includes culturing microorganisms (bacteria, fungi, viruses, etc.) or parasites using the intestinal epithelial cells or cell sheet of the present invention. By culturing microorganisms or parasites using the intestinal epithelial cells or cell sheet of the present invention, the infectivity of the microorganisms or parasites to the intestinal epithelial cells can be analyzed. Furthermore, if infectivity is detected, the maintenance or proliferation ability of the microorganisms or parasites can be confirmed. Furthermore, by culturing microorganisms or parasites using the intestinal epithelial cells or cell sheet of the present invention, it is possible to obtain microorganisms or parasites that repeatedly proliferate. Furthermore, the infection state of pathogenic microorganisms can be reproduced and used for screening therapeutic drugs, or the effects of substances produced by microorganisms or parasites on intestinal cells can be reproduced and used for screening substances that are useful or harmful to the small intestine of a living organism.

[0110] Specific examples of microorganisms or parasites include, but are not limited to, Escherichia coli, lactic acid bacteria, bifidobacteria, Vibrio parahaemolyticus, Salmonella, various enterobacteria, influenza virus, rotavirus, adenovirus, astrovirus, Sapporo virus, norovirus, HIV, and coronavirus.

[0111] (Fourth Use) A fourth use of the intestinal epithelial cells obtained by the production method of the present invention is a co-culture model using the intestinal epithelial cells of the present invention. The co-culture model described above can reproduce the structure of biological tissue and analyze interactions with other cells. Furthermore, the co-culture model described above can reproduce the pathology of a disease and screen for therapeutic drugs.

[0112] The cells to be co-cultured are preferably cell types present in the living body of a primate or rodent, particularly cells present around the small intestine. Examples include, but are not limited to, hepatocytes, pancreas, vascular endothelial cells, immune cells, nerve cells, fibroblasts, muscle cells, and interstitial cells. The cells to be co-cultured may also be established cell lines derived from primates or rodents. Examples include, but are not limited to, immortalized cell lines such as HeLa cells and THP-1 cells, pluripotent stem cells such as ES cells and iPS cells, and cells differentiated therefrom.

[0113] The culture vessel used for co-culture is not particularly limited, and examples include cell culture inserts, dishes, flasks, multi-well plates, etc. Culture can also be performed on an organ-on-a-chip (MPS: biomimetic system).

[0114] Test substances that can be tested in the co-culture model include organic and inorganic compounds of various molecular sizes. Existing or potential components of pharmaceuticals, nutritional foods, etc. are also suitable test substances. Plant extracts, cell extracts, culture supernatants, etc. may also be used as test substances. Two or more of these may also be used in combination.

[0115] The present invention will be specifically explained below by way of examples, but the present invention should not be construed as being limited thereto.

[0116] <Methods> 1. Cells The Windy strain was used as induced pluripotent stem cells (iPS cells). The iPS cell line was kindly provided by Dr. Akihiro Umezawa of the National Center for Child Health and Development. Mouse embryonic fibroblasts (MEF) were used as feeder cells for the maintenance culture of iPS cells (on-feeder culture). F-hiSIEC (registered trademark), commercially available from FUJIFILM, was used as human iPS cell-derived intestinal epithelial cells.

[0117] 2. Culture Medium: MEFs were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 2 mM L-glutamine (L-Glu), 1% non-essential amino acids (NEAA), 100 units / mL penicillin G, and 100 μg / mL streptomycin. MEFs were detached using 0.05% trypsin-ethylenediaminetetraacetic acid (EDTA), and MEFs were preserved using Cellbanker 1. Human iPS cells were maintained in DMEM Ham's F-12 (DMEM / F12) containing 20% ​​knockout serum replacement (KSR), 0.8% NEAA, 2 mM L-Glu, 0.1 mM 2-mercaptoethanol (2-MeE), and 5 ng / mL fibroblast growth factor (FGF)2. The detachment solution for human iPS cells was Dulbecco's phosphate-buffered saline (PBS) containing 1 mg / mL collagenase IV, 0.25% trypsin, 20% KSR, and 1 mM calcium chloride. The cryopreservation solution for human iPS cells was primate ES / iPS cell cryopreservation medium.

[0118] 3. Culturing human iPS cells Human iPS cells were cultured in mitomycin C-treated MEFs (6 × 10 5 Human iPS cells were seeded onto a 100 mm dish of 100 iPS cells and cultured at 37°C in a CO2 incubator under 5% CO2 / 95% air conditions. Human iPS cells were passaged after 3-5 days of culture at a split ratio of 1:2-1:3. The medium for human iPS cells was changed 48 hours after thawing, and was changed daily thereafter.

[0119] 4. Differentiation of human iPS cells into intestinal epithelial cells. Differentiation of human iPS cells into intestinal epithelial cells was initiated when the undifferentiated colonies occupied approximately 70% of the culture dish. Endoderm differentiation was achieved by culturing the cells in Roswell Park Memorial Institute (RPMI) + Glutamax medium containing 0.5% FBS, 100 ng / mL activin A, 100 units / mL penicillin G, and 100 μg / mL streptomycin for two days (days 0-2), and then in RPMI + Glutamax medium containing 2% FBS, 100 ng / mL activin A, 100 units / mL penicillin G, and 100 μg / mL streptomycin for one day (days 2-3). The cells were then cultured in DMEM / F12 containing 2% FBS, 1% Glutamax, and 250 ng / mL FGF2 for 4 days (days 3 to 7 after the start of differentiation) to differentiate into intestinal stem cells. After FGF2 treatment, Y-27632 (a Rho-associated kinase inhibitor) was added to a concentration of 10 μM and incubated for 60 minutes at 37°C in a CO2 incubator under 5% CO2 / 95% air conditions. The cells were then detached with actase and plated at 1.5 × 10 cells onto 24-well cell culture inserts coated with Matrigel (growth factor-free) pre-diluted 30-fold in human iPS cell medium. 5The cells were seeded at a density of 100 cells / well. Then, the cells were cultured in DMEM / F12 containing 2% FBS, 2 mM GlutaMAX, 1% NEAA, 2% B27 supplement, 1% N2 supplement, 1% HepExtend, 100 units / mL penicillin G, 100 μg / mL streptomycin, 20 ng / mL epidermal growth factor (EGF), and 10 μM Y-27632 for 1 day (days 7 to 8 after the initiation of differentiation), or in DMEM / F12 containing 2% FBS, 2 mM GlutaMAX, 1% NEAA, 2% B27 supplement, 1% N2 supplement, 1% HepExtend, 100 units / mL penicillin G, 100 μg / mL streptomycin, 20 ng / mL EGF, and 30 μM forskolin for 6 days (days 8 to 14 after the initiation of differentiation). The cells were cultured for 12 days (14th to 26th days after the start of differentiation) in DMEM / F12 containing 100 μg / mL penicillin G, 100 μg / mL streptomycin, 20 ng / mL EGF, 30 μM forskolin, 20 μM PD98059, 5 μM 5-aza-2'-deoxycytidine, and 0.5 μM A-83-01 to differentiate into intestinal epithelial cells. This differentiation method was used as the conventional protocol (12 days 5aza).

[0120] To examine the duration of 5-aza-2'-deoxycytidine addition, three groups were prepared: a group in which no 5-aza-2'-deoxycytidine was added from day 14 to day 26 after the start of differentiation (0day 5aza), a group in which 5-aza was added for 4 days from day 14 to day 18 after the start of differentiation (4days 5aza), and a group in which 5-aza-2'-deoxycytidine was added for 8 days from day 14 to day 22 after the start of differentiation (8days 5aza).

[0121] To examine the duration of air-liquid interface culture (ALI culture), the following groups were prepared: no ALI culture (LL), ALI culture from day 22 to day 26 after the start of differentiation (4-day ALI), ALI culture from day 18 to day 26 after the start of differentiation (8-day ALI), and ALI culture for 12 days from day 14 to day 26 after the start of differentiation (12-day ALI). Note that when examining ALI culture, 5 μM 5-aza-2'-deoxycytidine was added for 4 days from day 14 to day 18 after the start of differentiation.

[0122] After examining these conditions, the optimal differentiation conditions were determined to be the addition of 5 μM 5-aza-2'-deoxycytidine for 4 days from day 14 to day 18 after the start of differentiation, and the ALI culture for 8 days from day 18 to day 26 after the start of differentiation.

[0123] 5. Culturing F-hiSIEC® F-hiSIEC® was thawed and suspended in F-hiSIEC® Seeding Medium, and then 1.0 × 10 cells were placed on a 24-well cell culture insert coated with growth factor-free Matrigel, which had been previously diluted 30-fold with DMEM / F12. 5 Cells were seeded at a density of 1000 cells / well and cultured at 37°C in a CO2 incubator under 5% CO2 / 95% air conditions. The next day, the medium was replaced with F-hiSIEC® Culture Medium containing or not containing 5-aza-2'-deoxycytidine, and thereafter medium replacement was performed every 2-3 days. When performing ALI culture, the medium on the apical side was removed from day 4 after cell seeding.

[0124] 6. Total Ribonucleic Acid (RNA) Extraction Total RNA was extracted using Agencourt RNAdvance Tissue or RNeasy Mini Kit according to the attached manual.

[0125] 7. Reverse transcription reaction Complementary DNA (cDNA) was synthesized using the ReverTra Ace qPCR RT Master Mix with gDNA Remover Kit according to the attached manual.

[0126] 8. Real-time RT-PCR: Real-time RT-PCR was performed using the KAPA SYBR Fast qPCR Kit, cDNA as a template, according to the attached manual. Results were normalized using hypoxanthine phosphoribosyltransferase (HPRT) as an endogenous control.

[0127] 9. RNA Sequencing. Total RNA was extracted using the RNeasy Mini Kit, QIAshredder, and RNase-Free DNase Set. The total RNA was then subjected to paired-end Illumina sequencing at Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). The resulting data were analyzed using RaNA-Seq (https: / / ranaseq.eu / index.php). Specifically, transcripts per million (TPM) were calculated based on the GRCh38 reads, and differential expression analysis was performed using edgeR (quasi-likelihood F-tests). Subsequent clustering and Gene Ontology (GO) enrichment analyses were performed using R (4.0.3). Specifically, for clustering analysis, TPMs were converted to Z-scores using the genescale function in the Genefilter package (1.72.1), and clustering analysis was performed using the Heatmap function in the ComplexHeatmap package (2.6.2). For GO enrichment analysis, genes with a probability (p) value of 0.05 or less and a Log2 [TPM in ALI & new medium group / TPM in LL & Conventional medium group] of less than 1 were extracted, and analysis was performed using the enrichGO function of the clusterProfiler package (3.18.1) with the following settings: keyType = “ENSEMBL,” OrgDb = org.Hs.eg.db, ont = “ALL,” pAdjustMethod = “BH,” qvalueCutoff = 0.05.

[0128] 10. Drug Metabolism Test After differentiation induction, cells were used on day 26 after the start of differentiation. The apical medium was replaced with HBSS containing 10 mM HEPES (HBSS-HEPES), and the basal medium was left unincubated for 20 minutes. The apical medium was then replaced with HBSS-HEPES containing 5 μM diclofenac, 50 μM S-mephenytoin, 10 μM bufuralol, and 5 μM midazolam, and the metabolism test was initiated. After the specified time, samples were taken, and the amount of metabolites in the sample solution was quantified by LC-MS / MS.

[0129] 11. Drug Metabolism and Permeation Tests. Using F-hiSIEC® (registered trademark) cells 11 days after cell seeding, the apical medium was replaced with Hanks' balanced salt solution (HBSS) (HBSS-MES) containing 10 mM MES and 0.45 w / v% D-glucose, adjusted to pH 6.5, and the basal medium was replaced with HBSS (HBSS-HEPES) containing 10 mM HEPES and 0.45 w / v% D-glucose, adjusted to pH 7.4, for 60 minutes. The apical medium was then replaced with HBSS-MES containing 50 μM phenacetin, 5 μM diclofenac, 50 μM S-mephenytoin, 10 μM bufuralol, 5 μM midazolam, and 0.75% DMSO, and the basal medium was replaced with HBSS-HEPES containing 0.75% DMSO to initiate the permeation test. Samples were taken from the apical side immediately after the start of the permeation test and 60 minutes later, and from the basal side 60 minutes later. The amount of compound contained in the sampled solution was quantified by LC-MS / MS. Metabolic activity was calculated by adding the amount of metabolites contained in the apical and basal solutions after 60 minutes. The estimated Fg value was calculated using the following formula, with some modifications to the method proposed in Drug Metab Dispos 50, 204-213, 2022.

[0130]

[0131] <Results> The effects of 5-aza-2'-deoxycytidine and ALI culture on the culture of thawed F-hiSIEC® were examined. The results showed that ALI culture using a medium lacking 5-aza-2'-deoxycytidine significantly increased gene expression of the disaccharidase sucrase-isomaltase, the major drug-metabolizing enzyme CYP3A4 expressed in the small intestine, and the hydrolase carboxylesterase 2 (CES2) (Figure 1). The expression levels were elevated to the same level as those in the adult human small intestine.

[0132] We performed RNA sequencing on F-hiSIEC® cells cultured with or without 5-aza-2'-deoxycytidine in the medium and with or without ALI culture, and compared the resulting data. The results showed that ALI culture in 5-aza-2'-deoxycytidine-free medium resulted in higher expression of intestinal markers, primarily expressed in absorptive epithelial cells, compared with cells cultured using the conventional protocol or other combinations (Figure 2). We also analyzed genes highly expressed in the small intestinal epithelium or large intestinal epithelium. Again, ALI culture in 5-aza-2'-deoxycytidine-free medium resulted in higher expression of genes expressed in the small intestinal epithelium compared with cells cultured using the conventional protocol or other combinations (Figure 3). Furthermore, GO enrichment analysis of the RNA sequencing data revealed increased expression of genes involved in nutrient absorption (Figure 4). These results demonstrate that ALI culture in 5-aza-2'-deoxycytidine-free medium results in differentiation into cells exhibiting characteristics of small intestinal absorptive epithelial cells.

[0133] Metabolic enzyme activity was evaluated using a cocktail of drug-metabolizing enzyme substrates. Cells cultured using the conventional protocol showed high metabolic activity due to CYP1A1 / 2, which is highly expressed in the liver. However, in cells cultured at ALI using a medium that did not contain 5-aza-2'-deoxycytidine, metabolic activity due to CYP1A1 / 2 was suppressed. On the other hand, increased metabolic enzyme activity due to CYP3A4 / 5, CYP2C9, and CYP2C19, which are primarily expressed in the small intestine, was observed (Fig. 5, Table 1). Midazolam was metabolized by CYP3A4 and the proportion of drugs that escaped metabolism in the small intestine (F g ) has been reported to be 0.55 (J Med Chem, 53, 1098-1108, 2010). F estimated using cells cultured using the conventional protocol g However, the F estimated using cells cultured at ALI in a medium without 5-aza-2'-deoxycytidine was about 0.9, which was significantly different from the actual value in vivo. g The F value of drugs metabolized by CYP3A4 was approximately 0.6, which is almost the same as that in humans (Figure 6). g This may reflect the increased activity of CYP3A4 in the cells themselves.

[0134]

[0135] The effect of 5-aza-2'-deoxycytidine treatment duration on differentiation of human iPS cells into intestinal epithelial cells was investigated using the Windy strain. The results showed that the shorter the treatment duration, the higher the gene expression of intestinal epithelial cell markers and drug-metabolizing enzymes (Figure 7). However, when 5-aza-2'-deoxycytidine was not added at all, the expression tended to be the same or slightly lower compared to treatment with 4 days, and the expression of CYP3A4 was significantly lower.

[0136] The effect of ALI culture on the differentiation of human iPS cells into intestinal epithelial cells was also investigated using the Windy strain. In this case, the longer the ALI culture period, the higher the gene expression of small intestinal epithelial cell markers and drug-metabolizing enzymes (Figure 8). However, when the ALI culture period was 12 days (days 14 to 26) after the start of differentiation, the expression tended to be the same or slightly lower than when it was 8 days (days 18 to 26).

[0137] Based on these results, we conducted a more detailed study of the differentiation of human iPS cells into intestinal epithelial cells using a new protocol in which 5-aza-2'-deoxycytidine was added for four days from day 14 to day 18 after the start of differentiation and ALI culture was performed for eight days from day 8 to day 26 after the start of differentiation. The results showed that the expression of drug-metabolizing enzymes, transporters, and intestinal epithelial markers was significantly increased in intestinal epithelial cells induced by the new protocol compared to cells cultured using the conventional protocol (Figure 9). Functionally, significant increases in metabolic enzyme activity were observed in CYP3A4, CYP2C9, and CYP2C19, which are primarily expressed in the small intestine (Figure 10).

[0138] <Summary> By shortening the period of addition of 5-aza-2'-deoxycytidine (a DNA methyltransferase inhibitor), a differentiation-inducing factor, and combining this with ALI culture, we were able to create cells that exhibit characteristics closer to those of the human small intestine.

[0139] Increased gene expression and activity of CYP3A4, CYP2C9, and CYP2C19, which are major drug-metabolizing enzymes in the small intestine, was observed, as was increased expression of genes involved in nutrient absorption.

Claims

1. A method for producing intestinal epithelial cells from intestinal progenitor cells, comprising: (a-1) culturing intestinal progenitor cells in the presence of a DNA methyltransferase inhibitor, and subsequently (a-2) culturing in the absence of a DNA methyltransferase inhibitor, and switching from liquid culture to air-liquid interface culture midway through a culture period including the culture of (a-1) and the culture of (a-2).

2. The method according to claim 1, wherein the culture period of (a-1) is 1 to 12 days, the culture period of (a-2) is 1 to 12 days, the liquid culture period is 1 to 12 days, and the air-liquid interface culture period is 1 to 12 days.

3. The method according to claim 1, wherein the culture of (a-1) is liquid culture, and the culture of (a-2) is air-liquid interface culture.

4. The method of claim 1, wherein the DNA methyltransferase inhibitor is 5-aza-2'-deoxycytidine.

5. The method according to claim 1, wherein the culturing of (a-1) and the culturing of (a-2) comprise culturing in the presence of an epidermal growth factor, a cAMP signal activator, a MEK1 / 2 inhibitor, and a TGFβ receptor inhibitor.

6. The method according to claim 5, wherein the cAMP signal activator is forskolin, the MEK1 / 2 inhibitor is PD98059, and the TGFβ receptor inhibitor is A8301.

7. The method according to claim 1, wherein the intestinal progenitor cells are cells obtained by the following steps: (3) culturing intestinal stem cells in the presence of epidermal growth factor and cAMP signal activator.

8. The method according to claim 7, wherein the culture period in step (3) is 2 to 12 days.

9. The method according to claim 7, wherein the intestinal stem cells are cells obtained by the following steps: (1) differentiating pluripotent stem cells into endoderm cells; and (2) differentiating the endoderm cells obtained in step (1) into intestinal stem cells.

10. The method according to claim 9, wherein step (1) comprises culturing pluripotent stem cells in the presence of activin A, and step (2) comprises culturing endoderm cells in the presence of epidermal growth factor.

11. The method of claim 1, wherein the intestinal progenitor cells are derived from pluripotent stem cells.

12. The method according to claim 11, wherein the pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells.

13. The method of claim 11, wherein the pluripotent stem cells are human induced pluripotent stem cells.

14. A medium for producing intestinal epithelial cells to be used in the method according to claim 1, comprising an epidermal growth factor, a cAMP signal activator, a MEK1 / 2 inhibitor, a TGFβ receptor inhibitor and a DNA methyltransferase inhibitor.

15. A medium for producing intestinal epithelial cells to be used in the method according to claim 1, comprising an epidermal growth factor, a cAMP signal activator, a MEK1 / 2 inhibitor and a TGFβ receptor inhibitor, but not containing a DNA methyltransferase inhibitor.

16. A kit comprising the medium for preparing intestinal epithelial cells according to claim 14 and intestinal progenitor cells.

17. A kit comprising the medium for preparing intestinal epithelial cells according to claim 15 and intestinal progenitor cells.

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