WO2022168908A1 - Production method for intestinal tract cells derived from pluripotent stem cells and having crypt-villus-like structures, and use thereof - Google Patents

Production method for intestinal tract cells derived from pluripotent stem cells and having crypt-villus-like structures, and use thereof Download PDF

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WO2022168908A1
WO2022168908A1 PCT/JP2022/004220 JP2022004220W WO2022168908A1 WO 2022168908 A1 WO2022168908 A1 WO 2022168908A1 JP 2022004220 W JP2022004220 W JP 2022004220W WO 2022168908 A1 WO2022168908 A1 WO 2022168908A1
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cells
intestinal
culture
inhibitor
pluripotent stem
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民秀 松永
岳洋 岩尾
勇 小川
施萌 邱
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公立大学法人名古屋市立大学
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

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  • the present invention relates to a method for producing intestinal cells having crypt-villus-like structures derived from pluripotent stem cells and uses thereof.
  • Caco-2 cells derived from human colon cancer are frequently used as a small intestine model system.
  • the expression pattern of drug transporters in Caco-2 cells differs from that in the human small intestine.
  • expression of CYP3A4 a major drug-metabolizing enzyme in the small intestine, and induction of CYP3A4 by PXR ligands are hardly observed, making it difficult to accurately evaluate pharmacokinetics in the small intestine. .
  • iPSCs human induced pluripotent stem cells
  • hiPSC human induced pluripotent stem cells
  • Patent Document 1 discloses (1) a step of differentiating pluripotent stem cells into endoderm-like cells, (2) a step of differentiating the endoderm-like cells obtained in step (1) into intestinal stem cell-like cells, (3) culturing the intestinal stem cell-like cells obtained in step (2) in the presence of epidermal growth factor, fibroblast growth factor, TGF ⁇ receptor inhibitor, GSK-3 ⁇ inhibitor and ROCK inhibitor; (4) culturing the cells after step (3) to form spheroids, (5) differentiating the spheroids formed in step (4) to form intestinal organoids, comprising epidermal growth factor, Generation of intestinal organoids from pluripotent stem cells is described by a process involving culturing in the presence of a BMP inhibitor and a Wnt signal activator.
  • the present inventors have established a method for inducing differentiation of hiPSC-derived intestinal organoids (HIOs).
  • HIOs hiPSC-derived intestinal organoids
  • the present inventors further thought that by applying the culture technology, it would be possible to construct an intestinal evaluation system (two-dimensional HIOs) that mimics the crypt-villus structure peculiar to the intestine. been working on it.
  • the produced intestinal cells have a single-layer structure, and imitation of the three-dimensional structure of living intestinal tissue has not been realized.
  • the present invention is a pluripotent stem cell-derived crypt-villus that can acquire a crypt-villus-like structure similar to the intestinal tract in vivo, and can improve the efficiency of differentiation into intestinal epithelial cells and the function of the cells.
  • the problem to be solved was to provide a method for producing intestinal cells having a similar structure.
  • the present inventors improved the culture method by gas-liquid interface culture and addition of low-molecular compounds, etc., and in addition to morphology, barrier function, gene expression, pharmacokinetic We examined whether it would be useful as a new intestinal evaluation system by performing such evaluation. As a result, it was found that intestinal cells having pluripotent stem cell-derived crypt-villus-like structures can be produced by using gas-liquid interface culture.
  • the present invention has been completed based on the above findings. That is, according to the present invention, the following inventions are provided.
  • a method for producing intestinal cells from pluripotent stem cells comprising the following steps (1) to (6): (1) differentiating pluripotent stem cells into endoderm-like cells; (2) a step of differentiating the endoderm-like cells obtained in step (1) into intestinal stem cell-like cells; (3) culturing the intestinal stem cell-like cells obtained in step (2) in the presence of epidermal growth factor, fibroblast growth factor, TGF ⁇ receptor inhibitor, GSK-3 ⁇ inhibitor and ROCK inhibitor; (4) culturing the cells after step (3) to form spheroids; (5) differentiating the spheroids formed in step (4) to form intestinal organoids, comprising culturing in the presence of an epidermal growth factor, a BMP inhibitor and a Wnt signal activator; and (6) Cells composing the intestinal organoids formed in step (5) are cultured in gas-phase liquid phase in the presence of epidermal growth factor, cAMP signal activator, TGF ⁇ receptor inhibitor, and Wnt signal activator.
  • the fibroblast growth factor is FGF2, FGF4 or FGF10
  • the TGF ⁇ receptor inhibitor is A-83-01
  • the GSK-3 ⁇ inhibitor is CHIR99021, SB216763, CHIR98014, TWS119 , Tideglusib, SB415286, BIO, AZD2858, AZD1080, AR-A014418, TDZD-8, LY2090314, IM-12, Indirubin, Bikinin or 1-Azakenpaullone
  • the ROCK inhibitor is Y-27632, described in ⁇ 1> the method of.
  • step (6) The method according to ⁇ 1> or ⁇ 2>, wherein in step (5), the BMP inhibitor is Noggin and the Wnt signal activator is R-spondin-1.
  • step (6) the cAMP signal activator is forskolin, 8-Br-cAMP or IBMX
  • the TGF ⁇ receptor inhibitor is A-83-01
  • the Wnt signal activator is CHIR99021.
  • the gas-liquid phase culture in step (6) is performed in the presence of an epidermal growth factor, a cAMP signal activator, a TGF ⁇ receptor inhibitor, and a Wnt signal activator, as well as a Notch signal inhibitor.
  • ⁇ 6> The method according to any one of ⁇ 1> to ⁇ 4>.
  • ⁇ 6> The method according to ⁇ 5>, wherein the Notch signal inhibitor is DAPT.
  • ⁇ 7> The method according to ⁇ 5> or ⁇ 6>, wherein the gas-liquid phase culture in step (6) is performed in the presence of a Notch signal inhibitor and then in the absence of a Notch signal inhibitor.
  • ⁇ 8> The method according to any one of ⁇ 1> to ⁇ 7>, wherein the gas-liquid culture in step (6) is performed for 4 to 30 days.
  • step (4) and step (5), step (5), or part of the culture of step (5) A method for producing intestinal cells from pluripotent stem cells, which is a gas phase liquid phase culture: (1) differentiating pluripotent stem cells into endoderm-like cells; (2) a step of differentiating the endoderm-like cells obtained in step (1) into intestinal stem cell-like cells; (4) culturing the cells after step (2) in the presence of epidermal growth factor and cAMP signal activator; (5) A step of culturing the cells after step (4) in the presence of epidermal growth factor, MEK1/2 inhibitor, DNA methylation inhibitor, TGF ⁇ receptor inhibitor, and cAMP signal activator.
  • ⁇ 10> The method according to ⁇ 9>, further comprising step (3) between steps (2) and (4). (3) culturing the intestinal stem cell-like cells obtained in step (2) in the presence of epidermal growth factor and a ROCK inhibitor;
  • ⁇ 11> A method for producing intestinal cells, comprising culturing intestinal cells produced from pluripotent stem cells in a medium that does not contain 5-aza-2'-deoxycytidine.
  • ⁇ 12> The method according to ⁇ 11>, wherein at least part of the culture in the medium that does not contain 5-aza-2'-deoxycytidine is gas phase liquid phase culture.
  • the intestinal cells produced from pluripotent stem cells are cells obtained by a method comprising the following steps (11) to (14). (11) differentiating the pluripotent stem cells into endoderm-like cells; (12) differentiating the endoderm-like cells obtained in step (11) into intestinal stem cell-like cells; (14) culturing the intestinal stem cell-like cells obtained in step (12) in the presence of epidermal growth factor and cAMP signal activator; , a MEK1/2 inhibitor, a DNA methylation inhibitor, a TGF ⁇ receptor inhibitor, and a cAMP signal activator.
  • ⁇ 14> The method according to any one of ⁇ 1> to ⁇ 13>, wherein the pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells.
  • ⁇ 15> The method according to any one of ⁇ 1> to ⁇ 14>, wherein the pluripotent stem cells are human induced pluripotent stem cells.
  • ⁇ 16> The method according to any one of ⁇ 1> to ⁇ 15>, wherein the pluripotent stem cells are feederless cultured pluripotent stem cells.
  • ⁇ 17> Intestinal cells obtained by the method according to any one of ⁇ 1> to ⁇ 16>.
  • ⁇ 18> A method for evaluating pharmacokinetics or toxicity of a test substance using the intestinal cells of ⁇ 17>.
  • ⁇ 19> The method of ⁇ 18>, wherein the pharmacokinetics is metabolism, absorption, membrane permeability, drug interaction, induction of drug-metabolizing enzymes, or induction of drug transporters.
  • the method according to ⁇ 18> or ⁇ 19> comprising the following steps (i) and (ii): (i) contacting the test substance with the intestinal cells according to ⁇ 17>; (ii) evaluating the metabolism, absorption, membrane permeability, drug interaction, induction of drug-metabolizing enzymes, induction of drug transporters, or toxicity of the test substance;
  • a method for preparing an intestinal disease model comprising inducing an intestinal disease state in the intestinal cells obtained by the method according to any one of ⁇ 1> to ⁇ 16>.
  • intestinal cells having a crypt-villus-like structure close to the intestinal structure of a living body can be produced from pluripotent stem cells.
  • FIG. 1 shows the morphological observation of HIOs-derived intestinal cells prepared by gas-liquid interface culture and control of cAMP signal and TGF- ⁇ signal.
  • the culture period was 10 days, and the gas-liquid interface culture was performed from day 3 to day 10.
  • Air/liquid gas-liquid interfacial culture
  • liquid/liquid liquid phase culture
  • Forskolin 10 ⁇ M
  • A-83-01 0.5 ⁇ M
  • 8-Br-cAMP 1 mM
  • IBMX 500 ⁇ M.
  • Scale bar 100 ⁇ m.
  • Figure 2 shows the effect of Wnt and Notch signals in long-term culture.
  • the number of culture days was 22 days, and the addition period of CHIR99021 and DAPT was 4 days or 22 days.
  • FIG. 3 shows three-dimensional structure observation of HIOs-derived intestinal cells.
  • A Images of HIOs-derived intestinal cells on day 10 of culture taken using Cell3iMager Estier. Yellow dotted line indicates the cell surface. Scale bar: 100 ⁇ m.
  • B Z-direction signal extracted after filtering and binarizing the image in (A).
  • C Images from (B) stacked in the Z direction.
  • D H&E staining image after 10 days of culture. Scale bar: 50 ⁇ m.
  • FIG. 4 shows analysis of barrier function, gene expression and pharmacokinetic function of HIOs-derived intestinal cells.
  • A Intestinal barrier function evaluation by transepithelial electrical resistance measurement.
  • B Gene expression analysis of gut-related genes by RT-qPCR.
  • C Gene expression analysis of drug transporters.
  • D Gene expression analysis of immune system cell-related genes.
  • FIG. 6 shows changes over time in transepithelial electrical resistance (TEER) values in human iPSC-derived intestinal epithelial cells maintained and cultured in an on-feeder.
  • Mean ⁇ S.D. (n 3).
  • FIG. 7 shows the effects of cell number and gas phase culture on gene expression in human iPSC-derived intestinal epithelial cells.
  • Mean ⁇ SD (n 3); L-1: liquid phase culture, 1 ⁇ 10 5 cells/insert, L-1.5: liquid phase culture, 1.5 ⁇ 10 5 cells/insert, A-1: gas phase culture, 1 ⁇ 10 5 cells/insert, A-1.5: gas phase culture, 1.5 ⁇ 10 5 cells/insert, expression level is shown as a relative value with human adult small intestine as 100.
  • FIG. 8 shows a continuation of FIG.
  • FIG. 9 shows the effects on cell morphology of human iPSC-derived intestinal epithelial cells maintained and cultured in an on-feeder depending on the presence or absence of the addition of 5-aza-2′-deoxycytidine and differences in the gas phase culture period.
  • L-5+ liquid phase culture and 5-aza-2'-deoxycytidine addition group
  • L-5- liquid phase culture and 5-aza-2'-deoxycytidine non-addition group
  • A8-5+ differentiation initiation A group of gas phase culture and 5-aza-2'-deoxycytidine added from day 8, A8-5-: group of gas phase culture and no 5-aza-2'-deoxycytidine added from day 8 of differentiation
  • A14- 5+ group with gas phase culture and 5-aza-2'-deoxycytidine added from day 14 of differentiation
  • A14-5- group with gas phase culture and no 5-aza-2'-deoxycytidine from day 14 of differentiation Addition group
  • L-5+ Liquid phase culture and 5-aza-2'-deoxycytidine addition group
  • L-5- Liquid phase culture and 5-aza-2'-deoxycytidine non-addition group
  • A8-5+ Gas phase culture and 5-aza-2'-deoxycytidine addition group from day 8 of differentiation initiation
  • A8-5- Differentiation
  • A14-5+ gas phase culture and 5-aza-2'-deoxycytidine addition group from day 14 of differentiation
  • A14 -5- Group without addition of 5-aza-2'-deoxycytidine and gas-phase culture from 14 days after initiation of differentiation
  • FIG. 11 shows a continuation of FIG. FIG.
  • L-14 group added from day 14 of the start of differentiation of A83-01 in liquid phase culture
  • L-8 group added from day 8 of start of differentiation in liquid phase culture of A83-01
  • A14-14 start of differentiation
  • A14-8 Group of gas phase culture from day 14 and addition of A83-01 from day 14 of differentiation
  • A14-8 group of gas phase culture from day 14 of differentiation and addition of A83-01 from day 8 of differentiation
  • FIG. 12 shows the influence of the addition timing of A83-01 on cell morphology in human iPSC-derived intestinal epithelial cells maintained and cultured in an on-feeder.
  • FIG. 13 shows changes over time in transepithelial electrical resistance (TEER) values in human iPSC-derived intestinal epithelial cells maintained and cultured in an on-feeder, depending on the timing of addition of A83-01.
  • L-14 Group in which liquid-phase culture was added from day 14 of differentiation of A83-01
  • L-8 group of liquid-phase culture and addition of A83-01 from day 8 of differentiation
  • A14-14 differentiation initiation
  • A14-8 group gas phase culture from day 14 and addition from day 14 of A83-01 differentiation
  • FIG. 15 shows a continuation of FIG.
  • FIG. 16 shows the effect of addition of CHI99021 on cell morphology in human iPSC-derived intestinal epithelial cells maintained and cultured in an on-feeder.
  • FIG. 18 shows a continuation of FIG. FIG.
  • FIG. 19 shows a continuation of FIG.
  • FIG. 20 shows the effect of gas phase culture on gene expression in feederless cultured human iPSC-derived intestinal epithelial cells.
  • Mean ⁇ S.D. (n 3); L-: liquid phase culture, A12: group in which gas phase culture is performed from day 12 of differentiation, A18: group in which gas phase culture is performed from day 18 of differentiation, A24: gas phase Group where culture is started from the 24th day of differentiation
  • FIG. 21 shows a continuation of FIG.
  • FIG. 22 shows the results of TEER measurement in the first F-hiSIEC TM culture experiment.
  • FIG. 23 shows changes in midazolam concentration in the membrane permeation test of midazolam in the first F-hiSIEC TM culture experiment.
  • FIG. 24 shows the permeability coefficient of midazolam in the membrane permeation test of midazolam in the first F-hiSIEC TM culture experiment.
  • FIG. 25 shows changes in 1′-hydroxymidazolam concentration in the midazolam membrane permeation test in the first F-hiSIEC TM culture experiment.
  • FIG. 26 shows the measurement results of the production amount of 1′-hydroxymidazolam per unit protein amount in the midazolam membrane permeation test in the first F-hiSIEC TM culture experiment.
  • FIG. 31 shows the results of the Lucifer yellow membrane permeation test in the second F-hiSIEC TM culture experiment.
  • FIG. 32 shows the results of gene expression analysis in the second F-hiSIEC TM culture experiment.
  • FL Fetal liver total RNA.
  • HPH Human primary hepatocyte cultured for 48 hours.
  • the present invention relates to a method for producing intestinal cells from pluripotent stem cells (hereinafter also referred to as "the production method of the present invention”).
  • the production method of the present invention a method for producing intestinal cells from pluripotent stem cells.
  • Pluripotent stem cells have the ability to differentiate into all cells that make up the body (pluripotency) and the ability to produce daughter cells with the same differentiation potential as the self through cell division (self-renewal ability). ) refers to cells that have both Pluripotency can be evaluated by transplanting the cells to be evaluated into nude mice and examining the presence or absence of teratoma formation containing cells of each of the three germ layers (ectoderm, mesoderm, and endoderm). can.
  • pluripotent stem cells examples include embryonic stem cells (ES cells), embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells) and the like. As long as it is a cell, it is not limited to this. ES cells or iPS cells are preferably used. More preferably, iPS cells are used. Pluripotent stem cells are preferably mammalian cells (eg, primates such as humans, chimpanzees and cynomolgus monkeys, and rodents such as mice and rats), particularly preferably human cells.
  • ES cells embryonic stem cells
  • EG cells embryonic germ cells
  • iPS cells induced pluripotent stem cells
  • Pluripotent stem cells are preferably mammalian cells (eg, primates such as humans, chimpanzees and cynomolgus monkeys, and rodents such as mice and rats), particularly preferably human cells.
  • ES cells can be established, for example, by culturing an early embryo before implantation, an inner cell mass constituting the early embryo, a single blastomere, 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)).
  • an early embryo an early embryo produced by nuclear transfer of the nucleus of a somatic cell may be used (Wilmut et al. (Nature, 385, 810 (1997)), Cibelli et al. (Science, 280, 1256 (1998)), Akira Iriya et al.
  • ES cells are available from archives or are commercially available.
  • human ES cells are available from Kyoto University Institute for Frontier Medical Sciences (eg, KhES-1, KhES-2 and KhES-3), WiCell Research Institute, ESI BIO, and the like.
  • ES cells can be established by culturing primordial germ cells in the presence of LIF, bFGF, SCF, etc. (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)).
  • iPS cells are cells with pluripotency (multipotency) and proliferative potential, which are produced by reprogramming somatic cells through the introduction of reprogramming factors. Induced pluripotent stem cells exhibit properties similar to ES cells.
  • the somatic cells used for producing iPS cells are not particularly limited, and may be differentiated somatic cells or undifferentiated stem cells.
  • the origin is not particularly limited, but somatic cells of mammals (for example, primates such as humans, chimpanzees, and cynomolgus monkeys, and rodents such as mice and rats), particularly preferably somatic cells of humans, are used.
  • iPS cells can be produced by various methods reported so far. In addition, it is naturally envisioned that an iPS cell production method that will be developed in the future will be applied.
  • iPS cells derived from patients with intestinal disease iPS cells prepared from the patient's somatic cells
  • iPS cells are prepared from somatic cells (for example, skin, blood, mononuclear cells, etc.) collected from patients.
  • intestinal diseases include intractable inflammatory bowel diseases (Crohn's disease, ulcerative colitis), polyps, colon cancer, drug-induced enteritis, and the like.
  • Disease-specific intestinal organoids are useful as intestinal pathology models, and are expected to contribute to drug evaluation systems and elucidation of disease mechanisms (molecular mechanisms related to onset, pathogenesis, and progression).
  • the most basic method of producing iPS cells is to introduce four transcription factors, Oct3/4, Sox2, Klf4 and c-Myc, into cells using viruses (Takahashi K, Yamanaka S : Cell 126 (4), 663-676, 2006; Takahashi, K, et al.: Cell 131 (5), 861-72, 2007).
  • human iPS cells were established by introducing four factors, Oct4, Sox2, Lin28 and Nonog (Yu J, et al: Science 318(5858), 1917-1920, 2007). 3 factors excluding c-Myc (Nakagawa M, et al: Nat. Biotechnol.
  • Transformation into iPS cells i.e. cells that have undergone reprogramming (reprogramming), express pluripotent stem cell markers (undifferentiated markers) such as Fbxo15, Nanog, Oct/4, Fgf-4, Esg-1 and Cript etc. can be selected as an index. Selected cells are collected as iPS cells.
  • iPS cells can also be provided, for example, from Kyoto University or the RIKEN BioResource Center, a national research and development agency.
  • the terms “differentiate” and “induce” mean working to differentiate along a specific cell lineage.
  • pluripotent stem cells are differentiated into intestinal cells.
  • the first preparation method of the present invention includes the following six stages of culture steps. (1) a step of differentiating pluripotent stem cells into endoderm-like cells (step (1)); (2) a step of differentiating the endoderm-like cells obtained in step (1) into intestinal stem cell-like cells (step (2)); (3) culturing the intestinal stem cell-like cells obtained in step (2) in the presence of epidermal growth factor, fibroblast growth factor, TGF ⁇ receptor inhibitor, GSK-3 ⁇ inhibitor and ROCK inhibitor ( step (3)); (4) culturing the cells after step (3) to form spheroids (step (4)); (5) A step of differentiating the spheroids formed in step (4) to form intestinal organoids, comprising culturing in the presence of an epidermal growth factor, a BMP inhibitor and a Wnt signal activator (step (5)); and (6) cells composing the intestinal organoids formed in step (5) in the presence of epidermal growth factor, cAMP signal activ
  • pluripotent stem cells are cultured and differentiated into endoderm-like cells.
  • pluripotent stem cells are cultured under conditions that induce differentiation into endoderm-like cells.
  • Culture conditions are not particularly limited as long as the pluripotent stem cells differentiate into endoderm-like cells.
  • it is cultured in a medium supplemented with activin A according to a conventional method.
  • the concentration of activin A in the medium is, for example, 10 ng/mL to 200 ng/mL, preferably 20 ng/mL to 150 ng/mL.
  • serum or a serum substitute Knockout serum replacement (KSR), etc.
  • Serum is not limited to fetal bovine serum, and human serum, sheep serum, and the like can also be used.
  • the amount of serum or serum substitute added is, for example, 0.1% (v/v) to 10% (v/v).
  • Wnt ligands e.g., Wnt-3a
  • GSK-3 inhibitors e.g., CHIR99021, CHIR98014, BIO, SB415286, SB216763, TWS119, A1070722, etc.
  • Two or more stages of culture may be performed as step (1).
  • serum-free medium or medium with relatively low concentration of serum e.g., 0.1% (v / v) to 1% (v / v)
  • serum concentration for example, 1% (v/v) to 10% (v/v)
  • Adopting two or more stages of culture in this way is preferable in that the growth of undifferentiated cells is suppressed in the first stage of culture, and the differentiated cells are allowed to grow in the following two stages and beyond.
  • the period of step (1) is, for example, 1 to 10 days, preferably 2 to 7 days.
  • the first stage culture period is, for example, 1 to 7 days, preferably 2 to 5 days
  • the second stage culture period is, for example, 1 to 6 days. days, preferably 1 to 4 days.
  • Step (2) Differentiation into intestinal stem cell-like cells>
  • the endoderm-like cells obtained in step (1) are cultured and differentiated into intestinal stem cell-like cells.
  • endoderm cells are cultured under conditions that induce differentiation into intestinal stem cell-like cells. Culture conditions are not particularly limited as long as the endoderm-like cells differentiate into intestinal stem cell-like cells.
  • culture is preferably performed in the presence of FGF4 (fibroblast growth factor 4) and a GSK-3 inhibitor (e.g., CHIR99021, CHIR98014, BIO, SB415286, SB216763, TWS119, A1070722, etc.).
  • FGF4 fibroblast growth factor 4
  • GSK-3 inhibitor e.g., CHIR99021, CHIR98014, BIO, SB415286, SB216763, TWS119, A1070722, etc.
  • human FGF4 eg, human recombinant FGF4
  • Cultivation can also be performed in the presence of FGF2 (fibroblast growth factor 2).
  • step (2) may be performed after selecting endoderm-like cells from the cell population obtained through step (1). Selection of endoderm-like cells may be performed, for example, using a flow cytometer (cell sorter) using a cell surface marker as an index.
  • FGF4 and GSK-3 inhibitors are synonymous with the conditions in which FGF4 and GSK-3 inhibitors were added to the medium. Therefore, in order to perform culture in the presence of FGF4 and GSK-3 inhibitor, a medium supplemented with FGF4 and GSK-3 inhibitor may be used.
  • concentration of FGF4 added is 50 ng/mL to 2.5 ⁇ g/mL, preferably 150 ng/mL to 500 ng/mL.
  • concentration of the GSK-3 inhibitor added is 600 nmol/L to 60 ⁇ mol/L, preferably 1 ⁇ mol/L to 20 ⁇ mol/L.
  • FGF2 In the presence of FGF2 is synonymous with the condition in which FGF2 was added to the medium. Therefore, in order to perform culture in the presence of FGF2, a medium supplemented with FGF2 may be used.
  • concentration of FGF2 added is 50 ng/mL to 2.5 ⁇ g/mL, preferably 150 ng/mL to 500 ng/mL.
  • the addition concentration when using the exemplified compound that is, a compound different from CHIR99021, considering the characteristics of the compound used and the difference in the characteristics of the exemplified compound (especially the difference in activity), If so, it can be set according to the above density range. Also, whether or not the set concentration range is appropriate can be confirmed by a preliminary experiment according to the examples described later.
  • the period of step (2) (culture period) is, for example, 2 to 10 days, preferably 3 to 7 days. If the culture period is too short, the expected effects (increase in differentiation efficiency, promotion of acquisition of functions as intestinal stem cells) cannot be sufficiently obtained. On the other hand, if the culture period is too long, it causes a decrease in differentiation efficiency.
  • intestinal stem cell-like cells can be determined or evaluated, for example, using the expression of intestinal stem cell markers as an index.
  • intestinal stem cell markers are leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), ephrin B2 receptor (EphB2).
  • Step (3) Induction of direction of differentiation into intestinal tract>
  • the intestinal stem cell-like cells obtained in step (2) are cultured in the presence of epidermal growth factor, fibroblast growth factor, TGF ⁇ receptor inhibitor, GSK-3 ⁇ inhibitor and ROCK inhibitor.
  • this process can be understood as “the step of inducing differentiation toward the intestinal tract while maintaining stemness”. This process is important for the morphology (villus-crypt-like structure) and function (intestinal markers and drug transporters exhibit gene expression similar to that of the adult small intestine) of finally obtained intestinal organoids.
  • step (3) may be performed after intestinal stem cell-like cells are selected from the cell population obtained through step (2). Selection of intestinal stem cell-like cells may be performed, for example, by a flow cytometer (cell sorter) using a cell surface marker as an indicator.
  • a flow cytometer cell sorter
  • the presence of epidermal growth factor, fibroblast growth factor, TGF ⁇ receptor inhibitor, GSK-3 ⁇ inhibitor and ROCK inhibitor is synonymous with the conditions under which these compounds are added to the medium. Therefore, For culture in the presence of epidermal growth factor, fibroblast growth factor, TGF ⁇ receptor inhibitor, GSK-3 ⁇ inhibitor and ROCK inhibitor, a medium supplemented with these compounds may be used.
  • FGF2, FGF4 or FGF10 are fibroblast growth factors.
  • Two or three of these FGF families may be used in combination.
  • A-83-01 can be used as a TGF ⁇ receptor inhibitor.
  • GSK-3 ⁇ inhibitors include CHIR99021, SB216763, CHIR98014, TWS119, Tideglusib, SB415286, BIO, AZD2858, AZD1080, AR-A014418, TDZD-8, LY2090314, IM-12, Indirubin, Bikinin, and 1-Azakenpaullone.
  • Y-27632 for example, can be used as a ROCK inhibitor.
  • concentration of epidermal growth factor added is 10 ng/mL to 500 ng/mL, preferably 50 ng/mL to 200 ng/mL.
  • concentration of fibroblast growth factor added is 5 ng/mL to 200 ng/mL, preferably 20 ng/mL to 50 ng/mL.
  • An example of the addition concentration of (for A-83-01) is 0.1 ⁇ M to 5 ⁇ M, preferably 0.3 ⁇ M to 3 ⁇ M, and an example of the addition concentration of the GSK-3 ⁇ inhibitor (for CHIR99021) is 0.5 It is ⁇ M to 100 ⁇ M, preferably 1 ⁇ M to 30 ⁇ M, and an example of the addition concentration of the ROCK inhibitor (in the case of Y-27632) is 1 ⁇ M to 50 ⁇ M, preferably 3 ⁇ M to 30 ⁇ M.
  • the concentration to be added depends on the difference between the characteristics of the compound used and the characteristics of the exemplified compounds ( In particular, considering the difference in activity), a person skilled in the art can set the concentration according to the above range. Also, whether or not the set concentration range is appropriate can be confirmed by a preliminary experiment according to the examples described later.
  • serum or serum replacement should be used in combination. That is, culture is preferably carried out under conditions in which the above compound and serum or serum substitute are added to the medium.
  • a serum substitute is generally a composition that is used as a substitute for serum containing a differentiation-inducing factor in order to culture iPS cells, ES cells, etc. while maintaining their undifferentiated state.
  • KSR Knockout serum replacement
  • the serum concentration is 1% (v/v) to 10% (v/v), preferably 1% (v/v) to 5% (v/v).
  • concentration of serum replacement added (for KSR) is 5% (v/v) to 20% (v/v), preferably 5% (v/v) to 15% (v/v). be.
  • a culture vessel with a cell-adhesive culture surface is used for the culture in step (3).
  • the cell-adhesive culture surface one coated with a basement membrane component (eg, laminin, type IV collagen, entactin, vitronectin, fibronectin) or a fragment thereof is preferably used.
  • culture surfaces coated with laminin 511 or its E8 fragment may be employed.
  • E8 fragment of laminin 511 a product (product name: iMatrix-511, manufactured by Nippi Co., Ltd., sold by Matrixome Co., Ltd.), which is a highly purified recombinant (recombinant human laminin 511-E8 protein), is commercially available.
  • the culture vessel is not particularly limited, and for example, dishes, flasks, multi-well plates and the like can be used.
  • the period of step (3) (culture period) is, for example, 2 to 14 days, preferably 3 to 12 days. If the culture period is too short, the expected effect (ie, induction of the direction of differentiation necessary to form functional intestinal organoids with villus-crypt-like structures) cannot be obtained sufficiently. Incidentally, if the subculturing operation is repeated, the culture period can be set longer.
  • a subculture operation is performed in step (3). That is, subculture is performed in the middle of step (3).
  • Passage operation is considered to be particularly effective in purifying intestinal epithelial stem cell-like cells (improving purity and homogeneity) and more selectively inducing differentiation into the intestinal tract.
  • the number of subculture operations is, for example, 1 to 5 times, preferably 1 to 3 times, more preferably 1 or 2 times. Too many subcultures result in greater damage to the cells, leading to lower production efficiency.
  • Procedures for subculturing may follow conventional methods. For example, when the cells become confluent or subconfluent, some of the cells are harvested and transferred to another culture vessel to continue culturing.
  • a cell dissociation solution or the like may be used to collect cells.
  • trypsin-EDTA, collagenase IV, proteolytic enzymes such as metalloprotease, and the like can be used alone or in appropriate combination. Those with less cytotoxicity are preferred.
  • Commercial products such as Dispase (Edia), TrypLE (Invitrogen), and Accutase (MILLIPORE) are available as such cell dissociation solutions.
  • the cells that have undergone step (3) are cultured to form spheroids.
  • Suspension culture is suitable for forming spheroids.
  • Suspension culture usually comprises a culture surface with low cell adhesion or cell non-adhesion (for example, a culture surface imparted with low cell adhesion/cell non-adhesion by treatment/bonding with polymer materials, hydrogels, etc.)
  • a culture vessel is used to culture cells in a state away from the culture surface (ie, in a floating state).
  • Culture vessels used for suspension culture are not particularly limited, and for example, dishes, flasks, multiwell plates, tubes, trays, culture bags and the like can be used.
  • a culture vessel (generally referred to as a pattern plate) in which a plurality of wells of uniform shape and size are formed on a low cell adhesion or cell non-adhesion culture surface.
  • EZSPHERE registered trademark
  • Elplasia provided by Kuraray Co., Ltd.
  • spheroids can be formed efficiently, and the production efficiency of intestinal organoids is improved.
  • the cells/cell clusters may be statically cultured, swirl cultured, or shaken cultured.
  • the suspension culture here is performed by static culture. Static culture has many advantages, such as no need for a special device, less impact or damage to cells is expected, and the amount of culture solution can be reduced.
  • the culture conditions are not particularly limited as long as spheroids can be formed.
  • spheroids can be formed while maintaining stemness.
  • EGF epidermal growth factor
  • BMP inhibitors and Wnt signal activators BMP inhibitors and Wnt signal activators
  • ROCK inhibitors Y-27632, etc.
  • epidermal growth factor By using epidermal growth factor, the effect of promoting cell proliferation can be expected. Moreover, by using a BMP inhibitor, an effect of suppressing differentiation of stem cells and maintaining stemness can be expected. Wnt signal activators are expected to have the effect of maintaining stem cell proliferation and stemness.
  • Noggin can be used as a BMP inhibitor.
  • R-spondin-1 can be used as a Wnt signal activator.
  • concentration of epidermal growth factor added is 10 ng/mL to 500 ng/mL, preferably 50 ng/mL to 200 ng/mL.
  • concentration of the BMP inhibitor added is 10 ng/mL to 500 ng/mL, preferably 50 ng/mL to 200 ng/mL.
  • concentration of the Wnt signal activator added is 10 ng/mL to 1000 ng/mL, preferably 50 ng/mL to 500 g/mL.
  • addition concentration of the ROCK inhibitor in the case of Y-27632 is 1 ⁇ M to 50 ⁇ M, preferably 3 ⁇ M to 30 ⁇ M.
  • the concentration to be added should be determined according to the characteristics of the compound used and the difference in characteristics (especially the difference in activity) of the exemplified compounds. Taking into consideration, a person skilled in the art can set it according to the above concentration range. Also, whether or not the set concentration range is appropriate can be confirmed by a preliminary experiment according to the examples described later.
  • the period of step (4) (culture period) is, for example, 1 to 10 days, preferably 2 to 7 days. If the culture period is too short, spheroids of sufficient size will not be formed. On the other hand, if the culture period is too long, the spheroids become larger than necessary, and the cells inside may undergo necrosis.
  • spheroids with a diameter of about 100 ⁇ m to 200 ⁇ m are formed.
  • spheroids of this size can be formed by using a pattern plate in which wells having a diameter of 400 to 500 ⁇ m and a depth of 100 to 200 ⁇ m are uniformly formed.
  • the spheroids formed in step (4) are differentiated to form intestinal organoids.
  • Active induction of differentiation is not essential in this step, and in one aspect (first aspect), culture is performed under the same or similar medium conditions as in step (4). Specifically, it is cultured in the presence of epidermal growth factor, BMP inhibitor and Wnt signal activator, that is, using a medium supplemented with these components. Specific examples and concentration of each compound are the same as in the step (4).
  • the period of step (5) (culture period) is, for example, 6 to 36 days, preferably 8 to 18 days.
  • the culture in step (5) is performed in suspension culture.
  • a liquid medium to which a material that forms a three-dimensional network structure is added in an aqueous solution is used, and the plurality of spheroids formed in step (4) are collected together (that is, one (with multiple spheroids coexisting in the culture vessel). That is, in a preferred embodiment, part (but two or more) or all of the spheroids formed in step (4) are subjected to suspension culture using a characteristic liquid medium.
  • viscous material a material that forms a three-dimensional network structure in an aqueous solution
  • spheroids are captured or trapped in the network structure, or the viscosity of the medium is increased to restrict the movement of spheroids. , can prevent spheroid association and aggregation. Therefore, a plurality of spheroids can be collectively cultured in suspension, enabling efficient formation of intestinal organoids.
  • polymer gels and polysaccharides can be used as viscous materials.
  • polymer gels are collagen, polymer hydrogels, Matrigel TM (regular Matrigel, Growth Factor Reduced (GFR) Matrigel with reduced content of growth factors, etc.).
  • polysaccharides are gellan gum, crystalline cellulose, nanocellulose, carboxycellulose, carboxymethylcellulose, and the like. Two or more kinds of materials may be used together.
  • a polymer compound having an anionic functional group is used as the viscous material.
  • anionic functional groups include a carboxy group, a sulfo group, a phosphoric acid group and salts thereof, with a carboxy group or a salt thereof being preferred.
  • the polymer compound used in the present invention those having one or more selected from the group of anionic functional groups can be used.
  • Preferred specific examples of the polymer compound herein include, but are not particularly limited to, polysaccharides in which 10 or more monosaccharides (e.g., triose, tetrose, pentose, hexose, heptose, etc.) are polymerized, Acidic polysaccharides having anionic functional groups are more preferred.
  • the acidic polysaccharide referred to here is not particularly limited as long as it has an anionic functional group in its structure.
  • polysaccharides having a sulfate group or a phosphate group in a part of the structure or polysaccharides having both structures, which are not only polysaccharides obtained from nature but also polysaccharides produced by microorganisms, genes Polysaccharides produced by engineering or polysaccharides artificially synthesized using enzymes are also included.
  • hyaluronic acid gellan gum, deacylated gellan gum, rhamsan gum, diutan gum, xanthan gum, carrageenan, xanthan gum, hexuronic acid, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparin, heparitin sulfate, kerato Examples include those composed of one or more selected from the group consisting of sulfuric acid, chondroitin sulfate, dermatan sulfate, rhamnan sulfate, and salts thereof.
  • the polysaccharide is preferably hyaluronic acid, deacylated gellan gum, diutan gum, xanthan gum, carrageenan, or a salt thereof.
  • Gellan gum The salt referred to here includes, for example, salts of alkali metals such as lithium, sodium and potassium, salts of alkaline earth metals such as calcium, barium and magnesium, salts of aluminum, zinc, copper, iron, ammonium, organic bases and amino acids. is mentioned.
  • the weight average molecular weight of the polymer compound is preferably 10,000 to 50,000,000, more preferably 100,000 to 20,000,000, and still more preferably 1,000,000. ⁇ 10,000,000.
  • the molecular weight can be measured in terms of pullulan by gel permeation chromatography (GPC).
  • phosphorylated deacylated gellan gum can also be used.
  • the phosphorylation can be performed by a known technique.
  • a combination of multiple types (preferably two types) of the above polysaccharides can be used.
  • the type of combination of polysaccharides is not particularly limited as long as it can prevent aggregation and aggregation of spheroids, but preferably the combination contains at least deacylated gellan gum or a salt thereof.
  • suitable polysaccharide combinations include deacylated gellan gum or salts thereof, and polysaccharides other than deacylated gellan gum or salts thereof (e.g., xanthan gum, alginic acid, carrageenan, diutan gum, methylcellulose, locust bean gum or their salts).
  • Specific polysaccharide combinations include deacylated gellan gum and rhamsan gum, deacylated gellan gum and diutan gum, deacylated gellan gum and xanthan gum, deacylated gellan gum and carrageenan, deacylated gellan gum and xanthan gum, deacylated Examples include, but are not limited to, gellan gum and locust bean gum, deacylated gellan gum and ⁇ -carrageenan, deacylated gellan gum and sodium alginate, deacylated gellan gum and methylcellulose, and the like.
  • More preferred specific examples of the viscous material used in the present invention include hyaluronic acid, deacylated gellan gum, diutan gum, carrageenan, xanthan gum, and salts thereof, and the most preferred example is deacylated gellan gum or a salt thereof. is mentioned.
  • deacylated gellan gum commercially available products such as "KELCOGEL (registered trademark of CP Kelco) CG-LA” manufactured by Sansho Co., Ltd., "Kelcogel (registered trademark of CP Kelco Co., Ltd.)” manufactured by Sanei Gen FFI Co. company's registered trademark)” etc. can be used.
  • the native type gellan gum San-Ei Gen FFI Co., Ltd. "Kelcogel (registered trademark of CP Kelco) HT” and the like can be used.
  • examples of particularly preferred viscous materials include Polymer FP001 and Polymer FP003 provided by Nissan Chemical Industries, Ltd.
  • the polymer FP001 is a compounding component of the three-dimensional culture medium FCeM (registered trademark) series manufactured by Nissan Chemical Industries, Ltd.
  • the polymer FP003 is a compounding component of the same FCeM (registered trademark) Advance Preparation Kit.
  • the amount of the viscous material used is not particularly limited as long as the expected effect can be exhibited. Adjust the usage of If the viscosity of the medium is too low, the effect of preventing association and aggregation of spheroids cannot be obtained. On the other hand, if the viscosity of the medium is too high, the operability (handling) is affected (for example, recovery operation becomes complicated), and the supply of medium components to cells may be affected.
  • Matrigel as a specific example of the amount of viscous material to be used, it should be about 1% to 10% of the amount used in normal use (that is, use as a base material for three-dimensional culture). In the case of deacylated gellan gum, the 0.01% to 0.05% (w/v), most preferably 0.01% to 0.03% (w/v) should be added to the medium.
  • the culture vessel used for floating culture is not particularly limited, and for example, dishes, flasks, multiwell plates, tubes, trays, culture bags, etc. can be used.
  • step (5) It is also possible to subculture (eg, 1 to 5 times) in step (5).
  • the subculture operation in this case may follow, for example, the following procedure. First, collect the cell aggregates (spheroids), wash them if necessary, and then disperse/dissociate the spheroids (e.g. Thermo Fisher Scientific EDTA, Roche Dispase II, STEMCELL Technologies Gentle Cell Dissociation Reagent). ). Subsequently, after dispersing and suspending by pipetting operation or the like, the cells are seeded and the culture is continued.
  • spheroids e.g. Thermo Fisher Scientific EDTA, Roche Dispase II, STEMCELL Technologies Gentle Cell Dissociation Reagent.
  • ROCK inhibitor Rho-associated coiled-coil forming kinase/Rho-associated kinase
  • Step (6) Preparation of intestinal cells by gas phase liquid phase culture>
  • the cells constituting the intestinal organoids formed in step (5) are subjected to gas phase in the presence of epidermal growth factor, cAMP signal activator, TGF ⁇ receptor inhibitor, and Wnt signal activator.
  • Liquid phase culture flat culture. Cells are collected from the formed intestinal organoids, transferred to a two-dimensional culture system, and cultured in gas-liquid phase. Typically, cell populations or portions thereof collected from intestinal organoids are subjected to air-liquid planar culture without sorting. A cell dissociation solution or the like may be used to collect cells from intestinal organoids.
  • cell dissociation solution for example, trypsin-EDTA, collagenase IV, proteolytic enzymes such as metalloprotease, and the like can be used alone or in appropriate combination. Those with less cytotoxicity are preferred. Commercial products such as dispase (Edia), TrypLE (Invitrogen) and Accutase (MILLIPORE) are available as such cell dissociation solutions. In addition, it is preferable to treat the cells with a cell strainer or the like to increase the dispersibility of the cells.
  • ROCK inhibitor Rho-associated coiled-coil forming kinase/Rho-associated kinase
  • the culture conditions in step (6) "in the presence of epidermal growth factor, cAMP signal activator, TGF ⁇ receptor inhibitor, and Wnt signal activator" are those compounds added to the medium. Synonymous with condition. Therefore, for culture in the presence of epidermal growth factor, cAMP signal activator, TGF ⁇ receptor inhibitor, and Wnt signal activator, a medium supplemented with these compounds may be used.
  • concentration of epidermal growth factor added is 10 ng/mL to 500 ng/mL, preferably 50 ng/mL to 200 ng/mL.
  • cAMP derivatives cAMP degrading enzyme inhibitors, or cAMP activators can be used as cAMP signal activators. Two or more of these substances may be used in combination.
  • PKA active agents as cAMP derivatives (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
  • concentration of the cAMP derivative added is 0.1 mM to 10 mM, preferably 0.2 mM to 5 mM, more preferably 0.5 mM to 2 mM.
  • concentration to be added depends on the difference between the characteristics of the compound used and the characteristics of the exemplified compound (8-Br-cAMP) (especially Considering the difference in activity), a person skilled in the art can set the concentration range according to the above. Also, whether or not the set concentration range is appropriate can be confirmed by a preliminary experiment.
  • IBMX (3-isobutyl-1-methylxanthine) (MIX), Theophylline, Papaverine, Pentoxifylline (Trental), KS-505, 8-Methoxymethyl-IBMX, Vinpocetine (TCV-3B), EHNA, Trequinsin as cAMPase inhibitors (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 115 BS), Enoximone (MDL 17043), Imazodan (CL 914), SKF-94120, Vesnarinone (OPC 8212),
  • concentration of the cAMP degrading enzyme inhibitor added is 0.05 mM to 5 mM, preferably 0.1 mM to 3 mM, more preferably 0.2 mM to 1 mM.
  • the addition concentration should be determined considering the difference in the characteristics of the compound used and the exemplified compound (IBMX) (especially the difference in activity). , can be set according to the above concentration range by those skilled in the art. Also, whether or not the set concentration range is appropriate can be confirmed by a preliminary experiment.
  • cAMP activators forskolin, indomethacin, NKH477 (colforsin daropate), cell-derived toxin proteins (pertussis toxin, cholera toxin), PACAP-27, PACAP-38, SKF83822, etc.
  • concentration of the cAMP activator added is 1 ⁇ M to 200 ⁇ M, preferably 5 ⁇ M to 100 ⁇ M.
  • the concentration to be added takes into account the difference between the characteristics of the compound used and the characteristics of the exemplified compound (forskolin) (especially the difference in activity). Then, a person skilled in the art can set the concentration according to the above concentration range. Also, whether or not the set concentration range is appropriate can be confirmed by a preliminary experiment according to the examples described later.
  • the TGF ⁇ receptor inhibitor it is preferable to use one that exhibits inhibitory activity against one or more of the TGF- ⁇ receptors ALK4, ALK5, and ALK7.
  • A-83-01, SB431542, SB-505124, SB525334, D4476, ALK5 inhibitor, LY2157299, LY364947, GW788388, and RepSox satisfy this condition.
  • An example of the added concentration of the TGF ⁇ receptor inhibitor is 0.1 ⁇ M to 50 ⁇ M, preferably 0.3 ⁇ M to 30 ⁇ M.
  • the addition concentration when using the exemplified compound that is, a compound different from A-83-01
  • the difference in the characteristics of the compound used and the exemplified compound should be considered.
  • a person skilled in the art can set the concentration according to the above concentration range. Also, whether or not the set concentration range is appropriate can be confirmed by a preliminary experiment according to the examples described later.
  • the Wnt signal activator may be any compound having activity to inhibit GSK-3, for example, CHIR99021, CHIR98014, BIO, SB415286, SB216763, TWS119, A1070722 and the like.
  • An example of the added concentration of the Wnt signal activator is 0.1 ⁇ M to 100 ⁇ M, preferably 1 ⁇ M to 10 ⁇ M.
  • the addition concentration should be determined in consideration of the difference between the characteristics of the compound used and the exemplified compound (forskolin) (especially the difference in activity). For example, a person skilled in the art can set the concentration according to the above concentration range. Also, whether or not the set concentration range is appropriate can be confirmed by a preliminary experiment according to the examples described later.
  • the gas-liquid phase culture in step (6) may be performed in the presence of Notch signal inhibitor in addition to epidermal growth factor, cAMP signal activator, TGF ⁇ receptor inhibitor, and Wnt signal activator.
  • Notch signal inhibitors include N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl-1,1-dimethylethylester-glycine (DAPT), L-685,458, Compound E (CAS 209986- 17-4), (R)-Flurbiprofen, BMS299897, JLK6, LY-411575, R04929097, MK-0752, SCP0004, SCP0025, gamma-Secretase Inhibitor XI, gamma-Secretase Inhibitor XVI, gamma-Secretase Inhibitor I, gamma-Secretase ⁇ -Secretase inhibitors such as Inhibitor VII, Semagacestat (LY450139), gamma-Secretase Inhibitor III, Compound 34, BMS-708163, Compound W, YO
  • concentration of the Notch signal inhibitor added in the case of DAPT
  • concentration range is 1 nM to 20 ⁇ M, preferably 0.1 ⁇ M to 10 ⁇ M, and when using a compound different from DAPT, the concentration added is
  • a person skilled in the art can set the concentration range according to the above concentration range, taking into account the characteristics of the compounds and the difference in characteristics (especially the difference in activity) of the exemplified compounds. Also, whether or not the set concentration range is appropriate can be confirmed by a preliminary experiment according to the examples described later.
  • the gas phase liquid phase culture in step (6) includes performing gas phase liquid phase culture in the presence of the Notch signal inhibitor, followed by gas phase liquid phase culture in the absence of the Notch signal inhibitor. You can stay.
  • the gas phase liquid phase culture in step (6) can be performed in the presence or absence of 5-aza-2'-deoxycytidine.
  • a culture vessel with a cell-adhesive culture surface is used for the culture in step (6).
  • the cell-adhesive culture surface one coated with a basement membrane component (eg, laminin, type IV collagen, entactin, vitronectin, fibronectin) or a fragment thereof is preferably used.
  • culture surfaces coated with laminin 511 or its E8 fragment may be employed.
  • E8 fragment of laminin 511 a product (product name: iMatrix-511, manufactured by Nippi Co., Ltd., sold by Matrixome Co., Ltd.), which is a highly purified recombinant (recombinant human laminin 511-E8 protein), is commercially available.
  • the culture vessel is not particularly limited, and for example, dishes, flasks, multi-well plates and the like can be used.
  • cells are cultured on a semipermeable membrane (porous membrane) to form a cell layer.
  • a cell layer is obtained by using a culture vessel equipped with an insert (for example, Transwell (registered trademark) provided by Corning) and seeding and culturing cells in the insert.
  • Transwell registered trademark
  • Cultivation in step (6) is carried out by gas phase liquid phase culture.
  • cells are seeded on a semi-permeable membrane, and after the cells adhere to the culture surface (ie, the semi-permeable membrane), the culture is switched to gas-liquid phase culture.
  • the culture is switched to gas phase liquid phase culture.
  • the approximate time (timing) for switching to gas phase liquid phase culture is 3 hours to 3 days after seeding (for example, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours later), but the time for cells to adhere to the culture surface and the time to form a cell layer may vary depending on the state of the cells and other conditions (for example, the culture substrate used). It is preferable to determine the timing of switching by a preliminary experiment, or to observe the cells after seeding and switch to the gas phase liquid phase culture at an appropriate timing.
  • gas phase liquid phase culture means that the entire cell surface (excluding the part that adheres to the culture surface, the part that contacts or adheres to other cells, etc.) does not contact the medium, but the cell surface It refers to culturing in a state in which a part is in contact with the gas phase.
  • a culture vessel with an insert typically composed of an insert and a well plate serving as a receiving plate
  • cells are seeded in the insert, and after the cells adhere
  • the culture medium in the insert is removed (preferably after the cell layer is formed), the gas phase exists on the upper surface of the cells and the liquid phase (medium) exists on the lower surface of the cells (i.e., the side adhered to the insert).
  • the gas phase liquid phase culture can be started.
  • the culture medium in the well is removed to form a state in which the liquid phase exists on the upper surface side of the cells and the gas phase exists on the lower surface side of the cells. Culture may be performed.
  • the gas-liquid culture in step (6) is performed, for example, for 4 to 30 days, preferably 7 to 30 days, more preferably 7 to 14 days.
  • the gas-liquid phase culture is typically performed under aerobic conditions, anaerobic conditions may be adopted.
  • Subculture may be performed in the middle of step (6).
  • the cells become confluent or subconfluent, some of the cells are harvested and transferred to another culture vessel to continue culturing.
  • a cell dissociation solution or the like may be used to collect cells.
  • the cell dissociation solution for example, trypsin-EDTA, collagenase IV, proteolytic enzymes such as metalloprotease, and the like can be used alone or in appropriate combination. Those with less cytotoxicity are preferred.
  • Commercial products such as Dispase (Edia), TrypLE (Invitrogen), and Accutase (MILLIPORE) are available as such cell dissociation solutions.
  • step (6) medium exchange is performed as necessary.
  • the medium may be replaced once every 24 hours to every 3 days.
  • a ROCK inhibitor Rho-associated coiled-coil forming kinase/Rho-associated kinase
  • culture temperature may be the conditions generally adopted for culturing animal cells. That is, it may be cultured in an environment of, for example, 37°C and 5% CO 2 .
  • the basal medium is not particularly limited, but preferably a basal medium suitable for culturing epithelial cells (for example, a mixed medium of D-MEM and Ham's F12 medium, D-MEM) is used.
  • a basal medium suitable for culturing epithelial cells for example, a mixed medium of D-MEM and Ham's F12 medium, D-MEM
  • components that can be added to media include bovine serum albumin (BSA), antibiotics, 2-mercaptoethanol, PVA, non-essential amino acids (NEAA), insulin, transferrin, and selenium.
  • BSA bovine serum albumin
  • NEAA non-essential amino acids
  • insulin transferrin, and selenium.
  • the second preparation method of the present invention roughly includes the following four stages of culture steps. (1) differentiating pluripotent stem cells into endoderm-like cells; (2) a step of differentiating the endoderm-like cells obtained in step (1) into intestinal stem cell-like cells; (4) culturing the cells after step (2) in the presence of epidermal growth factor and cAMP signal activator; (5) A step of culturing the cells after step (4) in the presence of epidermal growth factor, MEK1/2 inhibitor, DNA methylation inhibitor, TGF ⁇ receptor inhibitor, and cAMP signal activator.
  • a step (3) may be further included between the step (2) and the step (4).
  • (3) culturing the intestinal stem cell-like cells obtained in step (2) in the presence of epidermal growth factor and a ROCK inhibitor;
  • step (4) and step (5), step (5), or part of the culture in step (5) is gas-liquid phase culture.
  • the latter half of the culture in step (5) is preferably gas phase liquid phase culture.
  • the details of the gas phase liquid phase culture are as described above in this specification.
  • steps (1) and (2) in the second production method of the present invention are the same as steps (1) and (2) in the first production method of the present invention.
  • Y-27632 and the like can be used as the ROCK inhibitor in step (3).
  • An example of the concentration of epidermal growth factor added in step (3) is 5 ng/mL to 500 ng/mL, preferably 10 ng/mL to 200 ng/mL.
  • An example of the addition concentration of the ROCK inhibitor (in the case of Y-27632) is 1 ⁇ M to 50 ⁇ M, preferably 3 ⁇ M to 30 ⁇ M.
  • the cAMP signal activator in step (4) the cAMP derivative, cAMP degrading enzyme inhibitor, or cAMP activator described herein above can be used.
  • An example of the concentration of epidermal growth factor added in step (4) is 5 ng/mL to 500 ng/mL, preferably 10 ng/mL to 200 ng/mL.
  • An example of the concentration of the cAMP activator added (in the case of forskolin) is 1 ⁇ M to 200 ⁇ M, preferably 5 ⁇ M to 100 ⁇ M.
  • Examples of MEK1/2 inhibitors in step (5) include PD98059, PD184352, PD184161, PD0325901, U0126, MEK inhibitor I, MEK inhibitor II, MEK1/2 inhibitor II, and SL327.
  • Examples of DNA methylation inhibitors include 5-aza-2'-deoxycytidine, 5-azacytidine, RG108, and Zebularine.
  • the TGF ⁇ receptor inhibitor it is preferable to use one that exhibits inhibitory activity against one or more of the TGF- ⁇ receptors ALK4, ALK5 and ALK7.
  • A-83-01, SB431542, SB-505124, SB525334, D4476, ALK5 inhibitor, LY2157299, LY364947, GW788388, and RepSox satisfy this condition.
  • the cAMP signal activator the cAMP derivative, cAMP degrading enzyme inhibitor or cAMP activator described herein above can be used.
  • An example of the concentration of epidermal growth factor added in step (5) is 5 ng/mL to 500 ng/mL, preferably 10 ng/mL to 200 ng/mL.
  • An example of the concentration of the MEK1/2 inhibitor added (in the case of PD98059) is 4 ⁇ M to 100 ⁇ M, preferably 10 to 40 ⁇ M.
  • An example of the added concentration of the DNA methylation inhibitor (in the case of 5-aza-2'-deoxycytidine) is 1 ⁇ M to 25 ⁇ M, preferably 2.5 ⁇ M to 10 ⁇ M.
  • An example of the concentration of the TGF ⁇ receptor inhibitor added is 0.1 ⁇ M to 2.5 ⁇ M, preferably 0.2 ⁇ M to 1 ⁇ M.
  • An example of the concentration of the cAMP activator added (in the case of forskolin) is 1 ⁇ M to 200 ⁇ M, preferably 5 ⁇ M to 100 ⁇ M.
  • the concentration to be added depends on the characteristics of the compound used and the exemplified Considering the difference in the properties (especially the difference in activity) of the compounds described above, a person skilled in the art can set the concentration range according to the above. Also, whether or not the set concentration range is appropriate can be confirmed by a preliminary experiment according to the examples described later.
  • the period of culture in step (3) is, for example, 1 to 5 days.
  • the period of culture in step (4) is, for example, 3 to 15 days.
  • the period of culture in step (5) is, for example, 5 to 20 days.
  • a third production method of the present invention is a method for producing intestinal cells, comprising culturing intestinal cells produced from pluripotent stem cells in a medium that does not contain 5-aza-2'-deoxycytidine. .
  • a medium that does not contain 5-aza-2'-deoxycytidine is gas phase liquid phase culture.
  • cells obtained by a method including the following steps (11) to (14) can be used. (11) differentiating the pluripotent stem cells into endoderm-like cells; (12) differentiating the endoderm-like cells obtained in step (11) into intestinal stem cell-like cells; (14) culturing the intestinal stem cell-like cells obtained in step (12) in the presence of epidermal growth factor and cAMP signal activator; , a MEK1/2 inhibitor, a DNA methylation inhibitor, a TGF ⁇ receptor inhibitor, and a cAMP signal activator.
  • culture conditions in each step constituting the present invention (first production method, second production method, and third production method) are conditions generally employed in culturing animal cells. And it is sufficient. That is, it may be cultured in an environment of, for example, 37°C and 5% CO 2 .
  • IMDM Iscove's modified Dulbecco's medium
  • GEBCO Ham F12 medium
  • D-MEM Dulbecco's modified Eagle's medium
  • RPMI1640 medium Two or more basal media may be used in combination.
  • a basal medium suitable for culturing epithelial cells for example, a mixed medium of D-MEM and Ham's F12 medium, D-MEM.
  • components that can be added to media include bovine serum albumin (BSA), antibiotics, 2-mercaptoethanol, PVA, non-essential amino acids (NEAA), insulin, transferrin, and selenium.
  • BSA bovine serum albumin
  • NEAA non-essential amino acids
  • insulin transferrin, and selenium.
  • Subculture may be performed during the steps (1) and (2) constituting the present invention.
  • the cells become confluent or subconfluent, some of the cells are harvested and transferred to another culture vessel to continue culturing.
  • a cell dissociation solution or the like may be used to collect cells.
  • the cell dissociation solution for example, trypsin-EDTA, collagenase IV, proteolytic enzymes such as metalloprotease, and the like can be used alone or in appropriate combination. Those with less cytotoxicity are preferred.
  • Commercial products such as Dispase (Edia), TrypLE (Invitrogen), and Accutase (MILLIPORE) are available as such cell dissociation solutions.
  • the recovered cells After treating them with a cell strainer or the like so that they are in a dispersed (discrete) state.
  • medium exchange is performed as necessary.
  • the medium may be replaced once every 24 hours to every 3 days.
  • the present invention relates to intestinal cells obtained by the production method of the present invention.
  • various assays are provided.
  • the intestinal cells of the present invention can be used in model systems of the intestinal tract, particularly the small intestine, and are useful for evaluation of pharmacokinetics (absorption, metabolism, etc.) and toxicity in the intestinal tract, particularly the small intestine.
  • the intestinal cells of the present invention are intended to be used for evaluating pharmacokinetics and toxicity of compounds.
  • the assay using intestinal cells of the present invention becomes a two-dimensional evaluation system, enabling higher throughput analysis.
  • the intestinal cells of the present invention can be used to test the metabolism, absorption, membrane permeability, drug interaction, induction of drug-metabolizing enzymes, induction of drug transporters, toxicity, etc. of a test substance.
  • the present invention provides a method for evaluating the metabolism, absorption, membrane permeability, drug interaction, induction of drug-metabolizing enzymes, induction of drug transporters, toxicity, etc. of a test substance as one of the uses of intestinal cells. I will provide a.
  • (I) a step of contacting a test substance with intestinal cells obtained by the production method of the present invention; A step of measuring and evaluating enzyme induction, drug transporter induction, or toxicity is performed.
  • Contact in step (I) is typically performed by adding the test substance to the medium.
  • the timing of addition of the test substance is not particularly limited. Therefore, after starting culture in a medium containing no test substance, the test substance may be added at a certain point, or culture may be started in advance in a medium containing the test substance.
  • Organic or inorganic compounds with various molecular sizes can be used as test substances.
  • organic compounds include nucleic acids, peptides, proteins, lipids (simple lipids, complex lipids (phosphoglycerides, sphingolipids, glycosylglycerides, cerebrosides, etc.), prostaglandins, isoprenoids, terpenes, steroids, polyphenols, catechins, vitamins (B1, B2, B3, B5, B6, B7, B9, B12, C, A, D, E, etc.) can be exemplified.
  • Existing ingredients or candidate ingredients such as pharmaceuticals, nutritional foods, food additives, agricultural chemicals, cosmetics (cosmetics), etc.
  • test substances Plant extracts, cell extracts, culture supernatants, etc. may be used as test substances.By adding two or more test substances at the same time, Interactions, synergistic effects, etc. between substances may be investigated, and the substance to be tested may be of natural origin or may be of synthetic origin, in the latter case, for example, by means of combinatorial synthesis techniques. It is possible to construct an efficient assay system by
  • the period of contact with 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 divided into multiple times.
  • step (I) the metabolism, absorption, membrane permeability, drug interactions, induction of drug-metabolizing enzymes, induction of drug transporters, or toxicity of the test substance are measured and evaluated (step (II)). .
  • step (I) that is, after contact with the test substance, measurement and evaluation of metabolism etc. without a substantial time interval, or after a certain period of time (for example, 10 minutes to 5 hours) Metabolism and the like may be measured and evaluated after the passage of time.
  • Metabolic measurements can be made, for example, by detecting metabolites.
  • the expected metabolites are usually measured qualitatively or quantitatively using the culture solution after step (I) as a sample.
  • An appropriate measurement method may be selected depending on the metabolite, and examples include mass spectrometry, liquid chromatography, immunological techniques (e.g., fluorescence immunoassay (FIA method), enzyme immunoassay (EIA method), ), etc. can be adopted.
  • FIA method fluorescence immunoassay
  • EIA method enzyme immunoassay
  • the metabolic rate of the test substance can be evaluated according to the amount of the metabolite.
  • the metabolic efficiency of the test substance may be calculated based on the metabolite detection results and the amount of the test substance used (typically, the amount added to the medium).
  • cytochrome P450 especially CYP3A4 in humans, CYP3A8 in cynomolgus monkeys
  • uridine diphosphate-glucuronyltransferase especially UGT1A8, UGT1A10
  • sulfotransferase especially SULT1A3, etc.
  • the residual amount of the test substance in the culture medium is measured.
  • the test substance is quantified using the culture medium after step (I) as a sample.
  • An appropriate measurement method may be selected depending on the test substance. For example, mass spectrometry, liquid chromatography, immunological techniques (for example, fluorescence immunoassay (FIA method), enzyme immunoassay (EIA method)) and the like can be employed.
  • FFA method fluorescence immunoassay
  • EIA method enzyme immunoassay
  • the absorption amount or absorption efficiency of the test substance can be determined and evaluated according to the degree of decrease. The absorption can also be evaluated by measuring the amount of the test substance taken into the cells.
  • the measurement/evaluation of metabolism and the measurement/evaluation of absorption may be performed at the same time or in parallel.
  • the intestinal cells obtained by the production method of the present invention can reproduce the pathology of intestinal diseases. Therefore, as a second use of the intestinal organoid of the present invention, an intestinal disease model, a method for producing the model, an assay using the same, and the like are provided.
  • an intestinal disease model for example, a pathological model of inflammatory bowel disease, a pathological model that reproduces tissue fibrosis that progresses due to inflammation in inflammatory bowel disease (fibrosis model), cancer Pathological models and the like can be produced.
  • inflammatory cytokines such as TNF- ⁇ , IFN- ⁇ , IL-1, IL-6, IL-17a (one or a combination of two or more) to induce inflammation or injury by culturing the intestinal cells of the present invention.
  • a disease model may be prepared by co-culturing with immune cells.
  • TGF- ⁇ may be used, or TNF- ⁇ and TNF- ⁇ may be used in combination to induce fibrosis.
  • Intestinal disease models are useful for drug screening. That is, by using an intestinal disease model, it is possible to construct an in vitro evaluation system for screening substances (active ingredients of pharmaceuticals or lead compounds) that are effective against intestinal diseases. In this evaluation system, the action and influence of the test substance on the pathological condition reproduced by the intestinal disease model are investigated, and the effectiveness thereof is evaluated.
  • a transplant material containing intestinal organoids is provided.
  • the implantable material of the present invention can be applied to treat various intestinal diseases (eg, intractable inflammatory bowel disease).
  • various intestinal diseases eg, intractable inflammatory bowel disease
  • it is expected to be used as a material for regeneration/reconstruction of damaged (including dysfunctional) intestinal tissue.
  • the graft material of the present invention can be used as a graft material as it is or after processing such as matrigel or collagen gel embedding. It is also envisioned to be used for screening of therapeutic drug candidate compounds as pathological models of various intestinal diseases, and for clarification of pathological mechanisms.
  • DMSO Dimethyl sulfoxide
  • serum albumin are used to protect cells
  • antibiotics are used to prevent bacterial contamination
  • various ingredients vitamins , cytokines, growth factors, steroids, etc.
  • other pharmaceutically acceptable components e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological saline, etc. It may be contained in the implant material of the present invention.
  • transplant material of the present invention can also be used to construct an in vivo experimental system.
  • transplant materials containing intestinal cells prepared using human pluripotent stem cells are transplanted into experimental animals such as mice, rats, guinea pigs, hamsters, pigs, cynomolgus monkeys, rhesus monkeys, and chimpanzees, and humanized animals (human intestinal model).
  • humanized animals are particularly useful for experiments such as pharmacokinetics and toxicity tests, and are expected to contribute to studies on the effects of the first-pass effect on oral drugs and drug-induced enteritis.
  • Intestinal cells prepared using iPS cells derived from patients with intestinal disease can be used as an intestinal disease model for drug evaluation systems. It can also be used for various experiments in research.
  • hiPSC Human induced pluripotent stem cells
  • iPS-51 Windy
  • hiPSC human fetal lung fibroblast MRC-5
  • octamer binding protein 3/4 OCT3/4
  • SOX2 sex determining region Y-box 2
  • KLF4 kruppel-like factor 4
  • c-MYC v-myc myelocytomatosis viral oncogene homolog
  • c-MYC v-myc myelocytomatosis viral oncogene homolog
  • Mouse embryonic fibroblasts (MEF) were used as feeder cells.
  • HiPSC-derived intestinal organoids HIOs
  • Matrigel growth factor removed
  • RPMI Roswell Park Memorial Institute
  • human iPSCs were cultured in RPMI + glutamax medium containing 2% FBS, 500 ng/mL FGF4, 3 ⁇ mol/L CHIR99021, 100 units/mL penicillin G, and 100 ⁇ g/mL streptomycin for 4 days to transform them into intestinal stem cells. differentiated. After FGF4 and CHIR99021 treatment, Y-27632 (Rho-binding kinase inhibitor) was added to 10 ⁇ mol/L and treated for 60 minutes at 37°C in a CO 2 incubator under 5% CO 2 /95% air conditions.
  • Y-27632 Rho-binding kinase inhibitor
  • Cells were detached with 0.05% trypsin-EDTA, cell clumps were crushed with a 40 ⁇ m nylon mesh cell strainer, diluted to 0.16 ⁇ g/mL with PBS (-) and placed on a 100 mm dish coated with iMatrix 511. ⁇ 10 6 cells were seeded. Cells were treated with 1% glutamax, 2% FBS, 100 units/mL penicillin G, 100 ⁇ g/mL streptomycin, 100 ng/mL epidermal growth factor (EGF), 30 ng/mL FGF2, 0.5 ⁇ mol/L A-83- 01, 3 ⁇ mol/L CHIR99021 and 10 ⁇ mol/L Y-27632 were cultured in Advanced-DMEM/F12.
  • the cells were detached with Accutase up to twice every 3 days, and 2.0 ⁇ 10 6 cells were seeded on a 100 mm dish coated with iMatrix 511 diluted to 0.16 ⁇ g/mL with PBS(-).
  • Cells were treated with 1% glutamax, 2% FBS, 100 units/mL penicillin G, 100 ⁇ g/mL streptomycin, 100 ng/mL epidermal growth factor (EGF), 30 ng/mL FGF2, 0.5 ⁇ mol/L A-83- 01, 3 ⁇ mol/L CHIR99021 and 10 ⁇ mol/L Y-27632 were cultured in Advanced-DMEM/F12.
  • Advanced-DMEM/F12 containing 10 ⁇ mol/L Y-27632 After 3 days of culture in Advanced-DMEM/F12 containing 10 ⁇ mol/L Y-27632, add 1% glutamax, 2% B27 supplement, 1% N2 supplement, 100 units/mL penicillin G, 100 ⁇ g/mL streptomycin, 100 ng/mL EGF, 100 ng/mL Noggin, 200 ng/mL R-spondin-1, Advanced-DMEM/F12 containing 3% growth factor-free matrigel for 9 to 12 days on a 100 mm dish with ultra-low adhesion They were differentiated into HIOs by floating culture at .
  • HIOs-Derived Intestinal Cells Twenty-four hours before the end of HIOs culture, 10 ⁇ M Y-27632 was applied. HIOs were collected in a centrifuge tube and washed twice with D-PBS(-). After centrifugation at 160 xg for 5 minutes, the supernatant was removed by aspiration. 2 mL of 0.5 nM EDTA was added and suspended. After standing at room temperature for 5 minutes, it was washed with D-PBS(-). After centrifugation at 160 xg for 5 minutes, the supernatant was removed by aspiration.
  • RNA extraction Total ribonucleic acid (RNA) extraction.
  • RNA was extracted according to the attached manual of Agencourt RNAdvence Tissue after completion of differentiation induction.
  • HE Hematoxylin-Eosin Staining
  • the intestinal organoids were fixed with 4% paraformaldehyde and cryo-embedded with OCT compound. Cryosections with a thickness of 10 ⁇ m were prepared, attached to glass slides, and stained with Mayer's hematoxylin and eosin alcohol.
  • HIOs-derived intestinal cells produced by air-liquid interface culture have a lower transepithelial electrical resistance and are more stable than those produced by liquid-phase culture.
  • Fig. 4A The TEER of living intestinal tissue ranged from 80 ⁇ cm 2 to 150 ⁇ cm 2 , which were very close values.
  • RT-qPCR revealed that the expression levels of gut-related genes such as intestinal epithelial marker villin, goblet cell marker MUC2, intestinal stem cell marker LGR5, and Paneth cell marker LYZ were higher than in living intestinal tissue. did.
  • CYP3A4 a major metabolic enzyme in the intestinal tract
  • nuclear receptor PXR nuclear receptor that induces its expression
  • PEPT1 peptide transporter 1
  • FIG. 4C An induction test of the CYP3A4 gene via PXR and VDR using rifampicin and activated vitamin D 3 (VD3) confirmed a significant increase in the expression level (Fig. 4C).
  • Rhodamine123 via MDR1, a multidrug efflux transporter showed preferential transport in the efflux direction, and the inhibitor verapamil significantly reduced the Efflux ratio (ER) (Fig. 4D).
  • Example A HIOs were developed in two-dimensional culture, and intestinal cells having a crypt-villus-like three-dimensional structure were successfully produced.
  • gas-liquid interface culture is necessary for this conformation, and that cAMP, TGF- ⁇ , Wnt and Notch signals are involved.
  • CHIR99021 and DAPT which are low-molecular-weight compounds involved in Wnt and Nocth signals, is also important.
  • the transepithelial electrical resistance of the prepared intestinal cells was similar to that of living intestinal tissue compared to conventional liquid-phase culture, suggesting that the development of excessive barrier function could be suppressed.
  • Example B> ⁇ Method> (1) Cells Windy strain was used as human iPSCs. Both cell lines were donated by Dr. Akihiro Umezawa of the National Center for Child Health and Development. MEFs were used as feeder cells for maintenance culture of iPSCs (on-feeder culture).
  • MEF cultures contain 10% fetal bovine serum (FBS), 2 mmol/L L-glutamine (L-Glu), 1% non-essential amino acids (NEAA), 100 units/mL penicillin G, 100 ⁇ g/mL streptomycin Dulbecco's Modified Eagle Medium (DMEM) was used. 0.05% trypsin-ethylenediaminetetraacetic acid (EDTA) was used as the MEF stripping solution, and Cellbanker 1 was used as the MEF preserving solution.
  • FBS fetal bovine serum
  • L-Glu L-glutamine
  • NEAA non-essential amino acids
  • EDTA trypsin-ethylenediaminetetraacetic acid
  • Feederless culture The medium used was mTeSR1. 0.5 mmol/L EDTA was used as a stripping solution for human iPSCs. StemSure (R) cryopreservation solution was used as the preservation solution for human iPSCs.
  • Feederless culture Matrigel from which growth factors were removed was diluted 30-fold with DMEM/F12, and a cell culture plate was coated with this diluted solution. The coated plate was placed at 4°C for 10 hours or longer. 30 minutes before use, it was brought back to room temperature.
  • Human iPSCs were seeded on matrigel-coated plates and cultured at 37°C in a CO 2 incubator under 5% CO 2 /95% air conditions. Human iPSCs were passaged at a split ratio of 1:6 after 3-5 days of culture. When human iPSCs were thawed, Y-27632 was added to 10 ⁇ mol/L, the medium was changed 24 hours later, and thereafter changed every day.
  • Differentiation into intestinal epithelial cells was performed using a standard protocol for the differentiation method. 5 ⁇ mol/L 5-aza-2'-deoxycytidine for 12 days (days 14-26 after initiation of differentiation), 0.5 ⁇ mol/L A-83-01 for 18 days (days 8-26 after initiation of differentiation) ) or for 12 days (days 14 to 26 after the initiation of differentiation) and 2 ⁇ mol/L CHIR-99021 for 18 days (days 8 to 26 after the initiation of differentiation) to differentiate into intestinal epithelial cells. . The medium on the apical side of the insert was removed, and gas phase liquid phase culture was performed from day 8 or day 14 of differentiation to the end of differentiation.
  • RPMI medium containing 0.2% B27 supplement 100 ng/mL activin A, 50 nmol/L PI-103, 2 ⁇ mol/L CHIR99021, 100 units/mL penicillin G, 100 ⁇ g/mL streptomycin, 1% NEAA for 2 days ( 0 to 1 days after initiation of differentiation)
  • RPMI medium containing 0.2% B27 supplement 100 ng/mL activin A, 20 ng/mL bFGF, 100 units/mL penicillin G, 100 ⁇ g/mL streptomycin, and 1% NEAA for 1 day (days 2-3 after initiation of differentiation)
  • 0.2% B27 supplement 100 ng/mL activin A, 20 ng/mL bFGF, 100 units/mL penicillin G, 100 ⁇ g/mL streptomycin,
  • Differentiation of endoderm into intestinal epithelial cells was performed using human iPSC-derived endoderm in DMEM/F12 containing 2% FBS, 2% B27 supplement, 1% glutamax, and 250 ng/mL FGF2 for 4 days (after initiation of differentiation). Days 8 to 11)
  • the cells were differentiated into intestinal stem cells by culturing. After FGF2 treatment, Y-27632 was added to 10 ⁇ mol/L, and the cells were treated for 60 minutes at 37°C in a CO 2 incubator under 5% CO 2 /95% air conditions.
  • the cells were seeded in a 24-well-insert for cell culture coated with matrigel from which growth factors were removed and diluted 30-fold with DMEM/F12.
  • RNA Total Ribonucleic Acid
  • -aza-2'-deoxycytidine addition group (Figs. 10 and 11).
  • the TEER value in the gas phase culture group was lower than that in the liquid phase culture group, but the transepithelial electrical resistance value was higher than 150 ⁇ cm 2 in both groups (Fig. 13). ).
  • the gene expression levels of Sucrase-isomaltase, CYP3A4, Muc2, Pgp and OLFM4 were higher in the gas-phase culture group than in the liquid-phase culture group.
  • the amount of gene expression of BCRP decreased by performing gas phase culture, it had a higher expression level than that of human adult small intestine (Figs. 14 and 15).
  • human iPSC-derived small intestinal epithelial cells can have a crypt-villus-like structure under conditions without addition of 5-aza-2'-deoxycytidine and addition of CHIR99021. became possible. It was found that differentiated intestinal epithelial cells could form tight junctions under gas phase culture conditions. Regardless of the maintenance culture method of human iPSCs, human iPSC-derived intestinal epithelial cells can enhance the gene-level expression of small intestinal epithelial cell markers, drug transporters, and drug-metabolizing enzymes by gas-phase culture under similar differentiation conditions. shown.
  • gas phase culture for 6 days prior to the maturation of differentiated cells contributes to the improvement of the expression level of each marker gene. became. Therefore, it was suggested that gas-phase culture is useful for inducing the differentiation of human iPSCs into small intestinal epithelial cells.
  • Example C> ⁇ Method> (1) Cells F-hiSIEC TM commercially available from FUJIFILM was used as human iPS cell-derived intestinal epithelial cells.
  • F-hiSIEC TM After thawing F-hiSIEC TM and suspending it in F-hiSIEC TM Seeding Medium, it was inoculated onto a 24-well cell culture insert pre-coated with Matrigel and placed in 5% CO 2 /95. It was cultured at 37°C in a CO 2 incubator under % air conditions. The next day, the medium was replaced with F-hiSIEC TM Culture Medium containing or not containing 5-aza-2'-deoxycitidine, and thereafter the medium was changed three times a week. The medium on the apical side was removed from day 8 after cell seeding when gas phase culture was performed for 3 days, and from day 4 after cell seeding when gas phase culture was performed for 7 days.
  • TEER value was measured at the timing of medium exchange from day 4 after cell seeding. Millicell ERS-2 was used for the measurement.
  • HBSS-MES Hanks' balanced salt solution
  • HBSS-HEPES Hanks' balanced salt solution
  • dQ/dt is the amount of compound permeated per unit time
  • A is the surface area of the cell culture insert
  • C0 is the initial concentration of the compound in the donor chamber.
  • RNA Total ribonucleic acid extraction
  • FIGS. 23 to 26 The results of the membrane permeation test and metabolic test of midazolam in the first F-hiSIEC TM culture experiment are shown in FIGS. 23 to 26.
  • FIG. 24 a decrease in apparent permeability coefficient was observed in the group subjected to gas phase culture.
  • FIG. 26 in the gas phase culture group, the amount of metabolites produced was large, and the group without addition of 5-aza-2'-deoxycitidine produced more metabolites than the group with addition of 5-aza-2'-deoxycitidine. increased the production of
  • FIG. 36 shows the results of the midazolam metabolism test in the second F-hiSIEC TM culture experiment.
  • an increase in CYP3A4 metabolic activity was observed in the group to which 5-aza-2'-deoxycitidine was not added.
  • Lucifer yellow Membrane Permeation Test The results of the Lucifer yellow membrane permeation test in the second F-hiSIEC TM culture experiment are shown in FIG. In FIG. 31, a decrease in the apparent membrane permeability coefficient of Lucifer yellow (increase in barrier function) was observed in the group to which 5-aza-2'-deoxycitidine was not added.

Abstract

The purpose of the present invention is to provide a production method for intestinal tract cells derived from pluripotent stem cells and having crypt-villus-like structures, said method making it possible to achieve crypt-villus-like structures like those in a living intestinal tract as well as further achieve efficient differentiation into intestinal tract epithelial cells and functional improvement of said cells. According to the present invention, provided is a method for producing intestinal tract cells from pluripotent stem cells, said method including (1) a step for differentiating pluripotent stem cells into endoderm-like cells, (2) a step for differentiating the endoderm-like cells into intestinal tract stem cell-like cells, (3) a step for culturing the intestinal tract stem cell-like cells in the presence of certain factors, (4) a step for culturing the aforementioned cells and forming spheroids, (5) a step for differentiating the spheroids and forming intestinal tract organoids, and (6) a step for gas-phase/liquid-phase culturing of cells constituting the intestinal tract organoids in the presence of certain factors.

Description

多能性幹細胞由来の陰窩－絨毛様構造を有する腸管細胞の製造方法及びその用途Method for producing intestinal cells having crypt-villus-like structure derived from pluripotent stem cells and uses thereof
　本発明は、多能性幹細胞由来の陰窩－絨毛様構造を有する腸管細胞の製造方法及びその用途に関する。 The present invention relates to a method for producing intestinal cells having crypt-villus-like structures derived from pluripotent stem cells and uses thereof.
　医薬品の多くは経口投与薬であり、新薬開発において、腸管での薬物の吸収及び代謝の正確な予測が重要である。現在、小腸のモデル系としてはヒト結腸癌由来のCaco-2細胞が多用されている。しかし、Caco-2細胞における薬物トランスポーターの発現パターンはヒト小腸とは異なる。また、Caco-2細胞においては、小腸の主要な薬物代謝酵素であるCYP3A4の発現およびPXRのリガンドによるCYP3A4の誘導はほとんど認められないことから、正確に小腸での薬物動態を評価することは難しい。 Many pharmaceuticals are orally administered drugs, and accurate prediction of drug absorption and metabolism in the intestinal tract is important in new drug development. Currently, Caco-2 cells derived from human colon cancer are frequently used as a small intestine model system. However, the expression pattern of drug transporters in Caco-2 cells differs from that in the human small intestine. In Caco-2 cells, expression of CYP3A4, a major drug-metabolizing enzyme in the small intestine, and induction of CYP3A4 by PXR ligands are hardly observed, making it difficult to accurately evaluate pharmacokinetics in the small intestine. .
　また、比較的ヒトのin vivoに近い評価系として動物モデルを利用した評価系もあるが、ヒトとの種差や倫理的な問題があるため、動物を用いない代替法への置換や動物数の削減が推進されている。 In addition, there are evaluation systems that use animal models as evaluation systems that are relatively close to human in vivo, but due to species differences from humans and ethical problems, it is necessary to replace them with alternative methods that do not use animals, and to increase the number of animals. Reduction is being promoted.
　したがって、小腸における薬物代謝および膜透過性を総合的に評価するためには初代小腸上皮細胞の利用が望ましいが、これは入手自体が困難である。そこで我々は、このような評価系への応用を目指し、ヒト人工多能性幹細胞（induced pluripotent stem cells：iPSC）から腸管細胞への分化誘導研究を進めている。 Therefore, it is desirable to use primary small intestinal epithelial cells to comprehensively evaluate drug metabolism and membrane permeability in the small intestine, but they are difficult to obtain. Therefore, we are conducting research on induction of differentiation from human induced pluripotent stem cells (iPSCs) to intestinal cells with the aim of applying them to such an evaluation system.
　近年ヒト人工多能性幹細胞（hiPSC）研究の発展が著しく、腸上皮細胞（二次元培養）や腸管オルガノイド（三次元培養）への分化誘導法が数多く報告されている。特に腸上皮細胞は薬物動態研究への応用も進められており、従来の評価系よりも精度の高い評価が可能になってきている。一方で、腸粘膜層や腸内細菌叢も薬物の代謝吸収に関与していることも明らかになってきており、より生体内環境を模倣した腸管評価系の構築が必要とされている。 In recent years, research on human induced pluripotent stem cells (hiPSC) has made remarkable progress, and many methods for inducing differentiation into intestinal epithelial cells (two-dimensional culture) and intestinal organoids (three-dimensional culture) have been reported. In particular, intestinal epithelial cells are being applied to pharmacokinetic studies, and evaluation with higher accuracy than conventional evaluation systems is becoming possible. On the other hand, it has become clear that the intestinal mucosal layer and intestinal microbiota are also involved in drug metabolism and absorption.
　特許文献１には、（１）多能性幹細胞を内胚葉様細胞へと分化させる工程、（２）工程（１）で得られた内胚葉様細胞を腸管幹細胞様細胞へと分化させる工程、（３）工程（２）で得られた腸管幹細胞様細胞を、上皮成長因子、線維芽細胞増殖因子、TGFβ受容体阻害剤、GSK-3β阻害剤及びROCK阻害剤の存在下で培養する工程、（４）工程（３）後の細胞を培養し、スフェロイドを形成させる工程、（５）工程（４）で形成されたスフェロイドを分化させ、腸管オルガノイドを形成させる工程であって、上皮成長因子、BMP阻害剤及びWntシグナル活性化剤の存在下での培養を含む工程によって、多能性幹細胞から腸管オルガノイドを作製することが記載されている。 Patent Document 1 discloses (1) a step of differentiating pluripotent stem cells into endoderm-like cells, (2) a step of differentiating the endoderm-like cells obtained in step (1) into intestinal stem cell-like cells, (3) culturing the intestinal stem cell-like cells obtained in step (2) in the presence of epidermal growth factor, fibroblast growth factor, TGFβ receptor inhibitor, GSK-3β inhibitor and ROCK inhibitor; (4) culturing the cells after step (3) to form spheroids, (5) differentiating the spheroids formed in step (4) to form intestinal organoids, comprising epidermal growth factor, Generation of intestinal organoids from pluripotent stem cells is described by a process involving culturing in the presence of a BMP inhibitor and a Wnt signal activator.
国際公開ＷＯ２０２０／０９１０２０号公報International publication WO2020/091020
　本発明者らは、これまでｈｉＰＳＣ由来腸管オルガノイド（ＨＩＯｓ）の分化誘導法を確立している。本発明者らはさらに、その培養技術を応用することで、腸管特有の陰窩－絨毛構造を模倣した腸管評価系（二次元ＨＩＯｓ）の構築が可能になると考え、腸管オルガノイドの二次元培養に取り組んできた。しかし、作製できた腸管細胞は単層構造であり、生体腸管組織の立体構造の模倣は実現していない。 The present inventors have established a method for inducing differentiation of hiPSC-derived intestinal organoids (HIOs). The present inventors further thought that by applying the culture technology, it would be possible to construct an intestinal evaluation system (two-dimensional HIOs) that mimics the crypt-villus structure peculiar to the intestine. been working on it. However, the produced intestinal cells have a single-layer structure, and imitation of the three-dimensional structure of living intestinal tissue has not been realized.
　本発明は、生体腸管様な陰窩－絨毛様構造を獲得し、さらに腸管上皮細胞への分化効率及びその細胞の機能向上を図ることができるような、多能性幹細胞由来の陰窩－絨毛様構造を有する腸管細胞の製造方法を提供することを解決すべき課題とした。 The present invention is a pluripotent stem cell-derived crypt-villus that can acquire a crypt-villus-like structure similar to the intestinal tract in vivo, and can improve the efficiency of differentiation into intestinal epithelial cells and the function of the cells. The problem to be solved was to provide a method for producing intestinal cells having a similar structure.
　本発明者らは上記課題を解決することを目的として、気相液相界面培養や低分子化合物の添加等で培養方法の改良を行い、形態やバリア機能、遺伝子発現に加え、薬物動態学的な評価を行うことで、新たな腸管評価系として有用であるかを検討した。その結果、気相液相界面培養を使用することにより、多能性幹細胞由来の陰窩－絨毛様構造を有する腸管細胞を製造できることを見出した。本発明は上記知見に基づいて完成したものである。即ち、本発明によれば以下の発明が提供される。 In order to solve the above problems, the present inventors improved the culture method by gas-liquid interface culture and addition of low-molecular compounds, etc., and in addition to morphology, barrier function, gene expression, pharmacokinetic We examined whether it would be useful as a new intestinal evaluation system by performing such evaluation. As a result, it was found that intestinal cells having pluripotent stem cell-derived crypt-villus-like structures can be produced by using gas-liquid interface culture. The present invention has been completed based on the above findings. That is, according to the present invention, the following inventions are provided.
＜１＞　以下の工程（１）～（６）を含む、多能性幹細胞から腸管細胞を作製する方法：
（１）多能性幹細胞を内胚葉様細胞へと分化させる工程；
（２）工程（１）で得られた内胚葉様細胞を腸管幹細胞様細胞へと分化させる工程；
（３）工程（２）で得られた腸管幹細胞様細胞を、上皮成長因子、線維芽細胞増殖因子、TGFβ受容体阻害剤、GSK-3β阻害剤及びROCK阻害剤の存在下で培養する工程；
（４）工程（３）後の細胞を培養し、スフェロイドを形成させる工程；
（５）工程（４）で形成されたスフェロイドを分化させ、腸管オルガノイドを形成させる工程であって、上皮成長因子、BMP阻害剤及びWntシグナル活性化剤の存在下での培養を含む工程；及び
（６）工程（５）で形成された腸管オルガノイドを構成する細胞を、上皮成長因子、cAMPシグナル活性化因子、TGFβ受容体阻害剤、及びWntシグナル活性化剤の存在下で気相液相培養する工程。
＜２＞　工程（３）において、線維芽細胞増殖因子がFGF2、FGF4又はFGF10であり、TGFβ受容体阻害剤がA-83-01であり、GSK-3β阻害剤がCHIR99021、SB216763、CHIR98014、TWS119、Tideglusib、SB415286、BIO、AZD2858、AZD1080、AR-A014418、TDZD-8、LY2090314、IM-12、Indirubin、Bikinin又は1-Azakenpaulloneであり、ROCK阻害剤がY-27632である、＜１＞に記載の方法。
＜３＞　工程（５）において、BMP阻害剤がNogginであり、Wntシグナル活性化剤がR-spondin-1である、＜１＞　又は＜２＞に記載の方法。
＜４＞　工程（６）において、cAMPシグナル活性化因子がフォルスコリン、8-Br-cAMP又はIBMXであり、TGFβ受容体阻害剤がA-83-01であり、Wntシグナル活性化剤がCHIR99021である、＜１＞～＜３＞のいずれか一に記載の方法。
＜５＞　工程（６）の気相液相培養を、上皮成長因子、cAMPシグナル活性化因子、TGFβ受容体阻害剤、及びWntシグナル活性化剤に加え、Notchシグナル阻害剤の存在下で行う、＜１＞～＜４＞のいずれか一に記載の方法。
＜６＞　Notchシグナル阻害剤が、DAPTである、＜５＞　に記載の方法。
＜７＞　工程（６）の気相液相培養を、Notchシグナル阻害剤の存在下で行った後に、Notchシグナル阻害剤の非存在下で行う、＜５＞　又は＜６＞に記載の方法。
＜８＞　工程（６）の気相液相培養を、４日～３０日間行う、＜１＞～＜７＞のいずれか一に記載の方法。
＜９＞　以下の工程（１）、（２）、（４）および（５）を含み、工程（４）及び工程（５）、工程（５）、又は工程（５）の培養の一部が気相液相培養である、多能性幹細胞から腸管細胞を作製する方法：
（１）多能性幹細胞を内胚葉様細胞へと分化させる工程；
（２）工程（１）で得られた内胚葉様細胞を腸管幹細胞様細胞へと分化させる工程；
（４）工程（２）後の細胞を、上皮成長因子及びcAMPシグナル活性化因子の存在下で培養する工程；
（５）工程（４）後の細胞を、上皮成長因子、MEK1/2阻害剤、DNAメチル化阻害剤、TGFβ受容体阻害剤、及びcAMPシグナル活性化因子の存在下で培養する工程。
＜１０＞　工程（２）と工程（４）の間に、工程（３）をさらに含む、＜９＞に記載の方法。
（３）工程（２）で得られた腸管幹細胞様細胞を、上皮成長因子、及びROCK阻害剤の存在下で培養する工程；
＜１１＞　多能性幹細胞から作製された腸管細胞を、5-アザ-2'-デオキシシチジンを含まない培地で培養することを含む、腸管細胞を作製する方法。
＜１２＞　5-アザ-2'-デオキシシチジンを含まない培地での培養の少なくとも一部が、気相液相培養である、＜１１＞に記載の方法。
＜１３＞　多能性幹細胞から作製された腸管細胞が、以下の工程（１１）～（１４）を含む方法により得られた細胞である、＜１１＞又は＜１２＞に記載の方法。
（１１）多能性幹細胞を内胚葉様細胞へと分化させる工程；
（１２）工程（１１）で得られた内胚葉様細胞を腸管幹細胞様細胞へと分化させる工程；
（１４）工程（１２）で得られた腸管幹細胞様細胞を、上皮成長因子及びcAMPシグナル活性化因子の存在下で培養する工程；および
（１５）工程（１４）後の細胞を、上皮成長因子、MEK1/2阻害剤、DNAメチル化阻害剤、TGFβ受容体阻害剤、及びcAMPシグナル活性化因子の存在下で培養する工程。
＜１４＞　多能性幹細胞が人工多能性幹細胞又は胚性幹細胞である、＜１＞～＜１３＞のいずれか一に記載の方法。
＜１５＞　多能性幹細胞がヒト人工多能性幹細胞である、＜１＞～＜１４＞のいずれか一に記載の方法。
＜１６＞　多能性幹細胞が、フィーダーレスで培養した多能性幹細胞である、＜１＞～＜１５＞のいずれか一に記載の方法。
＜１７＞　＜１＞～＜１６＞のいずれか一に記載の方法で得られた腸管細胞。
＜１８＞　＜１７＞に記載の腸管細胞を用いた、被検物質の体内動態又は毒性を評価する方法。
＜１９＞　前記体内動態が、代謝、吸収性、膜透過性、薬物相互作用、薬物代謝酵素の誘導、又は薬物トランスポーターの誘導である、＜１８＞に記載の方法。
＜２０＞　以下の工程（ｉ）及び（ｉｉ）を含む、＜１８＞又は＜１９＞に記載の方法：
（ｉ）＜１７＞に記載の腸管細胞に被検物質を接触させる工程；
（ｉｉ）被検物質の代謝、吸収性、膜透過性、薬物相互作用、薬物代謝酵素の誘導、又は薬物トランスポーターの誘導、或いは毒性を評価する工程。
＜２１＞　＜１＞～＜１６＞のいずれか一に記載の方法で得られた腸管細胞に腸疾患の病態を誘導することを特徴とする、腸疾患モデルの作製方法。 <1> A method for producing intestinal cells from pluripotent stem cells, comprising the following steps (1) to (6):
(1) differentiating pluripotent stem cells into endoderm-like cells;
(2) a step of differentiating the endoderm-like cells obtained in step (1) into intestinal stem cell-like cells;
(3) culturing the intestinal stem cell-like cells obtained in step (2) in the presence of epidermal growth factor, fibroblast growth factor, TGFβ receptor inhibitor, GSK-3β inhibitor and ROCK inhibitor;
(4) culturing the cells after step (3) to form spheroids;
(5) differentiating the spheroids formed in step (4) to form intestinal organoids, comprising culturing in the presence of an epidermal growth factor, a BMP inhibitor and a Wnt signal activator; and (6) Cells composing the intestinal organoids formed in step (5) are cultured in gas-phase liquid phase in the presence of epidermal growth factor, cAMP signal activator, TGFβ receptor inhibitor, and Wnt signal activator. process to do.
<2> In step (3), the fibroblast growth factor is FGF2, FGF4 or FGF10, the TGFβ receptor inhibitor is A-83-01, and the GSK-3β inhibitor is CHIR99021, SB216763, CHIR98014, TWS119 , Tideglusib, SB415286, BIO, AZD2858, AZD1080, AR-A014418, TDZD-8, LY2090314, IM-12, Indirubin, Bikinin or 1-Azakenpaullone, and the ROCK inhibitor is Y-27632, described in <1> the method of.
<3> The method according to <1> or <2>, wherein in step (5), the BMP inhibitor is Noggin and the Wnt signal activator is R-spondin-1.
<4> In step (6), the cAMP signal activator is forskolin, 8-Br-cAMP or IBMX, the TGFβ receptor inhibitor is A-83-01, and the Wnt signal activator is CHIR99021. The method according to any one of <1> to <3>.
<5> The gas-liquid phase culture in step (6) is performed in the presence of an epidermal growth factor, a cAMP signal activator, a TGFβ receptor inhibitor, and a Wnt signal activator, as well as a Notch signal inhibitor. The method according to any one of <1> to <4>.
<6> The method according to <5>, wherein the Notch signal inhibitor is DAPT.
<7> The method according to <5> or <6>, wherein the gas-liquid phase culture in step (6) is performed in the presence of a Notch signal inhibitor and then in the absence of a Notch signal inhibitor.
<8> The method according to any one of <1> to <7>, wherein the gas-liquid culture in step (6) is performed for 4 to 30 days.
<9> including the following steps (1), (2), (4) and (5), wherein step (4) and step (5), step (5), or part of the culture of step (5) A method for producing intestinal cells from pluripotent stem cells, which is a gas phase liquid phase culture:
(1) differentiating pluripotent stem cells into endoderm-like cells;
(2) a step of differentiating the endoderm-like cells obtained in step (1) into intestinal stem cell-like cells;
(4) culturing the cells after step (2) in the presence of epidermal growth factor and cAMP signal activator;
(5) A step of culturing the cells after step (4) in the presence of epidermal growth factor, MEK1/2 inhibitor, DNA methylation inhibitor, TGFβ receptor inhibitor, and cAMP signal activator.
<10> The method according to <9>, further comprising step (3) between steps (2) and (4).
(3) culturing the intestinal stem cell-like cells obtained in step (2) in the presence of epidermal growth factor and a ROCK inhibitor;
<11> A method for producing intestinal cells, comprising culturing intestinal cells produced from pluripotent stem cells in a medium that does not contain 5-aza-2'-deoxycytidine.
<12> The method according to <11>, wherein at least part of the culture in the medium that does not contain 5-aza-2'-deoxycytidine is gas phase liquid phase culture.
<13> The method according to <11> or <12>, wherein the intestinal cells produced from pluripotent stem cells are cells obtained by a method comprising the following steps (11) to (14).
(11) differentiating the pluripotent stem cells into endoderm-like cells;
(12) differentiating the endoderm-like cells obtained in step (11) into intestinal stem cell-like cells;
(14) culturing the intestinal stem cell-like cells obtained in step (12) in the presence of epidermal growth factor and cAMP signal activator; , a MEK1/2 inhibitor, a DNA methylation inhibitor, a TGFβ receptor inhibitor, and a cAMP signal activator.
<14> The method according to any one of <1> to <13>, wherein the pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells.
<15> The method according to any one of <1> to <14>, wherein the pluripotent stem cells are human induced pluripotent stem cells.
<16> The method according to any one of <1> to <15>, wherein the pluripotent stem cells are feederless cultured pluripotent stem cells.
<17> Intestinal cells obtained by the method according to any one of <1> to <16>.
<18> A method for evaluating pharmacokinetics or toxicity of a test substance using the intestinal cells of <17>.
<19> The method of <18>, wherein the pharmacokinetics is metabolism, absorption, membrane permeability, drug interaction, induction of drug-metabolizing enzymes, or induction of drug transporters.
<20> The method according to <18> or <19>, comprising the following steps (i) and (ii):
(i) contacting the test substance with the intestinal cells according to <17>;
(ii) evaluating the metabolism, absorption, membrane permeability, drug interaction, induction of drug-metabolizing enzymes, induction of drug transporters, or toxicity of the test substance;
<21> A method for preparing an intestinal disease model, comprising inducing an intestinal disease state in the intestinal cells obtained by the method according to any one of <1> to <16>.
　本発明によれば、生体腸管構造に近い、陰窩－絨毛様構造を有する腸管細胞を多能性幹細胞由来から製造することができる。 According to the present invention, intestinal cells having a crypt-villus-like structure close to the intestinal structure of a living body can be produced from pluripotent stem cells.
図１は、気相液相界面培養およびcAMPシグナル、TGF-βシグナル制御を行い作製したHIOs由来腸管細胞の形態観察を示す。培養期間は10日間で、気相液相界面培養はday3からday10まで行った。気/液：気相液相界面培養、液/液：液相培養、Forskolin：10 μM, A-83-01 :0.5 μM, 8-Br-cAMP：1 mM, IBMX：500 μM. スケールバー：100 μm.FIG. 1 shows the morphological observation of HIOs-derived intestinal cells prepared by gas-liquid interface culture and control of cAMP signal and TGF-β signal. The culture period was 10 days, and the gas-liquid interface culture was performed from day 3 to day 10. Air/liquid: gas-liquid interfacial culture, liquid/liquid: liquid phase culture, Forskolin: 10 μM, A-83-01: 0.5 μM, 8-Br-cAMP: 1 mM, IBMX: 500 μM. Scale bar: 100μm. 図２は、長期培養におけるWntシグナルおよびNotchシグナルの影響を示す。培養日数は22日間で、CHIR99021とDAPTの添加期間を４日間もしくは22日間とした。CHIR99021：3 μM, DAPT：2.5 μM. スケールバー：200 μm.Figure 2 shows the effect of Wnt and Notch signals in long-term culture. The number of culture days was 22 days, and the addition period of CHIR99021 and DAPT was 4 days or 22 days. CHIR99021: 3 μM, DAPT: 2.5 μM. Scale bar: 200 μm. 図３は、HIOs由来腸管細胞の三次元的な構造観察を示す。(A) 培養10日目のHIOs由来腸管細胞をCell3iMager Estierを用いて撮像した画像。黄色の点線は細胞表面を示す。スケールバー：100 μm. (B) (A)の画像をフィルター処理し二値化した後、Z方向のシグナルを抽出したもの。(C) (B)の画像をZ方向にスタックしたもの。(D) 10日間の培養終了後のH&E染色画像。スケールバー：50 μm.FIG. 3 shows three-dimensional structure observation of HIOs-derived intestinal cells. (A) Images of HIOs-derived intestinal cells on day 10 of culture taken using Cell3iMager Estier. Yellow dotted line indicates the cell surface. Scale bar: 100 μm. (B) Z-direction signal extracted after filtering and binarizing the image in (A). (C) Images from (B) stacked in the Z direction. (D) H&E staining image after 10 days of culture. Scale bar: 50 μm. 図４は、HIOs由来腸管細胞のバリア機能、遺伝子発現および薬物動態学的機能の解析を示す。(A) 経上皮電気抵抗値測定による腸管バリア機能評価。Mean±S.D. 液/液：n = 2, 気/気：n = 3. (B) RT-qPCRによる遺伝子発現解析。Mean±S.D. (n = 3). 生体腸管組織 = 100. (C) 誘導剤処理時間：48時間、Rifampicin：20 μM, VD3：0.1 μM. Mean±S.D. (n = 4). (D) Mean±S.D. (n = 4). 基質処理時間：30 min、基質：Rhodamine123（10 μM）、阻害剤：ベラパミル（100 μM）、A to B：apicalからbasalへの透過量、B to A：basalからapicalへの透過。Efflux ratio（ER）＝B to AのPapp/A to BのPapp FIG. 4 shows analysis of barrier function, gene expression and pharmacokinetic function of HIOs-derived intestinal cells. (A) Evaluation of intestinal barrier function by transepithelial electrical resistance measurement. Mean±SD liquid/liquid: n = 2, gas/gas: n = 3. (B) Gene expression analysis by RT-qPCR. Mean±SD (n = 3). Living intestinal tissue = 100. (C) Inducing agent treatment time: 48 hours, Rifampicin: 20 μM, VD3: 0.1 μM. Mean±SD (n = 4). (D) Mean± SD (n = 4). Substrate treatment time: 30 min, substrate: Rhodamine123 (10 μM), inhibitor: verapamil (100 μM), A to B: apical to basal penetration, B to A: basal to apical transmission to Efflux ratio (ER) = P app of B to A / P app of A to B 図５は、経上皮電気抵抗および遺伝子発現の経時的解析を示す。Mean±S.D. (n = 3). 生体腸管組織（ASI） = 1.  (A) 経上皮電気抵抗値測定による腸管バリア機能評価。(B) RT-qPCRによる腸管関連遺伝子の遺伝子発現解析。(C) 薬物トランスポーターの遺伝子発現解析。  (D) 免疫系細胞関連遺伝子の遺伝子発現解析。FIG. 5 shows the time course analysis of transepithelial electrical resistance and gene expression. Mean±S.D. (n = 3). Living intestinal tissue (ASI) = 1. (A) Intestinal barrier function evaluation by transepithelial electrical resistance measurement. (B) Gene expression analysis of gut-related genes by RT-qPCR. (C) Gene expression analysis of drug transporters. (D) Gene expression analysis of immune system cell-related genes. 図６は、オンフィーダーで維持培養したヒトiPSC由来腸管上皮細胞における経上皮電気抵抗（TEER）値の経時変化を示す。Mean ± S.D. (n = 3).FIG. 6 shows changes over time in transepithelial electrical resistance (TEER) values in human iPSC-derived intestinal epithelial cells maintained and cultured in an on-feeder. Mean ± S.D. (n = 3). 図７は、ヒトiPSC由来腸管上皮細胞における遺伝子発現に対する細胞数及び気相培養の影響を示す。Mean ± S.D. (n = 3); L-1: 液相培養、1×105 cells/insert,  L-1.5: 液相培養、1.5×105 cells/insert, A-1: 気相培養、1×105cells/insert, A-1.5: 気相培養、1.5×105 cells/insert, 発現量はヒト成人小腸を100とした相対値で示す。FIG. 7 shows the effects of cell number and gas phase culture on gene expression in human iPSC-derived intestinal epithelial cells. Mean ± SD (n = 3); L-1: liquid phase culture, 1×10 5 cells/insert, L-1.5: liquid phase culture, 1.5×10 5 cells/insert, A-1: gas phase culture, 1 ×10 5 cells/insert, A-1.5: gas phase culture, 1.5 × 10 5 cells/insert, expression level is shown as a relative value with human adult small intestine as 100. 図８は、図７の続きを示す。FIG. 8 shows a continuation of FIG. 図９は、オンフィーダーで維持培養したヒトiPSC由来腸管上皮細胞における5-アザ-2'-デオキシシチジンの添加有無及び気相培養期間の違いによる細胞形態への影響を示す。L-5+ : 液相培養かつ５-アザ-2'-デオキシシチジン添加群、 L-5- : 液相培養かつ５-アザ-2'-デオキシシチジン非添加群、 A8-5+ : 分化開始8日目より気相培養かつ５-アザ-2'-デオキシシチジン添加群、 A8-5- : 分化開始8日目より気相培養かつ５-アザ-2'-デオキシシチジン非添加群、 A14-5+ : 分化開始14日目より気相培養かつ５-アザ-2'-デオキシシチジン添加群、 A14-5- : 分化開始14日目より気相培養かつ５-アザ-2'-デオキシシチジン非添加群FIG. 9 shows the effects on cell morphology of human iPSC-derived intestinal epithelial cells maintained and cultured in an on-feeder depending on the presence or absence of the addition of 5-aza-2′-deoxycytidine and differences in the gas phase culture period. L-5+: liquid phase culture and 5-aza-2'-deoxycytidine addition group, L-5-: liquid phase culture and 5-aza-2'-deoxycytidine non-addition group, A8-5+: differentiation initiation A group of gas phase culture and 5-aza-2'-deoxycytidine added from day 8, A8-5-: group of gas phase culture and no 5-aza-2'-deoxycytidine added from day 8 of differentiation, A14- 5+ : group with gas phase culture and 5-aza-2'-deoxycytidine added from day 14 of differentiation, A14-5- : group with gas phase culture and no 5-aza-2'-deoxycytidine from day 14 of differentiation Addition group 図１０は、オンフィーダーで維持培養したヒトiPSC由来腸管上皮細胞における5-アザ-2'-デオキシシチジンの添加有無及び気相培養期間の違いによるマーカー遺伝子の発現への影響を示す。Mean ± S.D. (n = 3), 発現量はヒト成人小腸を100とした相対値で示す.L-5+ : 液相培養かつ５-アザ-2'-デオキシシチジン添加群、L-5- : 液相培養かつ５-アザ-2'-デオキシシチジン非添加群、 A8-5+ : 分化開始8日目より気相培養かつ５-アザ-2'-デオキシシチジン添加群、 A8-5- : 分化開始8日目より気相培養かつ５-アザ-2'-デオキシシチジン非添加群、 A14-5+ : 分化開始14日目より気相培養かつ５-アザ-2'-デオキシシチジン添加群、 A14-5- : 分化開始14日目より気相培養かつ５-アザ-2'-デオキシシチジン非添加群FIG. 10 shows the effects on marker gene expression of human iPSC-derived intestinal epithelial cells maintained and cultured in an on-feeder, depending on whether 5-aza-2'-deoxycytidine was added or not, and on differences in the gas phase culture period. Mean ± S.D. (n = 3), The expression level is shown relative to the human adult small intestine as 100. L-5+: Liquid phase culture and 5-aza-2'-deoxycytidine addition group, L-5-: Liquid phase culture and 5-aza-2'-deoxycytidine non-addition group, A8-5+: Gas phase culture and 5-aza-2'-deoxycytidine addition group from day 8 of differentiation initiation, A8-5-: Differentiation A14-5+: gas phase culture and 5-aza-2'-deoxycytidine addition group from day 14 of differentiation, A14 -5- : Group without addition of 5-aza-2'-deoxycytidine and gas-phase culture from 14 days after initiation of differentiation 図１１は、図１０の続きを示す。FIG. 11 shows a continuation of FIG. 図１２は、オンフィーダーで維持培養したヒトiPSC由来腸管上皮細胞におけるA83-01の添加時期による細胞形態への影響を示す。L-14 : 液相培養かつA83-01の分化開始14日目より添加した群、  L-8 : 液相培養かつA83-01の分化開始8日目より添加した群、A14-14 : 分化開始14日目より気相培養かつA83-01の分化開始14日目より添加した群、 A14-８ : 分化開始14日目より気相培養かつA83-01の分化開始8日目より添加した群FIG. 12 shows the influence of the addition timing of A83-01 on cell morphology in human iPSC-derived intestinal epithelial cells maintained and cultured in an on-feeder. L-14: group added from day 14 of the start of differentiation of A83-01 in liquid phase culture, L-8: group added from day 8 of start of differentiation in liquid phase culture of A83-01, A14-14: start of differentiation A14-8: Group of gas phase culture from day 14 and addition of A83-01 from day 14 of differentiation, A14-8: group of gas phase culture from day 14 of differentiation and addition of A83-01 from day 8 of differentiation 図１３は、オンフィーダーで維持培養したヒトiPSC由来腸管上皮細胞におけるA83-01の添加時期による経上皮電気抵抗（TEER）値の経時変化を示す。L-14 : 液相培養かつA83-01の分化開始14日目より添加した群、  L-8 : 液相培養かつA83-01の分化開始8日目より添加した群、 A14-14 : 分化開始14日目より気相培養かつA83-01の分化開始14日目より添加した群、A14-８ : 分化開始14日目より気相培養かつA83-01の分化開始8日目より添加した群. Mean ± S.D. (n = 3).FIG. 13 shows changes over time in transepithelial electrical resistance (TEER) values in human iPSC-derived intestinal epithelial cells maintained and cultured in an on-feeder, depending on the timing of addition of A83-01. L-14: Group in which liquid-phase culture was added from day 14 of differentiation of A83-01, L-8: group of liquid-phase culture and addition of A83-01 from day 8 of differentiation, A14-14: differentiation initiation A14-8 group: gas phase culture from day 14 and addition from day 14 of A83-01 differentiation; A14-8: group of gas phase culture from day 14 of differentiation and addition from day 8 of differentiation of A83-01 Mean ± S.D. (n = 3). 図１４は、オンフィーダーで維持培養したヒトiPSC由来腸管上皮細胞におけるA83-01の添加時期による腸管関連マーカー、薬物代謝酵素及び薬物トランスポーターの遺伝子発現量への影響を示す。Mean ± S.D. (n = 3), 発現量はヒト成人小腸を100とした相対値で示す。L-14 : 液相培養かつA83-01の分化開始14日目より添加した群、  L-8 : 液相培養かつA83-01の分化開始8日目より添加した群、A14-14 : 分化開始14日目より気相培養かつA83-01の分化開始14日目より添加した群、 A14-８ : 分化開始14日目より気相培養かつA83-01の分化開始8日目より添加した群FIG. 14 shows the influence of the addition timing of A83-01 on the gene expression levels of intestinal-related markers, drug-metabolizing enzymes, and drug transporters in human iPSC-derived intestinal epithelial cells maintained and cultured in an on-feeder. Mean ± S.D. (n = 3), The expression level is shown as a relative value with the human adult small intestine set to 100. L-14: group added from day 14 of the start of differentiation of A83-01 in liquid phase culture, L-8: group added from day 8 of start of differentiation in liquid phase culture of A83-01, A14-14: start of differentiation A14-8: Group of gas phase culture from day 14 and addition of A83-01 from day 14 of differentiation, A14-8: group of gas phase culture from day 14 of differentiation and addition of A83-01 from day 8 of differentiation 図１５は、図１４の続きを示す。FIG. 15 shows a continuation of FIG. 図１６は、オンフィーダーで維持培養したヒトiPSC由来腸管上皮細胞におけるCHI99021添加による細胞形態への影響を示す。L-: 液相培養かつCHIR99021の非添加群、 L-C: 液相培養かつCHIR99021の添加群、A-: 気相培養かつCHIR99021の非添加群、 A-C: 気相培養かつCHIR99021の添加群FIG. 16 shows the effect of addition of CHI99021 on cell morphology in human iPSC-derived intestinal epithelial cells maintained and cultured in an on-feeder. L-: Liquid-phase culture and CHIR99021-free group, L-C: Liquid-phase culture and CHIR99021-added group, A-: Gas-phase culture and CHIR99021-free group, A-C: Gas-phase culture and CHIR99021-added group 図１７は、ヒトiPSC由来腸管上皮細胞における遺伝子発現に対する細胞数及び気相培養の影響を示す。Mean ± S.D. (n = 3);  L-: 液相培養かつCHIR99021の非添加群、 L-C: 液相培養かつCHIR99021の添加群、A-: 気相培養かつCHIR99021の非添加群、 A-C: 気相培養かつCHIR99021の添加群FIG. 17 shows the effects of cell number and gas phase culture on gene expression in human iPSC-derived intestinal epithelial cells. Mean ± S.D. (n = 3); L-: Liquid phase culture without CHIR99021 addition group, L-C: Liquid phase culture with CHIR99021 addition group, A-: Gas phase culture without CHIR99021 addition group, A-C: Gas phase Cultured and CHIR99021 addition group 図１８は、図１７の続きを示す。FIG. 18 shows a continuation of FIG. 図１９は、図１７の続きを示す。FIG. 19 shows a continuation of FIG. 図２０は、フィーダーレスで培養したヒトiPSC由来腸管上皮細胞における遺伝子発現に対する気相培養の影響を示す。Mean ± S.D. (n = 3);  L-: 液相培養、A12: 気相培養を分化開始12日目から行う群、A18: 気相培養を分化開始18日目から行う群、A24: 気相培養を分化開始24日目から行う群FIG. 20 shows the effect of gas phase culture on gene expression in feederless cultured human iPSC-derived intestinal epithelial cells. Mean ± S.D. (n = 3); L-: liquid phase culture, A12: group in which gas phase culture is performed from day 12 of differentiation, A18: group in which gas phase culture is performed from day 18 of differentiation, A24: gas phase Group where culture is started from the 24th day of differentiation 図２１は、図２０の続きを示す。FIG. 21 shows a continuation of FIG. 図２２は、１回目のF-hiSIECTMの培養実験におけるTEER値の測定の結果を示す。FIG. 22 shows the results of TEER measurement in the first F-hiSIEC ™ culture experiment. 図２３は、１回目のF-hiSIECTMの培養実験におけるミダゾラムの膜透過試験におけるミダゾラム濃度の変動を示す。FIG. 23 shows changes in midazolam concentration in the membrane permeation test of midazolam in the first F-hiSIEC ™ culture experiment. 図２４は、１回目のF-hiSIECTMの培養実験におけるミダゾラムの膜透過試験におけるミダゾラムの透過係数を示す。FIG. 24 shows the permeability coefficient of midazolam in the membrane permeation test of midazolam in the first F-hiSIEC ™ culture experiment. 図２５は、１回目のF-hiSIECTMの培養実験におけるミダゾラムの膜透過試験における1’-ヒドロキシミダゾラム濃度の変動を示す。FIG. 25 shows changes in 1′-hydroxymidazolam concentration in the midazolam membrane permeation test in the first F-hiSIEC ™ culture experiment. 図２６は、１回目のF-hiSIECTMの培養実験におけるミダゾラムの膜透過試験における単位タンパク質量あたりの1’-ヒドロキシミダゾラムの生成量の測定結果を示す。FIG. 26 shows the measurement results of the production amount of 1′-hydroxymidazolam per unit protein amount in the midazolam membrane permeation test in the first F-hiSIEC ™ culture experiment. 図２７は、１回目のF-hiSIECTMの培養実験における遺伝子発現の解析結果を示す。n=2～3。SI(small intestine) = 1 としたときの相対値。FIG. 27 shows the analysis results of gene expression in the first F-hiSIEC ™ culture experiment. n=2-3. Relative value when SI(small intestine) = 1. 図２８は、１回目のF-hiSIECTMの培養実験における遺伝子発現の解析結果を示す。n = 3、SIのみ n = 1。SI(small intestine) = 1 としたときの相対値。FIG. 28 shows the analysis results of gene expression in the first F-hiSIEC ™ culture experiment. n = 3, n = 1 for SI only. Relative value when SI(small intestine) = 1. 図２９は、１回目のF-hiSIECTMの培養実験における遺伝子発現の解析結果を示す。n=2～3。SI(small intestine) = 1 としたときの相対値。FIG. 29 shows the analysis results of gene expression in the first F-hiSIEC ™ culture experiment. n=2-3. Relative value when SI(small intestine) = 1. 図３０は、２回目のF-hiSIECTMの培養実験におけるTEER値の測定の結果を示す。n = 8、Mean ± S.D.　conventional = conventional method(5aza有)FIG. 30 shows the results of TEER measurement in the second F-hiSIEC ™ culture experiment. n = 8, Mean ± SD conventional = conventional method (with 5aza) 図３１は、２回目のF-hiSIECTMの培養実験におけるLucifer yellowの膜透過試験の結果を示す。FIG. 31 shows the results of the Lucifer yellow membrane permeation test in the second F-hiSIEC ™ culture experiment. 図３２は、２回目のF-hiSIECTMの培養実験における遺伝子発現の解析結果を示す。n = 1。SI (small intestine) = 1 としたときの相対値FIG. 32 shows the results of gene expression analysis in the second F-hiSIEC ™ culture experiment. n=1. Relative value when SI (small intestine) = 1 図３３は、２回目のF-hiSIECTMの培養実験における遺伝子発現の解析結果を示す。n = 1。SI (small intestine) = 1 としたときの相対値FIG. 33 shows the results of gene expression analysis in the second F-hiSIEC ™ culture experiment. n=1. Relative value when SI (small intestine) = 1 図３４は、２回目のF-hiSIECTMの培養実験における遺伝子発現の解析結果を示す。n = 1。SI (small intestine) = 1 としたときの相対値。FL = Fetal liver total RNA。HPH = Human primary hepatocyteを48時間培養したもの。FIG. 34 shows the results of gene expression analysis in the second F-hiSIEC ™ culture experiment. n=1. Relative value when SI (small intestine) = 1. FL = Fetal liver total RNA. HPH = Human primary hepatocyte cultured for 48 hours. 図３５は、２回目のF-hiSIECTMの培養実験における遺伝子発現の解析結果を示す。n = 1。SI (small intestine) = 1 としたときの相対値FIG. 35 shows the results of gene expression analysis in the second F-hiSIEC ™ culture experiment. n=1. Relative value when SI (small intestine) = 1 図３６は、２回目のF-hiSIECTMの培養実験におけるミダゾラム代謝試験の結果を示す。n = 3。Mean ± S.D.FIG. 36 shows the results of the midazolam metabolism study in the second F-hiSIEC ™ culture experiment. n=3. Mean±SD
＜腸管細胞を作製する方法＞
　本発明は、多能性幹細胞から腸管細胞を作製する方法（以下、「本発明の作製方法」とも呼ぶ。）に関する。本発明によれば、生体の腸管組織と類似の特性を示す（腸管組織を模倣した）腸管細胞が得られる。 <Method for producing intestinal cells>
The present invention relates to a method for producing intestinal cells from pluripotent stem cells (hereinafter also referred to as "the production method of the present invention"). INDUSTRIAL APPLICABILITY According to the present invention, intestinal cells exhibiting properties similar to those of intestinal tissue in vivo (mimicking intestinal tissue) can be obtained.
　「多能性幹細胞」とは、生体を構成するすべての細胞に分化しうる能力（分化多能性）と、細胞分裂を経て自己と同一の分化能を有する娘細胞を生み出す能力（自己複製能）とを併せ持つ細胞をいう。分化多能性は、評価対象の細胞を、ヌードマウスに移植し、三胚葉（外胚葉、中胚葉、内胚葉）のそれぞれの細胞を含むテラトーマ形成の有無を試験することにより、評価することができる。 “Pluripotent stem cells” have the ability to differentiate into all cells that make up the body (pluripotency) and the ability to produce daughter cells with the same differentiation potential as the self through cell division (self-renewal ability). ) refers to cells that have both Pluripotency can be evaluated by transplanting the cells to be evaluated into nude mice and examining the presence or absence of teratoma formation containing cells of each of the three germ layers (ectoderm, mesoderm, and endoderm). can.
　多能性幹細胞として、胚性幹細胞（ES細胞）、胚性生殖細胞（EG細胞）、人工多能性幹細胞（iPS細胞）等を挙げることができるが、分化多能性及び自己複製能を併せ持つ細胞である限り、これに限定されない。好ましくはES細胞又はiPS細胞を用いる。更に好ましくはiPS細胞を用いる。多能性幹細胞は、好ましくは哺乳動物（例えば、ヒトやチンパンジー、カニクイザルなどの霊長類、マウスやラットなどのげっ歯類）の細胞、特に好ましくはヒトの細胞である。 Examples of pluripotent stem cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells) and the like. As long as it is a cell, it is not limited to this. ES cells or iPS cells are preferably used. More preferably, iPS cells are used. Pluripotent stem cells are preferably mammalian cells (eg, primates such as humans, chimpanzees and cynomolgus monkeys, and rodents such as mice and rats), particularly preferably human cells.
　ES細胞は、例えば、着床以前の初期胚、当該初期胚を構成する内部細胞塊、単一割球等を培養することによって樹立することができる（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））。初期胚として、体細胞の核を核移植することによって作製された初期胚を用いてもよい（Wilmut et al.（Nature， 385， 810（1997））、Cibelli et al. （Science， 280， 1256（1998））、入谷明ら（蛋白質核酸酵素， 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））。初期座として、単為発生胚を用いてもよい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））。上掲の論文の他、ES細胞の作製については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, P.M. et al. Methods in Enzymology, Vol.365, 2003等が参考になる。尚、ES細胞と体細胞の細胞融合によって得られる融合ES細胞も、本発明の作製方法に用いられる胚性幹細胞に含まれる。 ES cells can be established, for example, by culturing an early embryo before implantation, an inner cell mass constituting the early embryo, a single blastomere, 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 an early embryo, an early embryo produced by nuclear transfer of the nucleus of a somatic cell may be used (Wilmut et al. (Nature, 385, 810 (1997)), Cibelli et al. (Science, 280, 1256 (1998)), Akira Iriya 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)) Parthenogenic embryos may be used as the initial locus 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 (Cloning Stem Cells, 10, 11-24 (2008)), Strelchenko N., et al. 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, P.M. et al. Methods in Enzymology, Vol.365, 2003, etc. are helpful. Fusion 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.
　ES細胞の中には、保存機関から入手可能なもの、或いは市販されているものもある。例えば、ヒトES細胞については京都大学再生医科学研究所（例えばKhES-1、KhES-2及びKhES-3）、WiCell Research Institute、ESI BIOなどから入手可能である。 Some ES cells are available from archives or are commercially available. For example, human ES cells are available from Kyoto University Institute for Frontier Medical Sciences (eg, KhES-1, KhES-2 and KhES-3), WiCell Research Institute, ESI BIO, and the like.
　ES細胞は、始原生殖細胞を、LIF、bFGF、SCFの存在下で培養すること等により樹立することができる（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））。 ES cells can be established by culturing primordial germ cells in the presence of LIF, bFGF, SCF, etc. (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)).
　「人工多能性幹細胞（iPS細胞）」とは、初期化因子の導入などにより体細胞をリプログラミングすることによって作製される、多能性（多分化能）と増殖能を有する細胞である。人工多能性幹細胞はES細胞に近い性質を示す。iPS細胞の作製に使用する体細胞は特に限定されず、分化した体細胞でもよいし、未分化の幹細胞でもよい。また、その由来も特に限定されないが、好ましくは哺乳動物（例えば、ヒトやチンパンジー、カニクイザルなどの霊長類、マウスやラットなどのげっ歯類）の体細胞、特に好ましくはヒトの体細胞を用いる。iPS細胞は、これまでに報告された各種方法によって作製することができる。また、今後開発されるiPS細胞作製法を適用することも当然に想定される。 "Induced pluripotent stem cells (iPS cells)" are cells with pluripotency (multipotency) and proliferative potential, which are produced by reprogramming somatic cells through the introduction of reprogramming factors. Induced pluripotent stem cells exhibit properties similar to ES cells. The somatic cells used for producing iPS cells are not particularly limited, and may be differentiated somatic cells or undifferentiated stem cells. The origin is not particularly limited, but somatic cells of mammals (for example, primates such as humans, chimpanzees, and cynomolgus monkeys, and rodents such as mice and rats), particularly preferably somatic cells of humans, are used. iPS cells can be produced by various methods reported so far. In addition, it is naturally envisioned that an iPS cell production method that will be developed in the future will be applied.
　腸疾患の患者由来のiPS細胞（患者の体細胞から調製したiPS細胞）を用いることにすれば、疾患特異的な腸管細胞（腸管オルガノイド）を作製することが可能となる。当該iPS細胞は、患者から採取された体細胞（例えば皮膚、血液、単核球など）から調製される。ここでの腸疾患の例として難治性炎症性腸疾患（クローン病、潰瘍性大腸炎）、ポリープ、大腸がん、薬剤性腸炎などを挙げることができる。疾患特異的な腸管オルガノイドは腸管病態モデルとして有用であり、薬剤評価系への利用や、疾患の機序（発症、病態形成、進展に関する分子メカニズムなど）の解明への貢献を期待できる。 By using iPS cells derived from patients with intestinal disease (iPS cells prepared from the patient's somatic cells), it is possible to produce disease-specific intestinal cells (intestinal organoids). The iPS cells are prepared from somatic cells (for example, skin, blood, mononuclear cells, etc.) collected from patients. Examples of intestinal diseases here include intractable inflammatory bowel diseases (Crohn's disease, ulcerative colitis), polyps, colon cancer, drug-induced enteritis, and the like. Disease-specific intestinal organoids are useful as intestinal pathology models, and are expected to contribute to drug evaluation systems and elucidation of disease mechanisms (molecular mechanisms related to onset, pathogenesis, and progression).
　iPS細胞作製法の最も基本的な手法は、転写因子であるOct3/4、Sox2、Klf4及びc-Mycの４因子を、ウイルスを利用して細胞へ導入する方法である（Takahashi K, Yamanaka S: Cell 126 (4), 663-676, 2006; Takahashi, K, et al: Cell 131 (5), 861-72, 2007）。ヒトiPS細胞についてはOct4、Sox2、Lin28及びNonogの４因子の導入による樹立の報告がある（Yu J, et al: Science 318(5858), 1917-1920, 2007）。c-Mycを除く３因子（Nakagawa M, et al: Nat. Biotechnol. 26 (1), 101-106, 2008）、Oct3/4及びKlf4の２因子（Kim J B, et al: Nature 454 (7204), 646-650, 2008）、或いはOct3/4のみ（Kim J B, et al: Cell 136 (3), 411-419, 2009）の導入によるiPS細胞の樹立も報告されている。また、遺伝子の発現産物であるタンパク質を細胞に導入する手法（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）も報告されている。一方、ヒストンメチル基転移酵素G9aに対する阻害剤BIX-01294やヒストン脱アセチル化酵素阻害剤バルプロ酸（VPA）或いはBayK8644等を使用することによって作製効率の向上や導入する因子の低減などが可能であるとの報告もある（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）。遺伝子導入法についても検討が進められ、レトロウイルスの他、レンチウイルス（Yu J, et al: Science 318(5858), 1917-1920, 2007）、アデノウイルス（Stadtfeld M, et al: Science 322 (5903), 945-949, 2008）、プラスミド（Okita K, et al: Science 322 (5903), 949-953, 2008）、トランスポゾンベクター（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）、或いはエピソーマルベクター（Yu J, Hu K, Smuga-Otto K, Tian S, et al: Science 324, 797-801, 2009）を遺伝子導入に利用した技術が開発されている。 The most basic method of producing iPS cells is to introduce four transcription factors, Oct3/4, Sox2, Klf4 and c-Myc, into cells using viruses (Takahashi K, Yamanaka S : Cell 126 (4), 663-676, 2006; Takahashi, K, et al.: Cell 131 (5), 861-72, 2007). There is a report that human iPS cells were established by introducing four factors, Oct4, Sox2, Lin28 and Nonog (Yu J, et al: Science 318(5858), 1917-1920, 2007). 3 factors excluding c-Myc (Nakagawa M, et al: Nat. Biotechnol. 26 (1), 101-106, 2008), 2 factors Oct3/4 and Klf4 (Kim J B, et al: Nature 454 (7204 ), 646-650, 2008), or the establishment of iPS cells by introducing only Oct3/4 (Kim J B, et al: Cell 136 (3), 411-419, 2009) has also been reported. In addition, methods for introducing proteins, which are gene expression products, into cells (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 is possible to improve production efficiency and reduce factors to be introduced by using inhibitor BIX-01294 against histone methyltransferase G9a, histone deacetylase inhibitor valproic acid (VPA), BayK8644, etc. (Huangfu D, et al: Nat. Biotechnol. 26 (7), 795-797, 2008; Huangfu D, et al: Nat. Biotechnol. 26 (11), 1269-1275, 2008; Silva J 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), plasmid (Okita K, et al: Science 322 (5903), 949-953, 2008), transposon vector (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 Techniques using episomal vectors (Yu J, Hu K, Smuga-Otto K, Tian S, et al: Science 324, 797-801, 2009) for gene transfer have been developed.
　iPS細胞への形質転換、即ち初期化（リプログラミング）が生じた細胞はFbxo15、Nanog、Oct/4、Fgf-4、Esg-1及びCript等の多能性幹細胞マーカー（未分化マーカー）の発現などを指標として選択することができる。選択された細胞をiPS細胞として回収する。 Transformation into iPS cells, i.e. cells that have undergone reprogramming (reprogramming), express pluripotent stem cell markers (undifferentiated markers) such as Fbxo15, Nanog, Oct/4, Fgf-4, Esg-1 and Cript etc. can be selected as an index. Selected cells are collected as iPS cells.
　iPS細胞は、例えば、国立大学法人京都大学又は国立研究開発法人理化学研究所バイオリソースセンターから提供を受けることもできる。 iPS cells can also be provided, for example, from Kyoto University or the RIKEN BioResource Center, a national research and development agency.
　本明細書において「分化させる」、「誘導する」との用語は、特定の細胞系譜に沿って分化するように働きかけることを意味する。 As used herein, the terms "differentiate" and "induce" mean working to differentiate along a specific cell lineage.
　本発明では、多能性幹細胞を腸管細胞へと分化させる。
　本発明の第一の作製方法は、以下の６段階の培養工程を含む。
（１）多能性幹細胞を内胚葉様細胞へと分化させる工程（工程（１））；
（２）工程（１）で得られた内胚葉様細胞を腸管幹細胞様細胞へと分化させる工程（工程（２））；
（３）工程（２）で得られた腸管幹細胞様細胞を、上皮成長因子、線維芽細胞増殖因子、TGFβ受容体阻害剤、GSK-3β阻害剤及びROCK阻害剤の存在下で培養する工程（工程（３））；
（４）工程（３）後の細胞を培養し、スフェロイドを形成させる工程（工程（４））；
（５）工程（４）で形成されたスフェロイドを分化させ、腸管オルガノイドを形成させる工程であって、上皮成長因子、BMP阻害剤及びWntシグナル活性化剤の存在下での培養を含む工程（工程（５））；及び
（６）工程（５）で形成された腸管オルガノイドを構成する細胞を、上皮成長因子、cAMPシグナル活性化因子、TGFβ受容体阻害剤、及びWntシグナル活性化剤の存在下で気相液相培養する工程（工程（６））。 In the present invention, pluripotent stem cells are differentiated into intestinal cells.
The first preparation method of the present invention includes the following six stages of culture steps.
(1) a step of differentiating pluripotent stem cells into endoderm-like cells (step (1));
(2) a step of differentiating the endoderm-like cells obtained in step (1) into intestinal stem cell-like cells (step (2));
(3) culturing the intestinal stem cell-like cells obtained in step (2) in the presence of epidermal growth factor, fibroblast growth factor, TGFβ receptor inhibitor, GSK-3β inhibitor and ROCK inhibitor ( step (3));
(4) culturing the cells after step (3) to form spheroids (step (4));
(5) A step of differentiating the spheroids formed in step (4) to form intestinal organoids, comprising culturing in the presence of an epidermal growth factor, a BMP inhibitor and a Wnt signal activator (step (5)); and (6) cells composing the intestinal organoids formed in step (5) in the presence of epidermal growth factor, cAMP signal activator, TGFβ receptor inhibitor, and Wnt signal activator (step (6)).
＜工程（１）：内胚葉様細胞への分化＞ 
　この工程では多能性幹細胞を培養し、内胚葉様細胞へと分化させる。換言すれば、内胚葉様細胞への分化を誘導する条件下で多能性幹細胞を培養する。多能性幹細胞が内胚葉様細胞に分化する限り、培養条件は特に限定されない。例えば、常法に従い、アクチビンAを添加した培地で培養する。この場合、培地中のアクチビンAの濃度を例えば10 ng/mL～200 ng/mL、好ましくは20 ng/mL～150 ng/mLとする。細胞の増殖率や維持等の観点から、培地に血清又は血清代替物（Knockout serum replacement(KSR)など）を添加することが好ましい。血清はウシ胎仔血清に限られるものではなく、ヒト血清や羊血清等を用いることもできる。血清又は血清代替物の添加量は例えば0.1%(v/v)～10%(v/v)である。 <Step (1): Differentiation into endoderm-like cells>
In this step, pluripotent stem cells are cultured and differentiated into endoderm-like cells. In other words, pluripotent stem cells are cultured under conditions that induce differentiation into endoderm-like cells. Culture conditions are not particularly limited as long as the pluripotent stem cells differentiate into endoderm-like cells. For example, it is cultured in a medium supplemented with activin A according to a conventional method. In this case, the concentration of activin A in the medium is, for example, 10 ng/mL to 200 ng/mL, preferably 20 ng/mL to 150 ng/mL. From the viewpoint of cell growth rate, maintenance, etc., it is preferable to add serum or a serum substitute (Knockout serum replacement (KSR), etc.) to the medium. Serum is not limited to fetal bovine serum, and human serum, sheep serum, and the like can also be used. The amount of serum or serum substitute added is, for example, 0.1% (v/v) to 10% (v/v).
　Wntのリガンド（例えばWnt-3aなど）やGSK-3阻害剤（例えばCHIR99021、CHIR98014、BIO、SB415286、SB216763、TWS119、A1070722等）を培地に添加し、内胚葉様細胞への分化の促進を図ってもよい。  Wnt ligands (e.g., Wnt-3a) and GSK-3 inhibitors (e.g., CHIR99021, CHIR98014, BIO, SB415286, SB216763, TWS119, A1070722, etc.) were added to the medium to promote differentiation into endoderm-like cells. may 
　工程（１）として２段階以上の培養を行うことにしてもよい。１段階目の培養では、血清無しの培地、又は比較的低濃度の血清（例えば、0.1%(v/v)～1%(v/v)）を添加した培地で行い、続く２段階目以降の培養では一段階目の培養よりも血清濃度を高めた培地（血清濃度を例えば1%(v/v)～10%(v/v)）で行う。このように２段階以上の培養を採用することは、１段階目の培養により未分化細胞の増殖を抑制し、続く２段階目以降により分化した細胞を増殖させる点で好ましい。 Two or more stages of culture may be performed as step (1). In the first stage of culture, serum-free medium or medium with relatively low concentration of serum (e.g., 0.1% (v / v) to 1% (v / v)) was added, and the following second stage and later 2 culture is performed in a medium with a higher serum concentration than in the first-stage culture (serum concentration, for example, 1% (v/v) to 10% (v/v)). Adopting two or more stages of culture in this way is preferable in that the growth of undifferentiated cells is suppressed in the first stage of culture, and the differentiated cells are allowed to grow in the following two stages and beyond.
　工程（１）の期間（培養期間）は例えば１日間～１０日間、好ましくは２日間～７日間である。工程（１）として２段階の培養を採用する場合には１段階目の培養期間を例えば１日間～７日間、好ましくは２日間～５日間とし、２段階目の培養期間を例えば１日間～６日間、好ましくは１日間～４日間とする。  The period of step (1) (culture period) is, for example, 1 to 10 days, preferably 2 to 7 days. When a two-stage culture is employed as step (1), the first stage culture period is, for example, 1 to 7 days, preferably 2 to 5 days, and the second stage culture period is, for example, 1 to 6 days. days, preferably 1 to 4 days. 
＜工程（２）：腸管幹細胞様細胞への分化＞
　この工程では、工程（１）で得られた内胚葉様細胞を培養し、腸管幹細胞様細胞へと分化させる。換言すれば、腸管幹細胞様細胞への分化を誘導する条件下で内胚葉細胞を培養する。内胚葉様細胞が腸管幹細胞様細胞へ分化する限り、培養条件は特に限定されない。工程（２）は、好ましくは、FGF4（線維芽細胞増殖因子４）とGSK-3阻害剤（例えばCHIR99021、CHIR98014、BIO、SB415286、SB216763、TWS119、A1070722等）の存在下で培養を行うことができる。FGF4として例えばヒトFGF4（例えばヒト組換えFGF4）を用いる。また、 FGF2（線維芽細胞増殖因子２）の存在下で培養を行うこともできる。 <Step (2): Differentiation into intestinal stem cell-like cells>
In this step, the endoderm-like cells obtained in step (1) are cultured and differentiated into intestinal stem cell-like cells. In other words, endoderm cells are cultured under conditions that induce differentiation into intestinal stem cell-like cells. Culture conditions are not particularly limited as long as the endoderm-like cells differentiate into intestinal stem cell-like cells. In step (2), culture is preferably performed in the presence of FGF4 (fibroblast growth factor 4) and a GSK-3 inhibitor (e.g., CHIR99021, CHIR98014, BIO, SB415286, SB216763, TWS119, A1070722, etc.). can. For example, human FGF4 (eg, human recombinant FGF4) is used as FGF4. Cultivation can also be performed in the presence of FGF2 (fibroblast growth factor 2).
　典型的には、工程（１）を経て得られた細胞集団又はその一部を、選別することなく工程（２）に供する。一方で、工程（１）を経て得られた細胞集団の中から内胚葉様細胞を選別した上で工程（２）を実施することにしてもよい。内胚葉様細胞の選別は例えば、細胞表面マーカーを指標にしてフローサイトメーター（セルソーター）で行えばよい。 Typically, the cell population obtained through step (1) or a portion thereof is subjected to step (2) without selection. On the other hand, step (2) may be performed after selecting endoderm-like cells from the cell population obtained through step (1). Selection of endoderm-like cells may be performed, for example, using a flow cytometer (cell sorter) using a cell surface marker as an index.
　「FGF4とGSK-3阻害剤の存在下」とは、FGF4とGSK-3阻害剤が培地中に添加された条件と同義である。従って、FGF4とGSK-3阻害剤の存在下での培養を行うためには、FGF4とGSK-3阻害剤が添加された培地を用いればよい。FGF4の添加濃度の例を示すと50ng/mL～2.5μg/mL、好ましくは150ng/mL～500ng/mLである。また、GSK-3阻害剤の添加濃度の例（CHIR99021の場合）を示すと、600nmol/L～60μmol/L、好ましくは1μmol/L～20μmol/Lである。 "In the presence of FGF4 and GSK-3 inhibitors" is synonymous with the conditions in which FGF4 and GSK-3 inhibitors were added to the medium. Therefore, in order to perform culture in the presence of FGF4 and GSK-3 inhibitor, a medium supplemented with FGF4 and GSK-3 inhibitor may be used. An example of the concentration of FGF4 added is 50 ng/mL to 2.5 μg/mL, preferably 150 ng/mL to 500 ng/mL. An example of the concentration of the GSK-3 inhibitor added (in the case of CHIR99021) is 600 nmol/L to 60 μmol/L, preferably 1 μmol/L to 20 μmol/L.
　「FGF2の存在下」とは、FGF2が培地中に添加された条件と同義である。従って、FGF2の存在下での培養を行うためには、FGF2が添加された培地を用いればよい。FGF2の添加濃度の例を示すと50ng/mL～2.5μg/mL、好ましくは150ng/mL～500ng/mLである。 "In the presence of FGF2" is synonymous with the condition in which FGF2 was added to the medium. Therefore, in order to perform culture in the presence of FGF2, a medium supplemented with FGF2 may be used. An example of the concentration of FGF2 added is 50 ng/mL to 2.5 μg/mL, preferably 150 ng/mL to 500 ng/mL.
　尚、例示した化合物、即ち、CHIR99021とは異なる化合物を使用する場合の添加濃度については、使用する化合物の特性と、例示した化合物の特性の相違（特に活性の相違）を考慮すれば、当業者であれば上記濃度範囲に準じて設定することができる。また、設定した濃度範囲が適切であるか否かは、後述の実施例に準じた予備実験によって確認することができる。 Regarding the addition concentration when using the exemplified compound, that is, a compound different from CHIR99021, considering the characteristics of the compound used and the difference in the characteristics of the exemplified compound (especially the difference in activity), If so, it can be set according to the above density range. Also, whether or not the set concentration range is appropriate can be confirmed by a preliminary experiment according to the examples described later.
　工程（２）の期間（培養期間）は例えば２日間～１０日間、好ましくは３日間～７日間である。当該培養期間が短すぎると、期待される効果（分化効率の上昇、腸管幹細胞としての機能の獲得の促進）が十分に得られない。他方、当該培養期間が長すぎると、分化効率の低下を引き起こす。 The period of step (2) (culture period) is, for example, 2 to 10 days, preferably 3 to 7 days. If the culture period is too short, the expected effects (increase in differentiation efficiency, promotion of acquisition of functions as intestinal stem cells) cannot be sufficiently obtained. On the other hand, if the culture period is too long, it causes a decrease in differentiation efficiency.
　腸管幹細胞様細胞へ分化したことは、例えば、腸管幹細胞マーカーの発現を指標にして判定ないし評価することができる。腸管幹細胞マーカーの例を挙げると、ロイシンリッチリピートを含むGタンパク質共役受容体５（LGR5）、エフリンB2受容体（EphB2）である。 The differentiation into intestinal stem cell-like cells can be determined or evaluated, for example, using the expression of intestinal stem cell markers as an index. Examples of intestinal stem cell markers are leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), ephrin B2 receptor (EphB2).
＜工程（３）：腸管への分化方向の誘導＞
　この工程では、工程（２）で得られた腸管幹細胞様細胞を、上皮成長因子、線維芽細胞増殖因子、TGFβ受容体阻害剤、GSK-3β阻害剤及びROCK阻害剤の存在下で培養する。理論に拘泥する訳ではないが、後述の実験結果を踏まえると、当該工程を、「幹細胞性を維持させつつ腸管へと分化方向を誘導する段階」として理解することができる。この工程を経ることは、最終的に得られる腸管オルガノイドの形態（絨毛－クリプト様構造を示す）及び機能（腸管マーカーや薬物トランスポーターが成人小腸に近い遺伝子発現を示す）に重要である。 <Step (3): Induction of direction of differentiation into intestinal tract>
In this step, the intestinal stem cell-like cells obtained in step (2) are cultured in the presence of epidermal growth factor, fibroblast growth factor, TGFβ receptor inhibitor, GSK-3β inhibitor and ROCK inhibitor. Although not bound by theory, based on the experimental results described later, this process can be understood as "the step of inducing differentiation toward the intestinal tract while maintaining stemness". This process is important for the morphology (villus-crypt-like structure) and function (intestinal markers and drug transporters exhibit gene expression similar to that of the adult small intestine) of finally obtained intestinal organoids.
　典型的には、工程（２）を経て得られた細胞集団又はその一部を、選別することなく工程（３）に供する。一方で、工程（２）を経て得られた細胞集団の中から腸管幹細胞様細胞を選別した上で工程（３）を実施することにしてもよい。腸管幹細胞様細胞の選別は例えば、細胞表面マーカーを指標にしてフローサイトメーター（セルソーター）で行えばよい。  Typically, the cell population obtained through step (2) or a portion thereof is subjected to step (3) without selection. On the other hand, step (3) may be performed after intestinal stem cell-like cells are selected from the cell population obtained through step (2). Selection of intestinal stem cell-like cells may be performed, for example, by a flow cytometer (cell sorter) using a cell surface marker as an indicator. 
　「上皮成長因子、線維芽細胞増殖因子、TGFβ受容体阻害剤、GSK-3β阻害剤及びROCK阻害剤の存在下とは、これらの化合物が培地中に添加された条件と同義である。従って、上皮成長因子、線維芽細胞増殖因子、TGFβ受容体阻害剤、GSK-3β阻害剤及びROCK阻害剤の存在下での培養を行うためには、これらの化合物が添加された培地を用いればよい。 "The presence of epidermal growth factor, fibroblast growth factor, TGFβ receptor inhibitor, GSK-3β inhibitor and ROCK inhibitor is synonymous with the conditions under which these compounds are added to the medium. Therefore, For culture in the presence of epidermal growth factor, fibroblast growth factor, TGFβ receptor inhibitor, GSK-3β inhibitor and ROCK inhibitor, a medium supplemented with these compounds may be used.
　線維芽細胞増殖因子としてはFGF2、FGF4又はFGF10を採用するとよい。これらFGFファミリーの２つ又は３つを組み合わせて使用してもよい。TGFβ受容体阻害剤として例えばA-83-01を用いることができる。GSK-3β阻害剤としてCHIR99021、SB216763、CHIR98014、TWS119、Tideglusib、SB415286、BIO、AZD2858、AZD1080、AR-A014418、TDZD-8、LY2090314、IM-12、Indirubin、Bikinin、1-Azakenpaulloneを例示することができる。ROCK阻害剤として例えばY-27632を用いることができる。  It is recommended to adopt FGF2, FGF4 or FGF10 as fibroblast growth factors. Two or three of these FGF families may be used in combination. For example, A-83-01 can be used as a TGFβ receptor inhibitor. Examples of GSK-3β inhibitors include CHIR99021, SB216763, CHIR98014, TWS119, Tideglusib, SB415286, BIO, AZD2858, AZD1080, AR-A014418, TDZD-8, LY2090314, IM-12, Indirubin, Bikinin, and 1-Azakenpaullone. can. Y-27632, for example, can be used as a ROCK inhibitor. 
　上皮成長因子の添加濃度の例を示すと10 ng/mL～500 ng/mL、好ましくは50 ng/mL～200 ng/mLである。同様に、線維芽細胞増殖因子の添加濃度の例（FGF2の場合）を示すと5 ng/mL～200 ng/mL、好ましくは20 ng/mL～50 ng/mLであり、TGFβ受容体阻害剤の添加濃度の例（A-83-01の場合）を示すと0.1μM～5μM、好ましくは0.3μM～3μMであり、GSK-3β阻害剤の添加濃度の例（CHIR99021の場合）を示すと0.5μM～100μM、好ましくは1μM～30μMであり、ROCK阻害剤の添加濃度の例（Y-27632の場合）を示すと1μM～50μM、好ましくは3μM～30μMである。  An example of the concentration of epidermal growth factor added is 10 ng/mL to 500 ng/mL, preferably 50 ng/mL to 200 ng/mL. Similarly, an example of the concentration of fibroblast growth factor added (for FGF2) is 5 ng/mL to 200 ng/mL, preferably 20 ng/mL to 50 ng/mL. An example of the addition concentration of (for A-83-01) is 0.1 μM to 5 μM, preferably 0.3 μM to 3 μM, and an example of the addition concentration of the GSK-3β inhibitor (for CHIR99021) is 0.5 It is μM to 100 μM, preferably 1 μM to 30 μM, and an example of the addition concentration of the ROCK inhibitor (in the case of Y-27632) is 1 μM to 50 μM, preferably 3 μM to 30 μM. 
　尚、例示した化合物、即ち、FGF2、A-83-01、CHIR99021、Y-27632とは異なる化合物を使用する場合の添加濃度については、使用する化合物の特性と、例示した化合物の特性の相違（特に活性の相違）を考慮すれば、当業者であれば上記濃度範囲に準じて設定することができる。また、設定した濃度範囲が適切であるか否かは、後述の実施例に準じた予備実験によって確認することができる。 In addition, when using a compound different from the exemplified compounds, that is, FGF2, A-83-01, CHIR99021, and Y-27632, the concentration to be added depends on the difference between the characteristics of the compound used and the characteristics of the exemplified compounds ( In particular, considering the difference in activity), a person skilled in the art can set the concentration according to the above range. Also, whether or not the set concentration range is appropriate can be confirmed by a preliminary experiment according to the examples described later.
　上記の化合物（上皮成長因子、線維芽細胞増殖因子、TGFβ受容体阻害剤、GSK-3β阻害剤及びROCK阻害剤）に加え、血清又は血清代替物も併用するとよい。即ち、好ましくは、上記化合物及び血清又は血清代替物が培地中に添加された条件で培養する。血清代替物とは、一般にiPS細胞やES細胞等をその未分化な状態を維持させたままで培養するため、分化誘導因子を含む血清の代わりとして使用される組成物である。好ましくは、ノックアウト血清代替物（Knockout serum replacement(KSR)）を用いる。血清の添加濃度は、1%(v/v)～10%(v/v)、好ましくは1%(v/v)～5%(v/v)である。血清代替物の添加濃度の例（KSRの場合）を示すと5%(v/v)～20%(v/v)、好ましくは5%(v/v)～15%(v/v)である。  In addition to the above compounds (epidermal growth factor, fibroblast growth factor, TGFβ receptor inhibitor, GSK-3β inhibitor, and ROCK inhibitor), serum or serum replacement should be used in combination. That is, culture is preferably carried out under conditions in which the above compound and serum or serum substitute are added to the medium. A serum substitute is generally a composition that is used as a substitute for serum containing a differentiation-inducing factor in order to culture iPS cells, ES cells, etc. while maintaining their undifferentiated state. Preferably, Knockout serum replacement (KSR) is used. The serum concentration is 1% (v/v) to 10% (v/v), preferably 1% (v/v) to 5% (v/v). An example of the concentration of serum replacement added (for KSR) is 5% (v/v) to 20% (v/v), preferably 5% (v/v) to 15% (v/v). be. 
　工程（３）の培養には、細胞接着性の培養面を備える培養容器が使用される。細胞接着性の培養面として、好ましくは、基底膜成分（例えばラミニン、IV型コラーゲン、エンタクチン、ビトロネクチン、フィブロネクチン）又はその断片をコートしたものを用いるとよい。特に、ラミニン511又はそのE8断片でコートした培養面を採用するとよい。ラミニン511のE8断片については、その組換え体（組換えヒトラミニン511-E8タンパク質）を高純度に精製した製品（製品名：iMatrix-511、製造：株式会社ニッピ、販売：株式会社マトリクソーム）が市販されている。尚、培養容器は特に限定されず、例えば、ディッシュ、フラスコ、マルチウェルプレート等を用いることができる。  A culture vessel with a cell-adhesive culture surface is used for the culture in step (3). As the cell-adhesive culture surface, one coated with a basement membrane component (eg, laminin, type IV collagen, entactin, vitronectin, fibronectin) or a fragment thereof is preferably used. In particular, culture surfaces coated with laminin 511 or its E8 fragment may be employed. Regarding the E8 fragment of laminin 511, a product (product name: iMatrix-511, manufactured by Nippi Co., Ltd., sold by Matrixome Co., Ltd.), which is a highly purified recombinant (recombinant human laminin 511-E8 protein), is commercially available. It is The culture vessel is not particularly limited, and for example, dishes, flasks, multi-well plates and the like can be used. 
　工程（３）の期間（培養期間）は例えば２日間～１４日間、好ましくは３日間～１２日間である。当該培養期間が短すぎると、期待される効果（即ち、絨毛－クリプト様構造を有し、機能的な腸管オルガノイドを形成するために必要な分化方向の誘導）が十分に得られない。尚、継代操作を繰り返すことにすれば、当該培養期間をより長く設定することも可能である。  The period of step (3) (culture period) is, for example, 2 to 14 days, preferably 3 to 12 days. If the culture period is too short, the expected effect (ie, induction of the direction of differentiation necessary to form functional intestinal organoids with villus-crypt-like structures) cannot be obtained sufficiently. Incidentally, if the subculturing operation is repeated, the culture period can be set longer. 
　好ましい態様では、工程（３）において継代操作を行う。即ち、工程（３）の途中で継代培養を行う。継代操作は、特に、腸管上皮幹細胞様細胞の純化（純度、均一性の向上）及びより選択的に腸管へ分化誘導すること、に有効であると考えられる。 In a preferred embodiment, a subculture operation is performed in step (3). That is, subculture is performed in the middle of step (3). Passage operation is considered to be particularly effective in purifying intestinal epithelial stem cell-like cells (improving purity and homogeneity) and more selectively inducing differentiation into the intestinal tract.
　継代操作の回数は、例えば１～５回、好ましくは１～３回、更に好ましくは１回又は２回である。継代操作が多すぎれば細胞へのダメージが大きくなり、作製効率の低下に繋がる。継代操作の手順は常法に従えばよい。例えばコンフルエント又はサブコンフルエントになった際に細胞の一部を採取して別の培養容器に移し、培養を継続する。細胞の回収には細胞解離液などを利用すればよい。細胞解離液としては、例えば、トリプシン-EDTA、コラゲナーゼIV、メタロプロテアーゼ等のタンパク分解酵素等を単独で又は適宜組み合わせて用いることができる。細胞障害性が少ないものが好ましい。このような細胞解離液として、例えば、ディスパーゼ（エーディア）、TrypLE （Invitrogen）又はアキュターゼ（MILLIPORE）等の市販品が入手可能である。  The number of subculture operations is, for example, 1 to 5 times, preferably 1 to 3 times, more preferably 1 or 2 times. Too many subcultures result in greater damage to the cells, leading to lower production efficiency. Procedures for subculturing may follow conventional methods. For example, when the cells become confluent or subconfluent, some of the cells are harvested and transferred to another culture vessel to continue culturing. A cell dissociation solution or the like may be used to collect cells. As the cell dissociation solution, for example, trypsin-EDTA, collagenase IV, proteolytic enzymes such as metalloprotease, and the like can be used alone or in appropriate combination. Those with less cytotoxicity are preferred. Commercial products such as Dispase (Edia), TrypLE (Invitrogen), and Accutase (MILLIPORE) are available as such cell dissociation solutions. 
＜工程（４）：スフェロイドの形成＞
　この工程では、工程（３）を経た細胞を培養し、スフェロイドを形成させる。スフェロイドを形成させるためには浮遊培養が適する。浮遊培養では、通常、細胞低接着性又は細胞非接着性の培養面（例えば、ポリマー材料やハイドロゲル等の処理／結合により細胞低接着性／細胞非接着性が付与された培養面）を備える培養容器が使用され、培養面から離れた状態（即ち浮遊状態）で細胞を培養する。浮遊培養に用いる培養容器は特に限定されず、例えば、ディッシュ、フラスコ、マルチウェルプレート、チューブ、トレイ、培養バック等を用いることができる。好ましくは、細胞低接着性又は細胞非接着性の培養面に均一な形状及び大きさの複数のウェルが形成された培養容器（一般にパターンプレートと呼ばれる。具体例として、AGCテクノグラス株式会社が提供するEZSPHERE（登録商標）、株式会社クラレが提供するElplasia等を挙げることができる）を使用し、複数のスフェロイドをまとめて形成させる。このようにすれば、効率よくスフェロイドを形成させることができ、ひいては腸管オルガノイドの作製効率が向上する。  <Step (4): Formation of spheroids>
In this step, the cells that have undergone step (3) are cultured to form spheroids. Suspension culture is suitable for forming spheroids. Suspension culture usually comprises a culture surface with low cell adhesion or cell non-adhesion (for example, a culture surface imparted with low cell adhesion/cell non-adhesion by treatment/bonding with polymer materials, hydrogels, etc.) A culture vessel is used to culture cells in a state away from the culture surface (ie, in a floating state). Culture vessels used for suspension culture are not particularly limited, and for example, dishes, flasks, multiwell plates, tubes, trays, culture bags and the like can be used. Preferably, a culture vessel (generally referred to as a pattern plate) in which a plurality of wells of uniform shape and size are formed on a low cell adhesion or cell non-adhesion culture surface. EZSPHERE (registered trademark), Elplasia provided by Kuraray Co., Ltd.) is used to collectively form a plurality of spheroids. By doing so, spheroids can be formed efficiently, and the production efficiency of intestinal organoids is improved.
　浮遊培養に際しては、培養面に対する非接着状態を維持できる限り、細胞／細胞塊を静置培養してもよいし、旋回培養や振とう培養することにしてもよい。好ましくは、ここでの浮遊培養を静置培養により行う。静置培養は特別の装置が不要であること、細胞への衝撃ないしダメージも少ないことが期待されること、培養液の量も少なくできることなど、多くの利点を有する。  In the case of suspension culture, as long as the non-adhesive state to the culture surface can be maintained, the cells/cell clusters may be statically cultured, swirl cultured, or shaken cultured. Preferably, the suspension culture here is performed by static culture. Static culture has many advantages, such as no need for a special device, less impact or damage to cells is expected, and the amount of culture solution can be reduced. 
　スフェロイドを形成可能な限り、培養条件は特に限定されない。典型的には、幹細胞性を維持しつつスフェロイドを形成させるために、上皮成長因子（EGF）、BMP阻害剤及びWntシグナル活性化剤、ROCK阻害剤（Y-27632等）の存在下、即ち、これらの成分が添加された培地を用いて浮遊培養する。  The culture conditions are not particularly limited as long as spheroids can be formed. Typically, in order to form spheroids while maintaining stemness, in the presence of epidermal growth factor (EGF), BMP inhibitors and Wnt signal activators, ROCK inhibitors (Y-27632, etc.), that is, Suspension culture is performed using a medium to which these components have been added. 
　上皮成長因子を用いることにより、細胞増殖を促進させる効果を期待できる。また、BMP阻害剤を用いることにより、幹細胞の分化を抑制し、幹細胞性を維持する効果を期待できる。Wntシグナル活性化剤には幹細胞の増殖と幹細胞性を維持する効果を期待できる。  By using epidermal growth factor, the effect of promoting cell proliferation can be expected. Moreover, by using a BMP inhibitor, an effect of suppressing differentiation of stem cells and maintaining stemness can be expected. Wnt signal activators are expected to have the effect of maintaining stem cell proliferation and stemness. 
　BMP阻害剤として例えばNogginを用いることができる。また、Wntシグナル活性化剤として例えばR-spondin-1を用いることができる。  For example, Noggin can be used as a BMP inhibitor. Also, for example, R-spondin-1 can be used as a Wnt signal activator. 
　上皮成長因子の添加濃度の例を示すと10 ng/mL～500 ng/mL、好ましくは50 ng/mL～200 ng/mLである。BMP阻害剤の添加濃度の例（Nogginの場合）を示すと10 ng/mL～500 ng/mL、好ましくは50 ng/mL～200 ng/mLである。同様に、Wntシグナル活性化剤の添加濃度の例（R-spondin-1の場合）を示すと10 ng/mL～1000 ng/mL、好ましくは50 ng/mL～500 g/mLである。ROCK阻害剤の添加濃度の例（Y-27632の場合）を示すと1μM～50μM、好ましくは3μM～30μMである。 An example of the concentration of epidermal growth factor added is 10 ng/mL to 500 ng/mL, preferably 50 ng/mL to 200 ng/mL. An example of the concentration of the BMP inhibitor added (for Noggin) is 10 ng/mL to 500 ng/mL, preferably 50 ng/mL to 200 ng/mL. Similarly, an example concentration of the Wnt signal activator added (for R-spondin-1) is 10 ng/mL to 1000 ng/mL, preferably 50 ng/mL to 500 g/mL. An example of the addition concentration of the ROCK inhibitor (in the case of Y-27632) is 1 μM to 50 μM, preferably 3 μM to 30 μM.
　尚、例示した化合物、即ち、Noggin、R-spondin-1とは異なる化合物を使用する場合の添加濃度については、使用する化合物の特性と、例示した化合物の特性の相違（特に活性の相違）を考慮すれば、当業者であれば上記濃度範囲に準じて設定することができる。また、設定した濃度範囲が適切であるか否かは、後述の実施例に準じた予備実験によって確認することができる。  When using a compound different from the exemplified compounds, that is, Noggin and R-spondin-1, the concentration to be added should be determined according to the characteristics of the compound used and the difference in characteristics (especially the difference in activity) of the exemplified compounds. Taking into consideration, a person skilled in the art can set it according to the above concentration range. Also, whether or not the set concentration range is appropriate can be confirmed by a preliminary experiment according to the examples described later. 
　工程（４）の期間（培養期間）は例えば１日間～１０日間、好ましくは２日間～７日間である。当該培養期間が短すぎると、十分な大きさのスフェロイドが形成されない。他方、当該培養期間が長すぎると、必要以上にスフェロイドが大きくなり、内部の細胞がネクローシスを起こすおそれがある。好ましくは、直径が100μm～200μm程度のスフェロイドを形成させる。例えば、直径が400～500μm、深さが100～200μmのウェルが均一に形成されたパターンプレートの使用によって、当該サイズのスフェロイドを形成させることができる。 The period of step (4) (culture period) is, for example, 1 to 10 days, preferably 2 to 7 days. If the culture period is too short, spheroids of sufficient size will not be formed. On the other hand, if the culture period is too long, the spheroids become larger than necessary, and the cells inside may undergo necrosis. Preferably, spheroids with a diameter of about 100 μm to 200 μm are formed. For example, spheroids of this size can be formed by using a pattern plate in which wells having a diameter of 400 to 500 μm and a depth of 100 to 200 μm are uniformly formed.
＜工程（５）：腸管オルガノイドの形成＞ 
　この工程では、工程（４）で形成されたスフェロイドを分化させ、腸管オルガノイドを形成させる。この工程において積極的な分化誘導は必須ではなく、一態様（第１態様）では工程（４）と同一又は類似の培地条件で培養を行う。具体的には、上皮成長因子、BMP阻害剤及びWntシグナル活性化剤の存在下、即ち、これらの成分が添加された培地を用いて培養する。各化合物の具体例や添加濃度も工程（４）の場合と同様である。尚、工程（５）の期間（培養期間）は例えば６日間～３６日間、好ましくは８日間～１８日間である。 <Step (5): Formation of intestinal organoids>
In this step, the spheroids formed in step (4) are differentiated to form intestinal organoids. Active induction of differentiation is not essential in this step, and in one aspect (first aspect), culture is performed under the same or similar medium conditions as in step (4). Specifically, it is cultured in the presence of epidermal growth factor, BMP inhibitor and Wnt signal activator, that is, using a medium supplemented with these components. Specific examples and concentration of each compound are the same as in the step (4). The period of step (5) (culture period) is, for example, 6 to 36 days, preferably 8 to 18 days.
　スフェロイドから腸管オルガノイドを形成させるため、工程（５）の培養は浮遊培養で行う。ここでの浮遊培養には、好ましくは、水溶液中に３次元的網目構造を形成する材料を添加した液体培地を用い、工程（４）で形成させた複数のスフェロイドをまとめて（即ち、一つの培養容器内に複数のスフェロイドが併存した状態で）浮遊培養する。即ち、好ましい態様では、工程（４）で形成させたスフェロイドの一部（但し２個以上）又は全部が、特徴的な液体培地を使用した浮遊培養に供されることになる。  In order to form intestinal organoids from spheroids, the culture in step (5) is performed in suspension culture. For the suspension culture here, preferably, a liquid medium to which a material that forms a three-dimensional network structure is added in an aqueous solution is used, and the plurality of spheroids formed in step (4) are collected together (that is, one (with multiple spheroids coexisting in the culture vessel). That is, in a preferred embodiment, part (but two or more) or all of the spheroids formed in step (4) are subjected to suspension culture using a characteristic liquid medium. 
　水溶液中に３次元的網目構造を形成する材料（以下、「粘性材料」と呼ぶ）を用いることにより、網目構造にスフェロイドが捕捉ないしトラップされ、或いは培地の粘性が高まってスフェロイドの動きが制限され、スフェロイドの会合や凝集を防止できる。従って、複数のスフェロイドをまとめて浮遊培養することができ、効率的な腸管オルガノイドの形成が可能となる。 By using a material that forms a three-dimensional network structure in an aqueous solution (hereinafter referred to as "viscous material"), spheroids are captured or trapped in the network structure, or the viscosity of the medium is increased to restrict the movement of spheroids. , can prevent spheroid association and aggregation. Therefore, a plurality of spheroids can be collectively cultured in suspension, enabling efficient formation of intestinal organoids.
　粘性材料として、例えば、高分子ゲル、多糖を用いることができる。高分子ゲルの例は、コラーゲン、高分子ヒドロゲル、マトリゲル TM（通常のマトリゲル、成長因子の含有量を少なくしたグロースファクターリデュースト（GFR）マトリゲル等）である。多糖の例はジェランガムや、結晶セルロース、ナノセルロース、カルボキシセルロース、カルボキシメチルセルロースなどである。２種類以上の材料を併用することにしてもよい。  For example, polymer gels and polysaccharides can be used as viscous materials. Examples of polymer gels are collagen, polymer hydrogels, Matrigel TM (regular Matrigel, Growth Factor Reduced (GFR) Matrigel with reduced content of growth factors, etc.). Examples of polysaccharides are gellan gum, crystalline cellulose, nanocellulose, carboxycellulose, carboxymethylcellulose, and the like. Two or more kinds of materials may be used together. 
　好ましい一態様では、粘性材料として、アニオン性の官能基を有する高分子化合物が用いられる。アニオン性の官能基としては、カルボキシ基、スルホ基、リン酸基及びそれらの塩が挙げられ、カルボキシ基またはその塩が好ましい。本発明に用いる高分子化合物は、前記アニオン性の官能基の群より選択される１種又は２種以上を有するものを使用できる。ここでの高分子化合物の好ましい具体例としては、特に制限されるものではないが、単糖類（例えば、トリオース、テトロース、ペントース、ヘキソース、ヘプトース等）が１０個以上重合した多糖類が挙げられ、より好ましくは、アニオン性の官能基を有する酸性多糖類が挙げられる。ここにいう酸性多糖類とは、その構造中にアニオン性の官能基を有すれば特に制限されないが、例えば、ウロン酸（例えば、グルクロン酸、イズロン酸、ガラクツロン酸、マンヌロン酸）を有する多糖類、構造中の一部に硫酸基又はリン酸基を有する多糖類、或いはその両方の構造を持つ多糖類であって、天然から得られる多糖類のみならず、微生物により産生された多糖類、遺伝子工学的に産生された多糖類、或いは酵素を用いて人工的に合成された多糖類も含まれる。より具体的には、ヒアルロン酸、ジェランガム、脱アシル化ジェランガム、ラムザンガム、ダイユータンガム、キサンタンガム、カラギーナン、ザンタンガム、ヘキスロン酸、フコイダン、ペクチン、ペクチン酸、ペクチニン酸、ヘパラン硫酸、ヘパリン、ヘパリチン硫酸、ケラト硫酸、コンドロイチン硫酸、デルマタン硫酸、ラムナン硫酸及びそれらの塩からなる群より選択される１種又は２種以上から構成されるものが例示される。多糖類は、好ましくは、ヒアルロン酸、脱アシル化ジェランガム、ダイユータンガム、キサンタンガム、カラギーナン又はそれらの塩であり、低濃度の使用で目的を達成できることなどを考慮すると、最も好ましくは、脱アシル化ジェランガムである。ここでいう塩とは、例えば、リチウム、ナトリウム、カリウムといったアルカリ金属の塩、カルシウム、バリウム、マグネシウムといったアルカリ土類金属の塩又はアルミニウム、亜鉛、銅、鉄、アンモニウム、有機塩基及びアミノ酸等の塩が挙げられる。  In a preferred embodiment, a polymer compound having an anionic functional group is used as the viscous material. Examples of anionic functional groups include a carboxy group, a sulfo group, a phosphoric acid group and salts thereof, with a carboxy group or a salt thereof being preferred. As the polymer compound used in the present invention, those having one or more selected from the group of anionic functional groups can be used. Preferred specific examples of the polymer compound herein include, but are not particularly limited to, polysaccharides in which 10 or more monosaccharides (e.g., triose, tetrose, pentose, hexose, heptose, etc.) are polymerized, Acidic polysaccharides having anionic functional groups are more preferred. The acidic polysaccharide referred to here is not particularly limited as long as it has an anionic functional group in its structure. , polysaccharides having a sulfate group or a phosphate group in a part of the structure, or polysaccharides having both structures, which are not only polysaccharides obtained from nature but also polysaccharides produced by microorganisms, genes Polysaccharides produced by engineering or polysaccharides artificially synthesized using enzymes are also included. More specifically, hyaluronic acid, gellan gum, deacylated gellan gum, rhamsan gum, diutan gum, xanthan gum, carrageenan, xanthan gum, hexuronic acid, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparin, heparitin sulfate, kerato Examples include those composed of one or more selected from the group consisting of sulfuric acid, chondroitin sulfate, dermatan sulfate, rhamnan sulfate, and salts thereof. The polysaccharide is preferably hyaluronic acid, deacylated gellan gum, diutan gum, xanthan gum, carrageenan, or a salt thereof. Gellan gum. The salt referred to here includes, for example, salts of alkali metals such as lithium, sodium and potassium, salts of alkaline earth metals such as calcium, barium and magnesium, salts of aluminum, zinc, copper, iron, ammonium, organic bases and amino acids. is mentioned. 
　上記高分子化合物（多糖類等）の重量平均分子量は、好ましくは10，000～50，000，000であり、より好ましくは100，000～20，000，000、更に好ましくは1，000，000～10，000，000である。例えば、当該分子量は、ゲル浸透クロマトグラフィー（GPC）によるプルラン換算で測定できる。  The weight average molecular weight of the polymer compound (polysaccharide, etc.) is preferably 10,000 to 50,000,000, more preferably 100,000 to 20,000,000, and still more preferably 1,000,000. ~10,000,000. For example, the molecular weight can be measured in terms of pullulan by gel permeation chromatography (GPC). 
　更に、脱アシル化ジェランガムはリン酸化したものを使用することもできる。当該リン酸化は公知の手法で行うことができる。  Furthermore, phosphorylated deacylated gellan gum can also be used. The phosphorylation can be performed by a known technique. 
　本発明においては、上記多糖類を複数種（好ましくは２種）組み合わせて使用することができる。多糖類の組み合わせの種類は、スフェロイドの会合や凝集を防止できるものであれば特に限定されないが、好ましくは、当該組合せは少なくとも脱アシル化ジェランガム又はその塩を含む。即ち、好適な多糖類の組合せには、脱アシル化ジェランガム又はその塩、及び脱アシル化ジェランガム又はその塩以外の多糖類（例、キサンタンガム、アルギン酸、カラギーナン、ダイユータンガム、メチルセルロース、ローカストビーンガム又はそれらの塩）が含まれる。具体的な多糖類の組み合わせとしては、脱アシル化ジェランガムとラムザンガム、脱アシル化ジェランガムとダイユータンガム、脱アシル化ジェランガムとキサンタンガム、脱アシル化ジェランガムとカラギーナン、脱アシル化ジェランガムとザンタンガム、脱アシル化ジェランガムとローカストビーンガム、脱アシル化ジェランガムとκ－カラギーナン、脱アシル化ジェランガムとアルギン酸ナトリウム、脱アシル化ジェランガムとメチルセルロース等が挙げられるが、これらに限定されない。  In the present invention, a combination of multiple types (preferably two types) of the above polysaccharides can be used. The type of combination of polysaccharides is not particularly limited as long as it can prevent aggregation and aggregation of spheroids, but preferably the combination contains at least deacylated gellan gum or a salt thereof. Thus, suitable polysaccharide combinations include deacylated gellan gum or salts thereof, and polysaccharides other than deacylated gellan gum or salts thereof (e.g., xanthan gum, alginic acid, carrageenan, diutan gum, methylcellulose, locust bean gum or their salts). Specific polysaccharide combinations include deacylated gellan gum and rhamsan gum, deacylated gellan gum and diutan gum, deacylated gellan gum and xanthan gum, deacylated gellan gum and carrageenan, deacylated gellan gum and xanthan gum, deacylated Examples include, but are not limited to, gellan gum and locust bean gum, deacylated gellan gum and κ-carrageenan, deacylated gellan gum and sodium alginate, deacylated gellan gum and methylcellulose, and the like. 
　本発明に用いる粘性材料の更に好ましい具体例としては、ヒアルロン酸、脱アシル化ジェランガム、ダイユータンガム、カラギーナン及びキサンタンガム、並びにそれらの塩が挙げられ、最も好ましい例としては脱アシル化ジェランガムまたはその塩が挙げられる。脱アシル化ジェランガムの場合、市販のもの、例えば、三晶株式会社製「KELCOGEL（シーピー・ケルコ社の登録商標）CG-LA」、三栄源エフ・エフ・アイ株式会社製「ケルコゲル（シーピー・ケルコ社の登録商標）」等を使用することができる。また、ネイティブ型ジェランガムとして、三栄源エフ・エフ・アイ株式会社製「ケルコゲル（シーピー・ケルコ社の登録商標）HT」等を使用することができる。特に好ましい粘性材料の例として、日産化学工業株式会社が提供するポリマーFP001又はポリマーFP003を挙げることができる。尚、ポリマーFP001は日産化学工業株式会社製の三次元培養培地FCeM（登録商標）シリーズの配合成分であり、ポリマーFP003は同FCeM（登録商標）Advance Preparation Kitの配合成分である。  More preferred specific examples of the viscous material used in the present invention include hyaluronic acid, deacylated gellan gum, diutan gum, carrageenan, xanthan gum, and salts thereof, and the most preferred example is deacylated gellan gum or a salt thereof. is mentioned. In the case of deacylated gellan gum, commercially available products such as "KELCOGEL (registered trademark of CP Kelco) CG-LA" manufactured by Sansho Co., Ltd., "Kelcogel (registered trademark of CP Kelco Co., Ltd.)" manufactured by Sanei Gen FFI Co. company's registered trademark)" etc. can be used. Also, as the native type gellan gum, San-Ei Gen FFI Co., Ltd. "Kelcogel (registered trademark of CP Kelco) HT" and the like can be used. Examples of particularly preferred viscous materials include Polymer FP001 and Polymer FP003 provided by Nissan Chemical Industries, Ltd. The polymer FP001 is a compounding component of the three-dimensional culture medium FCeM (registered trademark) series manufactured by Nissan Chemical Industries, Ltd., and the polymer FP003 is a compounding component of the same FCeM (registered trademark) Advance Preparation Kit. 
　粘性材料の使用量、即ち、培地への添加量は、期待される上記効果を発揮できる限り特に限定されないが、例えば、培地の粘度が5 mPas・s～2000 mPas・sになるように粘性材料の使用量を調整する。培地の粘度が低すぎると、スフェロイドの会合や凝集を防止するという効果が得られなくなる。一方、培地の粘度が高すぎると、操作性（取扱い）に影響し（例えば、回収操作が煩雑となる）、培地成分の細胞への供給に影響が出るおそれもある。尚、粘性材料の使用量の具体例としてマトリゲルの場合を示すと、通常の使用（即ち３次元培養用の基材としての使用）における使用量の1％～10％程度にするとよい。また、脱アシル化ジェランガムの場合、0.001％～1.0％（w/v）、好ましくは0.003％～0.5％（w/v）、より好ましくは0.005％～0.3％（w/v）、更に好ましくは0.01％～0.05％（w/v）、最も好ましくは、0.01％～0.03％（w/v）培地中に添加するとよい。  The amount of the viscous material used, that is, the amount added to the medium is not particularly limited as long as the expected effect can be exhibited. Adjust the usage of If the viscosity of the medium is too low, the effect of preventing association and aggregation of spheroids cannot be obtained. On the other hand, if the viscosity of the medium is too high, the operability (handling) is affected (for example, recovery operation becomes complicated), and the supply of medium components to cells may be affected. In the case of Matrigel as a specific example of the amount of viscous material to be used, it should be about 1% to 10% of the amount used in normal use (that is, use as a base material for three-dimensional culture). In the case of deacylated gellan gum, the 0.01% to 0.05% (w/v), most preferably 0.01% to 0.03% (w/v) should be added to the medium. 
　浮遊培養に用いる培養容器は、特に限定されず、例えば、ディッシュ、フラスコ、マルチウェルプレート、チューブ、トレイ、培養バック等を用いることができる。  The culture vessel used for floating culture is not particularly limited, and for example, dishes, flasks, multiwell plates, tubes, trays, culture bags, etc. can be used. 
　工程（５）において継代培養（例えば１回～５回）を行うことも可能である。この場合の継代操作は例えば以下の手順に従えばよい。まず、細胞塊（スフェロイド）を回収し、必要に応じて洗浄した後、スフェロイド用の分散／解離液（例えばThermo Fisher Scientific社のEDTAや、Roche社のDispase II、STEMCELL Technologies社のGentle Cell Dissociation Reagent）で処理する。続いて、ピペッティング操作などによって分散、懸濁させた後、播種し、培養を継続する。  It is also possible to subculture (eg, 1 to 5 times) in step (5). The subculture operation in this case may follow, for example, the following procedure. First, collect the cell aggregates (spheroids), wash them if necessary, and then disperse/dissociate the spheroids (e.g. Thermo Fisher Scientific EDTA, Roche Dispase II, STEMCELL Technologies Gentle Cell Dissociation Reagent). ). Subsequently, after dispersing and suspending by pipetting operation or the like, the cells are seeded and the culture is continued. 
　継代培養や培地交換に伴う、細胞の回収の際には、細胞死を抑制するためにY-27632等のROCK阻害剤（Rho-associated coiled-coil forming kinase/Rho結合キナーゼ）で予め細胞を処理しておくとよい。  When harvesting cells during subculture or medium exchange, pre-treat the cells with a ROCK inhibitor (Rho-associated coiled-coil forming kinase/Rho-associated kinase) such as Y-27632 to suppress cell death. should be processed. 
＜工程（６）：気相液相培養による腸管細胞の作製＞ 
　工程（６）では、工程（５）で形成された腸管オルガノイドを構成する細胞を、上皮成長因子、cAMPシグナル活性化因子、TGFβ受容体阻害剤、及びWntシグナル活性化剤の存在下で気相液相培養（平面培養）する。形成された腸管オルガノイドから細胞を回収して二次元培養系に移し、気相液相培養する。典型的には、腸管オルガノイドから回収した細胞集団又はその一部を選別することなく気液平面培養に供する。腸管オルガノイドからの細胞の回収には細胞解離液などを利用すればよい。細胞解離液としては、例えば、トリプシン-EDTA、コラゲナーゼIV、メタロプロテアーゼ等のタンパク分解酵素等を単独で又は適宜組み合わせて用いることができる。細胞障害性が少ないものが好ましい。このような細胞解離液として、例えば、ディスパーゼ（エーディア）、TrypLE （Invitrogen）又はアキュターゼ（MILLIPORE）等の市販品が入手可能である。また、セルストレイナーなどで処理し、細胞の分散性を高めるとよい。尚、細胞の回収の際には、細胞死を抑制するためにY-27632等のROCK阻害剤（Rho-associated coiled-coil forming kinase/Rho結合キナーゼ）で予め細胞を処理しておくとよい。  <Step (6): Preparation of intestinal cells by gas phase liquid phase culture>
In step (6), the cells constituting the intestinal organoids formed in step (5) are subjected to gas phase in the presence of epidermal growth factor, cAMP signal activator, TGFβ receptor inhibitor, and Wnt signal activator. Liquid phase culture (flat culture). Cells are collected from the formed intestinal organoids, transferred to a two-dimensional culture system, and cultured in gas-liquid phase. Typically, cell populations or portions thereof collected from intestinal organoids are subjected to air-liquid planar culture without sorting. A cell dissociation solution or the like may be used to collect cells from intestinal organoids. As the cell dissociation solution, for example, trypsin-EDTA, collagenase IV, proteolytic enzymes such as metalloprotease, and the like can be used alone or in appropriate combination. Those with less cytotoxicity are preferred. Commercial products such as dispase (Edia), TrypLE (Invitrogen) and Accutase (MILLIPORE) are available as such cell dissociation solutions. In addition, it is preferable to treat the cells with a cell strainer or the like to increase the dispersibility of the cells. When collecting the cells, it is preferable to treat the cells in advance with a ROCK inhibitor (Rho-associated coiled-coil forming kinase/Rho-associated kinase) such as Y-27632 in order to suppress cell death.
　工程（６）の培養条件である、「上皮成長因子、cAMPシグナル活性化因子、TGFβ受容体阻害剤、及びWntシグナル活性化剤の存在下」とは、これらの化合物が培地中に添加された条件と同義である。従って、上皮成長因子、cAMPシグナル活性化因子、TGFβ受容体阻害剤、及びWntシグナル活性化剤の存在下での培養を行うためには、これらの化合物が添加された培地を用いればよい。 The culture conditions in step (6), "in the presence of epidermal growth factor, cAMP signal activator, TGFβ receptor inhibitor, and Wnt signal activator" are those compounds added to the medium. Synonymous with condition. Therefore, for culture in the presence of epidermal growth factor, cAMP signal activator, TGFβ receptor inhibitor, and Wnt signal activator, a medium supplemented with these compounds may be used.
　上皮成長因子の添加濃度の例を示すと10 ng/mL～500 ng/mL、好ましくは50 ng/mL～200 ng/mLである。 An example of the concentration of epidermal growth factor added is 10 ng/mL to 500 ng/mL, preferably 50 ng/mL to 200 ng/mL.
　cAMPシグナル活性化因子としてcAMP誘導体、cAMP分解酵素阻害剤又はcAMP活性化物質を用いることができる。これらの中の二つ以上の物質を併用してもよい。 cAMP derivatives, cAMP degrading enzyme inhibitors, or cAMP activators can be used as cAMP signal activators. Two or more of these substances may be used in combination.
　cAMP誘導体としてPKA活性剤（例えば、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活性剤（Rp-8-Br-cAMPS(8-Bromoadenosine 3',5'-cyclic Monophosphothioate, Rp-Isomer . sodium salt, CAS Number : 129735-00-8)、8-CPT-cAMP(8-(4-Chlorophenylthio)adenosine 3',5'-cyclic monophosphate, CAS Number : 93882-12-3)、8-pCPT-2'-O-Me-cAMP(8-(4-Chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium, CAS Number : 634207-53-7)等）を採用することができる。cAMP誘導体の添加濃度の例（8-Br-cAMPの場合）を示すと、0.1 mM～10 mM、好ましくは0.2 mM～5 mM、更に好ましくは0.5 mM～2 mMである。尚、例示した化合物、即ち、8-Br-cAMPとは異なる化合物を使用する場合の添加濃度については、使用する化合物の特性と、例示した化合物（8-Br-cAMP）の特性の相違（特に活性の相違）を考慮すれば、当業者であれば上記濃度範囲に準じて設定することができる。また、設定した濃度範囲が適切であるか否かは、予備実験によって確認することができる。 PKA active agents as cAMP derivatives (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 activator (Rp-8-Br-cAMPS (8-Bromoadenosine 3',5'-cyclic Monophosphothioate, Rp-Isomer. sodium salt, CAS Number: 129735-00-8), 8-CPT-cAMP (8 -(4-Chlorophenylthio)adenosine 3',5'-cyclic monophosphate, CAS Number: 93882-12-3), 8-pCPT-2'-O-Me-cAMP(8-(4-Chlorophenylthio)-2'- O-methyladenosine 3',5'-cyclic monophosphate monosodium, CAS Number: 634207-53-7), etc.) can be done. An example of the concentration of the cAMP derivative added (in the case of 8-Br-cAMP) is 0.1 mM to 10 mM, preferably 0.2 mM to 5 mM, more preferably 0.5 mM to 2 mM. When using the exemplified compound, that is, a compound different from 8-Br-cAMP, the concentration to be added depends on the difference between the characteristics of the compound used and the characteristics of the exemplified compound (8-Br-cAMP) (especially Considering the difference in activity), a person skilled in the art can set the concentration range according to the above. Also, whether or not the set concentration range is appropriate can be confirmed by a preliminary experiment.
　cAMP分解酵素阻害剤として、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 115 BS)、Enoximone (MDL 17043)、Imazodan (CL 914)、SKF-94120、Vesnarinone (OPC 8212)、Rolipram (Ro-20-1724)、(ZK-62711)、Denbufyll'ine、Zaprinast (M&B-22, 948)、Dipyridamole、Zaprinast (M&B-22, 948)、Dipyridamole、Zardaverine、AH-21-132、Sulmazol (AR-L 115 BS)を例示することができる。cAMP分解酵素阻害剤の添加濃度の例（IBMXの場合）を示すと、0.05 mM～5 mM、好ましくは0.1 mM～3 mM、更に好ましくは0.2 mM～1 mMである。尚、例示した化合物、即ち、IBMXとは異なる化合物を使用する場合の添加濃度については、使用する化合物の特性と、例示した化合物（IBMX）の特性の相違（特に活性の相違）を考慮すれば、当業者であれば上記濃度範囲に準じて設定することができる。また、設定した濃度範囲が適切であるか否かは、予備実験によって確認することができる。  IBMX (3-isobutyl-1-methylxanthine) (MIX), Theophylline, Papaverine, Pentoxifylline (Trental), KS-505, 8-Methoxymethyl-IBMX, Vinpocetine (TCV-3B), EHNA, Trequinsin as cAMPase inhibitors (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 115 BS), Enoximone (MDL 17043), Imazodan (CL 914), SKF-94120, Vesnarinone (OPC 8212), Rolipram (Ro-20-1724), (ZK-62711), Denbufyll'ine, Zaprinast (M&B-22, 948), Dipyridamole, Zaprinast (M&B-22, 948), Dipyridamole, Zardaverine, AH- 21-132 and Sulmazol (AR-L 115 BS). An example of the concentration of the cAMP degrading enzyme inhibitor added (in the case of IBMX) is 0.05 mM to 5 mM, preferably 0.1 mM to 3 mM, more preferably 0.2 mM to 1 mM. When using the exemplified compound, that is, a compound different from IBMX, the addition concentration should be determined considering the difference in the characteristics of the compound used and the exemplified compound (IBMX) (especially the difference in activity). , can be set according to the above concentration range by those skilled in the art. Also, whether or not the set concentration range is appropriate can be confirmed by a preliminary experiment. 
　cAMP活性化物質としては、フォルスコリン、インドメタシン、NKH477（コルホルシンダロパート）、細胞由来毒素タンパク質（百日咳毒素、コレラ毒素）、PACAP-27、PACAP-38、SKF83822等を用いることができる。cAMP活性化物質の添加濃度の例（フォルスコリンの場合）を示すと、1μM～200μM、好ましくは5μM～100μMである。尚、例示した化合物、即ち、フォルスコリンとは異なる化合物を使用する場合の添加濃度については、使用する化合物の特性と、例示した化合物（フォルスコリン）の特性の相違（特に活性の相違）を考慮すれば、当業者であれば上記濃度範囲に準じて設定することができる。また、設定した濃度範囲が適切であるか否かは、後述の実施例に準じた予備実験によって確認することができる。 As cAMP activators, forskolin, indomethacin, NKH477 (colforsin daropate), cell-derived toxin proteins (pertussis toxin, cholera toxin), PACAP-27, PACAP-38, SKF83822, etc. can be used. An example of the concentration of the cAMP activator added (in the case of forskolin) is 1 μM to 200 μM, preferably 5 μM to 100 μM. When using the exemplified compound, that is, a compound different from forskolin, the concentration to be added takes into account the difference between the characteristics of the compound used and the characteristics of the exemplified compound (forskolin) (especially the difference in activity). Then, a person skilled in the art can set the concentration according to the above concentration range. Also, whether or not the set concentration range is appropriate can be confirmed by a preliminary experiment according to the examples described later.
　TGFβ受容体阻害剤については、好ましくは、TGF-β受容体ALK4、ALK5、ALK7の一以上に対して阻害活性を示すものを用いるとよい。例えば、A-83-01、SB431542、SB-505124、SB525334、D4476、ALK5 inhibitor、LY2157299、LY364947、GW788388、RepSoxが当該条件を満たす。 As for the TGFβ receptor inhibitor, it is preferable to use one that exhibits inhibitory activity against one or more of the TGF-β receptors ALK4, ALK5, and ALK7. For example, A-83-01, SB431542, SB-505124, SB525334, D4476, ALK5 inhibitor, LY2157299, LY364947, GW788388, and RepSox satisfy this condition.
　TGFβ受容体阻害剤の添加濃度の例（A-83-01の場合）を示すと0.1μM～50μM、好ましくは0.3μM～30μMである。尚、例示した化合物、即ち、A-83-01とは異なる化合物を使用する場合の添加濃度については、使用する化合物の特性と、例示した化合物の特性の相違（特に活性の相違）を考慮すれば、当業者であれば上記濃度範囲に準じて設定することができる。また、設定した濃度範囲が適切であるか否かは、後述の実施例に準じた予備実験によって確認することができる。 An example of the added concentration of the TGFβ receptor inhibitor (in the case of A-83-01) is 0.1 μM to 50 μM, preferably 0.3 μM to 30 μM. Regarding the addition concentration when using the exemplified compound, that is, a compound different from A-83-01, the difference in the characteristics of the compound used and the exemplified compound (especially the difference in activity) should be considered. For example, a person skilled in the art can set the concentration according to the above concentration range. Also, whether or not the set concentration range is appropriate can be confirmed by a preliminary experiment according to the examples described later.
　Wntシグナル活性化剤は、GSK-3を阻害する活性を有する化合物であればよく、例えば、
CHIR99021、CHIR98014、BIO、SB415286、SB216763、TWS119、A1070722等が挙げられる。 The Wnt signal activator may be any compound having activity to inhibit GSK-3, for example,
CHIR99021, CHIR98014, BIO, SB415286, SB216763, TWS119, A1070722 and the like.
　Wntシグナル活性化剤の添加濃度の例（CHIR99021の場合）を示すと、0.1μM～100μM、好ましくは1μM～10μMである。尚、例示した化合物、即ち、CHIR99021とは異なる化合物を使用する場合の添加濃度については、使用する化合物の特性と、例示した化合物（フォルスコリン）の特性の相違（特に活性の相違）を考慮すれば、当業者であれば上記濃度範囲に準じて設定することができる。また、設定した濃度範囲が適切であるか否かは、後述の実施例に準じた予備実験によって確認することができる。 An example of the added concentration of the Wnt signal activator (in the case of CHIR99021) is 0.1 μM to 100 μM, preferably 1 μM to 10 μM. When using the exemplified compound, that is, a compound different from CHIR99021, the addition concentration should be determined in consideration of the difference between the characteristics of the compound used and the exemplified compound (forskolin) (especially the difference in activity). For example, a person skilled in the art can set the concentration according to the above concentration range. Also, whether or not the set concentration range is appropriate can be confirmed by a preliminary experiment according to the examples described later.
　工程（６）の気相液相培養は、上皮成長因子、cAMPシグナル活性化因子、TGFβ受容体阻害剤、及びWntシグナル活性化剤に加え、Notchシグナル阻害剤の存在下で行ってもよい。 The gas-liquid phase culture in step (6) may be performed in the presence of Notch signal inhibitor in addition to epidermal growth factor, cAMP signal activator, TGFβ receptor inhibitor, and Wnt signal activator.
　Notchシグナル阻害剤としては、N-[(3,5-difluorophenyl) acetyl]-L-alanyl-2-phenyl-1,1-dimethylethyl ester-glycine (DAPT)、L-685,458、Compound E (CAS 209986-17-4)、(R)-Flurbiprofen、BMS299897、JLK6、LY-411575、R04929097、MK-0752、SCP0004、SCP0025、gamma-Secretase Inhibitor XI、gamma-Secretase Inhibitor XVI、gamma-Secretase Inhibitor I、gamma-Secretase Inhibitor VII、Semagacestat (LY450139)、gamma-Secretase Inhibitor III、Compound 34、BMS-708163、Compound W、YO-01027 (Dibenzazepine)、Avagacestat (BMS-708163)などのγ－セクレターゼ阻害剤を使用することができる。 Notch signal inhibitors include N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl-1,1-dimethylethylester-glycine (DAPT), L-685,458, Compound E (CAS 209986- 17-4), (R)-Flurbiprofen, BMS299897, JLK6, LY-411575, R04929097, MK-0752, SCP0004, SCP0025, gamma-Secretase Inhibitor XI, gamma-Secretase Inhibitor XVI, gamma-Secretase Inhibitor I, gamma-Secretase γ-Secretase inhibitors such as Inhibitor VII, Semagacestat (LY450139), gamma-Secretase Inhibitor III, Compound 34, BMS-708163, Compound W, YO-01027 (Dibenzazepine), Avagacestat (BMS-708163) can be used. .
　Notchシグナル阻害剤の添加濃度の例（DAPTの場合）を示すと1 nM～20μM、好ましくは0.1μM～10μMであり、尚、DAPTとは異なる化合物を使用する場合の添加濃度については、使用する化合物の特性と、例示した化合物の特性の相違（特に活性の相違）を考慮すれば、当業者であれば上記濃度範囲に準じて設定することができる。また、設定した濃度範囲が適切であるか否かは、後述の実施例に準じた予備実験によって確認することができる。 An example of the concentration of the Notch signal inhibitor added (in the case of DAPT) is 1 nM to 20 μM, preferably 0.1 μM to 10 μM, and when using a compound different from DAPT, the concentration added is A person skilled in the art can set the concentration range according to the above concentration range, taking into account the characteristics of the compounds and the difference in characteristics (especially the difference in activity) of the exemplified compounds. Also, whether or not the set concentration range is appropriate can be confirmed by a preliminary experiment according to the examples described later.
　工程（６）の気相液相培養は、Notchシグナル阻害剤の存在下で気相液相培養を行った後に、Notchシグナル阻害剤の非存在下で行う気相液相培養を行うことを含んでいてもよい。 The gas phase liquid phase culture in step (6) includes performing gas phase liquid phase culture in the presence of the Notch signal inhibitor, followed by gas phase liquid phase culture in the absence of the Notch signal inhibitor. You can stay.
　工程（６）の気相液相培養を、5-アザ-2’-デオキシシチジンの存在下又は非存在下において行うことができる。 The gas phase liquid phase culture in step (6) can be performed in the presence or absence of 5-aza-2'-deoxycytidine.
　工程（６）の培養には、細胞接着性の培養面を備える培養容器が使用される。細胞接着性の培養面として、好ましくは、基底膜成分（例えばラミニン、IV型コラーゲン、エンタクチン、ビトロネクチン、フィブロネクチン）又はその断片をコートしたものを用いるとよい。特に、ラミニン511又はそのE8断片でコートした培養面を採用するとよい。ラミニン511のE8断片については、その組換え体（組換えヒトラミニン511-E8タンパク質）を高純度に精製した製品（製品名：iMatrix-511、製造：株式会社ニッピ、販売：株式会社マトリクソーム）が市販されている。尚、培養容器は特に限定されず、例えば、ディッシュ、フラスコ、マルチウェルプレート等を用いることができる。  A culture vessel with a cell-adhesive culture surface is used for the culture in step (6). As the cell-adhesive culture surface, one coated with a basement membrane component (eg, laminin, type IV collagen, entactin, vitronectin, fibronectin) or a fragment thereof is preferably used. In particular, culture surfaces coated with laminin 511 or its E8 fragment may be employed. Regarding the E8 fragment of laminin 511, a product (product name: iMatrix-511, manufactured by Nippi Co., Ltd., sold by Matrixome Co., Ltd.), which is a highly purified recombinant (recombinant human laminin 511-E8 protein), is commercially available. It is The culture vessel is not particularly limited, and for example, dishes, flasks, multi-well plates and the like can be used. 
　一態様では、半透過性膜（多孔性膜）の上で細胞を培養し、細胞層を形成させる。具体的には、例えば、インサートを備えた培養容器（例えば、コーニング社が提供するトランズウェル（登録商標））を使用し、インサート内に細胞を播種して培養することにより細胞層を得る。この態様によれば、細胞層を透過した被検物質の定量によって評価を行う評価系への利用に適した細胞層が得られることになる。尚、評価系の構築方法やその利用方法等は後述する。 In one aspect, cells are cultured on a semipermeable membrane (porous membrane) to form a cell layer. Specifically, for example, a cell layer is obtained by using a culture vessel equipped with an insert (for example, Transwell (registered trademark) provided by Corning) and seeding and culturing cells in the insert. According to this aspect, it is possible to obtain a cell layer suitable for use in an evaluation system in which evaluation is performed by quantifying the test substance that permeates the cell layer. A method of constructing the evaluation system and a method of using it will be described later.
　工程（６）の培養は、気相液相培養により行う。
　例えば、半透過性膜に細胞を播種し、細胞が培養面（即ち半透過性膜）に接着した後、気相液相培養へと切り替える。好ましくは、細胞層が形成されたのを確認後に気相液相培養へ切り替える。気相液相培養への切り替えの時期（タイミング）の目安は播種から３時間後～３日後（例えば６時間後、８時間後、１２時間後、２４時間後、３６時間後、４８時間後、６０時間後）であるが、細胞の状態やその他の条件（例えば使用する培養基材）によって細胞が培養面に接着するまでの時間や細胞層が形成されるまでの時間は変動し得るため、予備実験によって切り替えの時期を決定するか、或いは播種後に細胞を観察し、適切なタイミングで気相液相培養へ切り替えるとよい。  Cultivation in step (6) is carried out by gas phase liquid phase culture.
For example, cells are seeded on a semi-permeable membrane, and after the cells adhere to the culture surface (ie, the semi-permeable membrane), the culture is switched to gas-liquid phase culture. Preferably, after confirming that a cell layer has been formed, the culture is switched to gas phase liquid phase culture. The approximate time (timing) for switching to gas phase liquid phase culture is 3 hours to 3 days after seeding (for example, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours later), but the time for cells to adhere to the culture surface and the time to form a cell layer may vary depending on the state of the cells and other conditions (for example, the culture substrate used). It is preferable to determine the timing of switching by a preliminary experiment, or to observe the cells after seeding and switch to the gas phase liquid phase culture at an appropriate timing.
　用語「気相液相培養」とは、細胞表面の全体（但し、培養面への接着部分や他の細胞と接触ないし接着した部分等を除く）が培地に接触するのではなく、細胞表面の一部が気相に接触した状態で培養することをいう。例えば、上記のごとき、インサートを備えた培養容器（典型的には、インサートとその受け皿となるウェルプレートから構成される）を使用することにし、インサート内に細胞を播種し、細胞が接着した後（好ましくは細胞層が形成された後）にインサート内の培養液を除去すれば、細胞の上面側に気相、細胞の下面側（即ちインサートに接着した側）に液相（培地）が存在する状態となり、気相液相培養を開始することができる。一方、インサート内の培養液を除去するのではなく、ウェル内の培養液を除去し、細胞の上面側に液相、細胞の下面側に気相が存在する状態を形成させ、気相液相培養を行ってもよい。 The term "gas phase liquid phase culture" means that the entire cell surface (excluding the part that adheres to the culture surface, the part that contacts or adheres to other cells, etc.) does not contact the medium, but the cell surface It refers to culturing in a state in which a part is in contact with the gas phase. For example, as described above, a culture vessel with an insert (typically composed of an insert and a well plate serving as a receiving plate) is used, cells are seeded in the insert, and after the cells adhere If the culture medium in the insert is removed (preferably after the cell layer is formed), the gas phase exists on the upper surface of the cells and the liquid phase (medium) exists on the lower surface of the cells (i.e., the side adhered to the insert). Then, the gas phase liquid phase culture can be started. On the other hand, instead of removing the culture medium in the insert, the culture medium in the well is removed to form a state in which the liquid phase exists on the upper surface side of the cells and the gas phase exists on the lower surface side of the cells. Culture may be performed.
　工程（６）の気相液相培養は、例えば４日～３０日間、好ましくは７日～３０日間、更に好ましくは７日～１４日間行う。尚、典型的には好気的条件下で気相液相培養を行うが、嫌気的条件を採用することにしてもよい。 The gas-liquid culture in step (6) is performed, for example, for 4 to 30 days, preferably 7 to 30 days, more preferably 7 to 14 days. Although the gas-liquid phase culture is typically performed under aerobic conditions, anaerobic conditions may be adopted.
　工程（６）の途中で継代培養を行ってもよい。例えばコンフルエント又はサブコンフルエントになった際に細胞の一部を採取して別の培養容器に移し、培養を継続する。細胞の回収には細胞解離液などを利用すればよい。細胞解離液としては、例えば、トリプシン-EDTA、コラゲナーゼIV、メタロプロテアーゼ等のタンパク分解酵素等を単独で又は適宜組み合わせて用いることができる。細胞障害性が少ないものが好ましい。このような細胞解離液として、例えば、ディスパーゼ（エーディア）、TrypLE （Invitrogen）又はアキュターゼ（MILLIPORE）等の市販品が入手可能である。分散（離散）状態となるように、回収後の細胞をセルストレイナーなどで処理した後に継代培養に供するとよい。一方、工程（６）において、必要に応じて培地交換が行われる。例えば、２４時間～３日に１回の頻度で培地交換するとよい。継代培養や培地交換に伴う、細胞の回収の際には、細胞死を抑制するためにY-27632等のROCK阻害剤（Rho-associated coiled-coil forming kinase/Rho結合キナーゼ）で予め細胞を処理しておくとよい。  Subculture may be performed in the middle of step (6). For example, when the cells become confluent or subconfluent, some of the cells are harvested and transferred to another culture vessel to continue culturing. A cell dissociation solution or the like may be used to collect cells. As the cell dissociation solution, for example, trypsin-EDTA, collagenase IV, proteolytic enzymes such as metalloprotease, and the like can be used alone or in appropriate combination. Those with less cytotoxicity are preferred. Commercial products such as Dispase (Edia), TrypLE (Invitrogen), and Accutase (MILLIPORE) are available as such cell dissociation solutions. It is preferable to subculture the recovered cells after treating them with a cell strainer or the like so that they are in a dispersed (discrete) state. On the other hand, in step (6), medium exchange is performed as necessary. For example, the medium may be replaced once every 24 hours to every 3 days. When harvesting cells during subculture or medium exchange, pre-treat the cells with a ROCK inhibitor (Rho-associated coiled-coil forming kinase/Rho-associated kinase) such as Y-27632 to suppress cell death. should be processed. 
　その他の培養条件（培養温度など）は、動物細胞の培養において一般に採用されている条件とすればよい。即ち、例えば37℃、5%CO 2の環境下で培養すればよい。基本培地は特に限定されないが、好ましくは、上皮細胞の培養に適した基本培地（例えばD-MEMとハムF12培地の混合培地、D-MEM）を用いる。培地に添加可能な成分の例としてウシ血清アルブミン（BSA）、抗生物質、2-メルカプトエタノール、PVA、非必須アミノ酸（NEAA）、インスリン、トランスフェリン、セレニウムを挙げることができる。  Other culture conditions (culture temperature, etc.) may be the conditions generally adopted for culturing animal cells. That is, it may be cultured in an environment of, for example, 37°C and 5% CO 2 . The basal medium is not particularly limited, but preferably a basal medium suitable for culturing epithelial cells (for example, a mixed medium of D-MEM and Ham's F12 medium, D-MEM) is used. Examples of components that can be added to media include bovine serum albumin (BSA), antibiotics, 2-mercaptoethanol, PVA, non-essential amino acids (NEAA), insulin, transferrin, and selenium. 
　本発明の第二の作製方法は、大別して以下の４段階の培養工程を含む。
（１）多能性幹細胞を内胚葉様細胞へと分化させる工程；
（２）工程（１）で得られた内胚葉様細胞を腸管幹細胞様細胞へと分化させる工程；
（４）工程（２）後の細胞を、上皮成長因子及びcAMPシグナル活性化因子の存在下で培養する工程；
（５）工程（４）後の細胞を、上皮成長因子、MEK1/2阻害剤、DNAメチル化阻害剤、TGFβ受容体阻害剤、及びcAMPシグナル活性化因子の存在下で培養する工程。 The second preparation method of the present invention roughly includes the following four stages of culture steps.
(1) differentiating pluripotent stem cells into endoderm-like cells;
(2) a step of differentiating the endoderm-like cells obtained in step (1) into intestinal stem cell-like cells;
(4) culturing the cells after step (2) in the presence of epidermal growth factor and cAMP signal activator;
(5) A step of culturing the cells after step (4) in the presence of epidermal growth factor, MEK1/2 inhibitor, DNA methylation inhibitor, TGFβ receptor inhibitor, and cAMP signal activator.
　工程（２）と工程（４）の間に、工程（３）をさらに含んでいてもよい。
（３）工程（２）で得られた腸管幹細胞様細胞を、上皮成長因子、及びROCK阻害剤の存在下で培養する工程； A step (3) may be further included between the step (2) and the step (4).
(3) culturing the intestinal stem cell-like cells obtained in step (2) in the presence of epidermal growth factor and a ROCK inhibitor;
　本発明の上記の第二の作製方法においては、工程（４）及び工程（５）、工程（５）、又は工程（５）の培養の一部が気相液相培養である。工程（５）の培養の一部が気相液相培養である場合には、工程（５）の培養の後半が、気相液相培養であることが好ましい。気相液相培養の詳細については、本明細書中上記した通りである。 In the above-described second preparation method of the present invention, step (4) and step (5), step (5), or part of the culture in step (5) is gas-liquid phase culture. When part of the culture in step (5) is gas phase liquid phase culture, the latter half of the culture in step (5) is preferably gas phase liquid phase culture. The details of the gas phase liquid phase culture are as described above in this specification.
　本発明の第二の作製方法における工程（１）および工程（２）の詳細は、本発明の第一の作製方法における工程（１）及び工程（２）と同様である。 The details of steps (1) and (2) in the second production method of the present invention are the same as steps (1) and (2) in the first production method of the present invention.
　工程（３）におけるROCK阻害剤としては、Y-27632等を使用することができる。
　工程（３）における上皮成長因子の添加濃度の例を示すと5 ng/mL～500 ng/mL、好ましくは10 ng/mL～200 ng/mLである。ROCK阻害剤の添加濃度の例（Y-27632の場合）を示すと1μM～50μM、好ましくは3μM～30μMである。  Y-27632 and the like can be used as the ROCK inhibitor in step (3).
An example of the concentration of epidermal growth factor added in step (3) is 5 ng/mL to 500 ng/mL, preferably 10 ng/mL to 200 ng/mL. An example of the addition concentration of the ROCK inhibitor (in the case of Y-27632) is 1 μM to 50 μM, preferably 3 μM to 30 μM.
　工程（４）におけるcAMPシグナル活性化因子としては、本明細書中上記したcAMP誘導体、cAMP分解酵素阻害剤又はcAMP活性化物質を用いることができる。
　工程（４）における上皮成長因子の添加濃度の例を示すと5 ng/mL～500 ng/mL、好ましくは10 ng/mL～200 ng/mLである。cAMP活性化物質の添加濃度の例（フォルスコリンの場合）を示すと、1μM～200μM、好ましくは5μM～100μMである。 As the cAMP signal activator in step (4), the cAMP derivative, cAMP degrading enzyme inhibitor, or cAMP activator described herein above can be used.
An example of the concentration of epidermal growth factor added in step (4) is 5 ng/mL to 500 ng/mL, preferably 10 ng/mL to 200 ng/mL. An example of the concentration of the cAMP activator added (in the case of forskolin) is 1 μM to 200 μM, preferably 5 μM to 100 μM.
　工程（５）におけるMEK1/2阻害剤としては、PD98059、PD184352、PD184161、PD0325901、U0126、MEK inhibitor I、MEK inhibitor II、MEK1/2 inhibitor II、SL327を挙げることができる。
　DNAメチル化阻害剤として5-アザ-2’-デオキシシチジン、5-アザシチジン、RG108、ゼブラリンを挙げることができる。
　TGFβ受容体阻害剤については、好ましくは、TGF-β受容体ALK4、ALK5、ALK7の一以上に対して阻害活性を示すものを用いるとよい。例えば、A-83-01、SB431542、SB-505124、SB525334、D4476、ALK5 inhibitor、LY2157299、LY364947、GW788388、RepSoxが当該条件を満たす。
　cAMPシグナル活性化因子としては、本明細書中上記したcAMP誘導体、cAMP分解酵素阻害剤又はcAMP活性化物質を用いることができる。 Examples of MEK1/2 inhibitors in step (5) include PD98059, PD184352, PD184161, PD0325901, U0126, MEK inhibitor I, MEK inhibitor II, MEK1/2 inhibitor II, and SL327.
Examples of DNA methylation inhibitors include 5-aza-2'-deoxycytidine, 5-azacytidine, RG108, and Zebularine.
As for the TGFβ receptor inhibitor, it is preferable to use one that exhibits inhibitory activity against one or more of the TGF-β receptors ALK4, ALK5 and ALK7. For example, A-83-01, SB431542, SB-505124, SB525334, D4476, ALK5 inhibitor, LY2157299, LY364947, GW788388, and RepSox satisfy this condition.
As the cAMP signal activator, the cAMP derivative, cAMP degrading enzyme inhibitor or cAMP activator described herein above can be used.
　工程（５）における上皮成長因子の添加濃度の例を示すと5 ng/mL～500 ng/mL、好ましくは10 ng/mL～200 ng/mLである。
　MEK1/2阻害剤の添加濃度の例（PD98059の場合）を示すと4μM～100μM、好ましくは10～40μMである。
　DNAメチル化阻害剤の添加濃度の例（5-アザ-2’-デオキシシチジンの場合）を示すと、1μM～25μM、好ましくは2.5μM～10μMである。
　TGFβ受容体阻害剤の添加濃度の例（A-83-01の場合）を示すと0.1μM～2.5μM、好ましくは0.2μM～1μMである。
　cAMP活性化物質の添加濃度の例（フォルスコリンの場合）を示すと、1μM～200μM、好ましくは5μM～100μMである。 An example of the concentration of epidermal growth factor added in step (5) is 5 ng/mL to 500 ng/mL, preferably 10 ng/mL to 200 ng/mL.
An example of the concentration of the MEK1/2 inhibitor added (in the case of PD98059) is 4 μM to 100 μM, preferably 10 to 40 μM.
An example of the added concentration of the DNA methylation inhibitor (in the case of 5-aza-2'-deoxycytidine) is 1 μM to 25 μM, preferably 2.5 μM to 10 μM.
An example of the concentration of the TGFβ receptor inhibitor added (in the case of A-83-01) is 0.1 μM to 2.5 μM, preferably 0.2 μM to 1 μM.
An example of the concentration of the cAMP activator added (in the case of forskolin) is 1 μM to 200 μM, preferably 5 μM to 100 μM.
　尚、例示した化合物、即ち、PD98059、5-アザ-2’-デオキシシチジン、A-83-01、フォルスコリンとは異なる化合物を使用する場合の添加濃度については、使用する化合物の特性と、例示した化合物の特性の相違（特に活性の相違）を考慮すれば、当業者であれば上記濃度範囲に準じて設定することができる。また、設定した濃度範囲が適切であるか否かは、後述の実施例に準じた予備実験によって確認することができる。 When using a compound different from the exemplified compounds, that is, PD98059, 5-aza-2'-deoxycytidine, A-83-01, and forskolin, the concentration to be added depends on the characteristics of the compound used and the exemplified Considering the difference in the properties (especially the difference in activity) of the compounds described above, a person skilled in the art can set the concentration range according to the above. Also, whether or not the set concentration range is appropriate can be confirmed by a preliminary experiment according to the examples described later.
　工程（３）の培養の期間は、例えば１日間～５日間である。
　工程（４）の培養の期間は、例えば３日間～１５日間である。
　工程（５）の培養の期間は、例えば５日間～２０日間である。 The period of culture in step (3) is, for example, 1 to 5 days.
The period of culture in step (4) is, for example, 3 to 15 days.
The period of culture in step (5) is, for example, 5 to 20 days.
　本発明の第三の作製方法は、多能性幹細胞から作製された腸管細胞を、5-アザ-2'-デオキシシチジンを含まない培地で培養することを含む、腸管細胞を作製する方法である。
　好ましくは、5-アザ-2'-デオキシシチジンを含まない培地での培養の少なくとも一部が、気相液相培養である。 A third production method of the present invention is a method for producing intestinal cells, comprising culturing intestinal cells produced from pluripotent stem cells in a medium that does not contain 5-aza-2'-deoxycytidine. .
Preferably, at least part of the culture in the medium that does not contain 5-aza-2'-deoxycytidine is gas phase liquid phase culture.
　多能性幹細胞から作製された腸管細胞としては、以下の工程（１１）～（１４）を含む方法により得られた細胞を使用することができる。
（１１）多能性幹細胞を内胚葉様細胞へと分化させる工程；
（１２）工程（１１）で得られた内胚葉様細胞を腸管幹細胞様細胞へと分化させる工程；
（１４）工程（１２）で得られた腸管幹細胞様細胞を、上皮成長因子及びcAMPシグナル活性化因子の存在下で培養する工程；および
（１５）工程（１４）後の細胞を、上皮成長因子、MEK1/2阻害剤、DNAメチル化阻害剤、TGFβ受容体阻害剤、及びcAMPシグナル活性化因子の存在下で培養する工程。 As intestinal cells produced from pluripotent stem cells, cells obtained by a method including the following steps (11) to (14) can be used.
(11) differentiating the pluripotent stem cells into endoderm-like cells;
(12) differentiating the endoderm-like cells obtained in step (11) into intestinal stem cell-like cells;
(14) culturing the intestinal stem cell-like cells obtained in step (12) in the presence of epidermal growth factor and cAMP signal activator; , a MEK1/2 inhibitor, a DNA methylation inhibitor, a TGFβ receptor inhibitor, and a cAMP signal activator.
　本発明（第一の作製方法、第二の作製方法及び第三の作製方法）を構成する各工程における、その他の培養条件（培養温度など）は、動物細胞の培養において一般に採用されている条件とすればよい。即ち、例えば37℃、5%CO 2の環境下で培養すればよい。また、基本培地として、イスコフ改変ダルベッコ培地（IMDM）（GIBCO社等）、ハムF12培地（HamF12）（SIGMA社、Gibco社等）、ダルベッコ変法イーグル培地（D-MEM）（ナカライテスク株式会社、シグマ社、Gibco社等）、グラスゴー基本培地（Gibco社等）、RPMI1640培地等を用いることができる。二種以上の基本培地を併用することにしてもよい。工程（４）及び工程（５）においては、上皮細胞の培養に適した基本培地（例えばD-MEMとハムF12培地の混合培地、D-MEM）を用いることが好ましい。培地に添加可能な成分の例としてウシ血清アルブミン（BSA）、抗生物質、2-メルカプトエタノール、PVA、非必須アミノ酸（NEAA）、インスリン、トランスフェリン、セレニウムを挙げることができる。  Other culture conditions (culture temperature, etc.) in each step constituting the present invention (first production method, second production method, and third production method) are conditions generally employed in culturing animal cells. And it is sufficient. That is, it may be cultured in an environment of, for example, 37°C and 5% CO 2 . In addition, as a basal medium, Iscove's modified Dulbecco's medium (IMDM) (GIBCO, etc.), Ham F12 medium (HamF12) (SIGMA, Gibco, etc.), Dulbecco's modified Eagle's medium (D-MEM) (Nacalai Tesque Co., Ltd., Sigma, Gibco, etc.), Glasgow basal medium (Gibco, etc.), RPMI1640 medium, etc. can be used. Two or more basal media may be used in combination. In steps (4) and (5), it is preferable to use a basal medium suitable for culturing epithelial cells (for example, a mixed medium of D-MEM and Ham's F12 medium, D-MEM). Examples of components that can be added to media include bovine serum albumin (BSA), antibiotics, 2-mercaptoethanol, PVA, non-essential amino acids (NEAA), insulin, transferrin, and selenium. 
　本発明を構成する工程（１）、工程（２）の途中で継代培養を行ってもよい。例えばコンフルエント又はサブコンフルエントになった際に細胞の一部を採取して別の培養容器に移し、培養を継続する。細胞の回収には細胞解離液などを利用すればよい。細胞解離液としては、例えば、トリプシン-EDTA、コラゲナーゼIV、メタロプロテアーゼ等のタンパク分解酵素等を単独で又は適宜組み合わせて用いることができる。細胞障害性が少ないものが好ましい。このような細胞解離液として、例えば、ディスパーゼ（エーディア）、TrypLE （Invitrogen）又はアキュターゼ（MILLIPORE）等の市販品が入手可能である。分散（離散）状態となるように、回収後の細胞をセルストレイナーなどで処理した後に継代培養に供するとよい。一方、本発明を構成する各工程において、必要に応じて培地交換が行われる。例えば、２４時間～３日に１回の頻度で培地交換するとよい。  Subculture may be performed during the steps (1) and (2) constituting the present invention. For example, when the cells become confluent or subconfluent, some of the cells are harvested and transferred to another culture vessel to continue culturing. A cell dissociation solution or the like may be used to collect cells. As the cell dissociation solution, for example, trypsin-EDTA, collagenase IV, proteolytic enzymes such as metalloprotease, and the like can be used alone or in appropriate combination. Those with less cytotoxicity are preferred. Commercial products such as Dispase (Edia), TrypLE (Invitrogen), and Accutase (MILLIPORE) are available as such cell dissociation solutions. It is preferable to subculture the recovered cells after treating them with a cell strainer or the like so that they are in a dispersed (discrete) state. On the other hand, in each step constituting the present invention, medium exchange is performed as necessary. For example, the medium may be replaced once every 24 hours to every 3 days. 
＜腸管細胞の用途＞
　本発明は、本発明の作製方法で得られた腸管細胞に関する。第１の用途として各種アッセイが提供される。本発明の腸管細胞は、腸管、特に小腸のモデル系に利用可能であり、腸管、特に小腸での薬物動態（吸収、代謝など）の評価や毒性の評価に有用である。換言すれば、本発明の腸管細胞は、化合物の体内動態の評価や毒性の評価にその利用が図られる。本発明の腸管細胞を利用したアッセイは二次元評価系となり、よりハイスループットな解析を可能にする。 <Uses of intestinal cells>
The present invention relates to intestinal cells obtained by the production method of the present invention. As a first use, various assays are provided. The intestinal cells of the present invention can be used in model systems of the intestinal tract, particularly the small intestine, and are useful for evaluation of pharmacokinetics (absorption, metabolism, etc.) and toxicity in the intestinal tract, particularly the small intestine. In other words, the intestinal cells of the present invention are intended to be used for evaluating pharmacokinetics and toxicity of compounds. The assay using intestinal cells of the present invention becomes a two-dimensional evaluation system, enabling higher throughput analysis.
　具体的には、本発明の腸管細胞を用いて被検物質の代謝、吸収性、膜透過性、薬物相互作用、薬物代謝酵素の誘導、薬物トランスポーターの誘導、毒性等を試験することができる。即ち、本発明は、腸管細胞の用途の一つとして、被検物質の代謝、吸収性、膜透過性、薬物相互作用、薬物代謝酵素の誘導、薬物トランスポーターの誘導、毒性等を評価する方法を提供する。当該方法では、（Ｉ）本発明の作製方法で得られた腸管細胞に被検物質を接触させる工程と、（ＩＩ）被検物質の代謝、吸収性、膜透過性、薬物相互作用、薬物代謝酵素の誘導、又は薬物トランスポーターの誘導、或いは毒性を測定・評価する工程を行う。 Specifically, the intestinal cells of the present invention can be used to test the metabolism, absorption, membrane permeability, drug interaction, induction of drug-metabolizing enzymes, induction of drug transporters, toxicity, etc. of a test substance. . That is, the present invention provides a method for evaluating the metabolism, absorption, membrane permeability, drug interaction, induction of drug-metabolizing enzymes, induction of drug transporters, toxicity, etc. of a test substance as one of the uses of intestinal cells. I will provide a. In the method, (I) a step of contacting a test substance with intestinal cells obtained by the production method of the present invention; A step of measuring and evaluating enzyme induction, drug transporter induction, or toxicity is performed.
　工程（Ｉ）での「接触」は、典型的には、培地に被検物質を添加することによって行われる。被検物質の添加のタイミングは特に限定されない。従って、被検物質を含まない培地で培養を開始した後、ある時点で被検物質を添加することにしても、予め被検物質を含む培地で培養を開始することにしてもよい。  "Contact" in step (I) is typically performed by adding the test substance to the medium. The timing of addition of the test substance is not particularly limited. Therefore, after starting culture in a medium containing no test substance, the test substance may be added at a certain point, or culture may be started in advance in a medium containing the test substance. 
　被検物質には様々な分子サイズの有機化合物又は無機化合物を用いることができる。有機化合物の例として核酸、ペプチド、タンパク質、脂質（単純脂質、複合脂質（ホスホグリセリド、スフィンゴ脂質、グリコシルグリセリド、セレブロシド等）、プロスタグランジン、イソプレノイド、テルペン、ステロイド、ポリフェノール、カテキン、ビタミン（B1、B2、B3、B5、B6、B7、B9、B12、C、A、D、E等）を例示できる。医薬品、栄養食品、食品添加物、農薬、香粧品（化粧品）等の既存成分或いは候補成分も好ましい被検物質の一つである。植物抽出液、細胞抽出液、培養上清などを被検物質として用いてもよい。２種類以上の被検物質を同時に添加することにより、被検物質間の相互作用、相乗作用などを調べることにしてもよい。被検物質は天然物由来であっても、或いは合成によるものであってもよい。後者の場合には例えばコンビナトリアル合成の手法を利用して効率的なアッセイ系を構築することができる。  Organic or inorganic compounds with 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, vitamins (B1, B2, B3, B5, B6, B7, B9, B12, C, A, D, E, etc.) can be exemplified.Existing ingredients or candidate ingredients such as pharmaceuticals, nutritional foods, food additives, agricultural chemicals, cosmetics (cosmetics), etc. is also one of the preferred test substances.Plant extracts, cell extracts, culture supernatants, etc. may be used as test substances.By adding two or more test substances at the same time, Interactions, synergistic effects, etc. between substances may be investigated, and the substance to be tested may be of natural origin or may be of synthetic origin, in the latter case, for example, by means of combinatorial synthesis techniques. It is possible to construct an efficient assay system by
　被検物質を接触させる期間は任意に設定可能である。接触期間は例えば１０分間～３日間、好ましくは１時間～１日間である。接触を複数回に分けて行うことにしてもよい。  The period of contact with 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 divided into multiple times. 
　工程（Ｉ）の後、被検物質の代謝、吸収性、膜透過性、薬物相互作用、薬物代謝酵素の誘導、又は薬物トランスポーターの誘導、或いは毒性を測定・評価する（工程（ＩＩ））。工程（Ｉ）の直後、即ち、被検物質の接触の後、実質的な時間間隔を置かずに代謝等を測定・評価しても、或いは、一定の時間（例えば１０分～５時間）を経過した後に代謝等を測定・評価することにしてもよい。代謝の測定は、例えば、代謝産物の検出によって行うことができる。この場合には、通常、工程（Ｉ）後の培養液をサンプルとして、予想される代謝産物を定性的又は定量的に測定する。測定方法は代謝産物に応じて適切なものを選択すればよいが、例えば、質量分析、液体クロマトグラフィー、免疫学的手法（例えば蛍光免疫測定法（FIA法）、酵素免疫測定法（EIA法））等を採用可能である。  After step (I), the metabolism, absorption, membrane permeability, drug interactions, induction of drug-metabolizing enzymes, induction of drug transporters, or toxicity of the test substance are measured and evaluated (step (II)). . Immediately after step (I), that is, after contact with the test substance, measurement and evaluation of metabolism etc. without a substantial time interval, or after a certain period of time (for example, 10 minutes to 5 hours) Metabolism and the like may be measured and evaluated after the passage of time. Metabolic measurements can be made, for example, by detecting metabolites. In this case, the expected metabolites are usually measured qualitatively or quantitatively using the culture solution after step (I) as a sample. An appropriate measurement method may be selected depending on the metabolite, and examples include mass spectrometry, liquid chromatography, immunological techniques (e.g., fluorescence immunoassay (FIA method), enzyme immunoassay (EIA method), ), etc. can be adopted. 
　典型的には、被検物質の代謝産物が検出されたとき、「被検物質が代謝された」と判定ないし評価する。また、代謝産物の量に応じて被検物質の代謝量を評価することができる。代謝産物の検出結果と、被検物質の使用量（典型的には培地への添加量）に基づき、被検物質の代謝効率を算出することにしてもよい。  Typically, when metabolites of the test substance are detected, it is judged or evaluated that "the test substance has been metabolized." Moreover, the metabolic rate of the test substance can be evaluated according to the amount of the metabolite. The metabolic efficiency of the test substance may be calculated based on the metabolite detection results and the amount of the test substance used (typically, the amount added to the medium). 
　腸管細胞における薬物代謝酵素（シトクロムP450（特にヒトではCYP3A4、カニクイザルではCYP3A8）、ウリジン2リン酸-グルクロン酸転移酵素（特にUGT1A8、UGT1A10）、硫酸転移酵素（特にSULT1A3など））の発現を指標として被検物質の代謝を測定することも可能である。薬物代謝酵素の発現はmRNAレベル又はタンパク質レベルで評価することができる。例えば、薬物代謝酵素のmRNAレベルに上昇を認めたとき、「遺伝子レベルでの発現量が上昇した」と判定することができる。同様に、薬物代謝酵素の活性に上昇を認めたとき、「被検物質が代謝された」と判定することができる。代謝産物を指標として判定する場合と同様に、薬物代謝酵素の発現量に基づいて定量的な判定・評価を行うことにしてもよい。  Expression of drug-metabolizing enzymes (cytochrome P450 (especially CYP3A4 in humans, CYP3A8 in cynomolgus monkeys), uridine diphosphate-glucuronyltransferase (especially UGT1A8, UGT1A10), sulfotransferase (especially SULT1A3, etc.)) in intestinal cells as an index It is also possible to measure the metabolism of the test substance. Expression of drug-metabolizing enzymes can be assessed at the mRNA level or the protein level. For example, when an increase in the mRNA level of a drug-metabolizing enzyme is observed, it can be determined that "the expression level at the gene level has increased." Similarly, when an increase in the activity of drug-metabolizing enzymes is observed, it can be determined that "the test substance has been metabolized." As in the case of determination using metabolites as an index, quantitative determination/evaluation may be performed based on the expression level of drug-metabolizing enzymes. 
　被検物質の吸収を評価するためには、例えば、培養液中の被検物質の残存量を測定する。通常、工程（Ｉ）後の培養液をサンプルとして被検物質を定量する。測定方法は被検物質に応じて適切なものを選択すればよい。例えば、質量分析、液体クロマトグラフィー、免疫学的手法（例えば蛍光免疫測定法（FIA法）、酵素免疫測定法（EIA法））等を採用可能である。典型的には、培養液中の被検物質の含有量の低下を認めたとき、「被検物質が吸収された」と判定・評価する。また、低下の程度に応じて被検物質の吸収量ないし吸収効率を判定・評価することができる。尚、細胞内に取り込まれた被検物質の量を測定することによっても、吸収の評価は可能である。  In order to evaluate the absorption of the test substance, for example, the residual amount of the test substance in the culture medium is measured. Usually, the test substance is quantified using the culture medium after step (I) as a sample. An appropriate measurement method may be selected depending on the test substance. For example, mass spectrometry, liquid chromatography, immunological techniques (for example, fluorescence immunoassay (FIA method), enzyme immunoassay (EIA method)) and the like can be employed. 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." In addition, the absorption amount or absorption efficiency of the test substance can be determined and evaluated according to the degree of decrease. The absorption can also be evaluated by measuring the amount of the test substance taken into the cells. 
　尚、代謝の測定・評価と吸収の測定・評価を同時に又は並行して行うことにしてもよい。  It should be noted that the measurement/evaluation of metabolism and the measurement/evaluation of absorption may be performed at the same time or in parallel. 
　本発明の作製方法で得られた腸管細胞は、腸疾患の病態を再現し得る。そこで、本発明の腸管オルガノイドの第２の用途として、腸疾患モデル、その作製方法、及びそれを利用したアッセイ等が提供される。 The intestinal cells obtained by the production method of the present invention can reproduce the pathology of intestinal diseases. Therefore, as a second use of the intestinal organoid of the present invention, an intestinal disease model, a method for producing the model, an assay using the same, and the like are provided.
　本発明の腸管細胞を利用すれば腸疾患モデル、例えば、炎症性腸疾患の病態モデル、炎症性腸疾患の炎症によって進行する組織の線維化を再現する病態モデル（線維化症モデル）、がん病態モデル等を作製し得る。炎症性腸疾患の病態モデルの作製には、例えば、TNF-α、IFN-γ、IL-1、IL-6、IL-17a等の炎症性サイトカイン（一つ又は二以上の組合せ）の存在下で本発明の腸管細胞を培養し、炎症ないし障害を誘導すればよい。免疫細胞との共培養を利用して病態モデルを作製することにしてもよい。一方、線維化症モデルの作製には例えば、TGF-βを用いて、或いはTNF-αとTNF-βを併用して線維化を誘導すればよい。  If the intestinal cells of the present invention are used, an intestinal disease model, for example, a pathological model of inflammatory bowel disease, a pathological model that reproduces tissue fibrosis that progresses due to inflammation in inflammatory bowel disease (fibrosis model), cancer Pathological models and the like can be produced. In the presence of inflammatory cytokines such as TNF-α, IFN-γ, IL-1, IL-6, IL-17a (one or a combination of two or more) to induce inflammation or injury by culturing the intestinal cells of the present invention. A disease model may be prepared by co-culturing with immune cells. On the other hand, to prepare a fibrosis model, for example, TGF-β may be used, or TNF-α and TNF-β may be used in combination to induce fibrosis. 
　腸疾患モデルは薬物スクリーニングに有用である。即ち、腸疾患モデルを用いれば、腸疾患に有効な物質（医薬品の有効成分又はリード化合物）をスクリーニングするためのin vitro評価系を構築することができる。当該評価系では、腸疾患モデルが再現する病態に対する被検物質の作用や影響を調べ、その有効性が評価される。  Intestinal disease models are useful for drug screening. That is, by using an intestinal disease model, it is possible to construct an in vitro evaluation system for screening substances (active ingredients of pharmaceuticals or lead compounds) that are effective against intestinal diseases. In this evaluation system, the action and influence of the test substance on the pathological condition reproduced by the intestinal disease model are investigated, and the effectiveness thereof is evaluated. 
　本発明の作製方法で得られた腸管細胞の第３の用途として腸管オルガノイドを含む移植材料が提供される。本発明の移植材料は各種腸疾患（例えば難治性炎症性腸疾患）の治療に適用可能である。特に、障害された（機能不全を含む）腸管組織の再生・再建用の材料としての利用が想定される。即ち、再生医療への貢献を期待できる。本発明の移植材料はそのままで、或いはマトリゲルやコラーゲンゲル包埋等の処理をした後、移植材料として利用することができる。また、各種腸疾患病態モデルとしての治療薬候補化合物のスクリーニングや病態メカニズムの解明研究のような利用形態も想定される。細胞の保護を目的としてジメチルスルホキシド（DMSO）や血清アルブミン等を、細菌の混入を阻止することを目的として抗生物質等を、細胞の活性化、増殖又は分化誘導などを目的として各種の成分（ビタミン類、サイトカイン、成長因子、ステロイド等）を本発明の移植材料に含有させてもよい。さらに、製剤上許容される他の成分（例えば、担体、賦形剤、崩壊剤、緩衝剤、乳化剤、懸濁剤、無痛化剤、安定剤、保存剤、防腐剤、生理食塩水など）を本発明の移植材料に含有させてもよい。  As a third use of the intestinal cells obtained by the production method of the present invention, a transplant material containing intestinal organoids is provided. The implantable material of the present invention can be applied to treat various intestinal diseases (eg, intractable inflammatory bowel disease). In particular, it is expected to be used as a material for regeneration/reconstruction of damaged (including dysfunctional) intestinal tissue. In other words, it can be expected to contribute to regenerative medicine. The graft material of the present invention can be used as a graft material as it is or after processing such as matrigel or collagen gel embedding. It is also envisioned to be used for screening of therapeutic drug candidate compounds as pathological models of various intestinal diseases, and for clarification of pathological mechanisms. Dimethyl sulfoxide (DMSO) and serum albumin are used to protect cells, antibiotics are used to prevent bacterial contamination, and various ingredients (vitamin , cytokines, growth factors, steroids, etc.) may be included in the implantable material of the present invention. In addition, other pharmaceutically acceptable components (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological saline, etc.) It may be contained in the implant material of the present invention. 
　本発明の移植材料はin vivo実験系の構築にも利用可能である。例えば、ヒト多能性幹細胞を用いて作製した腸管細胞を含む移植材料をマウス、ラット、モルモット、ハムスター、ブタ、カニクイザル、アカゲザル、チンパンジー等の実験動物に移植し、ヒト化動物（ヒト腸管モデル）を作製することができる。このようなヒト化動物は薬物動態や毒性試験等の実験に特に有用であり、経口薬に対する初回通過効果の影響や薬剤性腸炎等の研究への貢献が期待される。  The transplant material of the present invention can also be used to construct an in vivo experimental system. For example, transplant materials containing intestinal cells prepared using human pluripotent stem cells are transplanted into experimental animals such as mice, rats, guinea pigs, hamsters, pigs, cynomolgus monkeys, rhesus monkeys, and chimpanzees, and humanized animals (human intestinal model). can be made. Such humanized animals are particularly useful for experiments such as pharmacokinetics and toxicity tests, and are expected to contribute to studies on the effects of the first-pass effect on oral drugs and drug-induced enteritis. 
　腸疾患の患者由来のiPS細胞を用いて作製した腸管細胞については、腸管病態モデルとして、薬剤評価系に利用できる他、腸疾患の発症、病態形成及び／又は進展の機序の解明を目指した研究における各種実験にも利用可能である。  Intestinal cells prepared using iPS cells derived from patients with intestinal disease can be used as an intestinal disease model for drug evaluation systems. It can also be used for various experiments in research. 
＜実施例Ａ＞
＜方法＞
（１）細胞
　ヒト人工多能性幹細胞（hiPSC）（iPS-51：Windy）は、ヒト胎児肺線維芽細胞MRC-5にoctamer binding protein 3/4（OCT3/4）、sex determining region Y-box 2（SOX2）、kruppel-like factor 4（KLF4）、v-myc myelocytomatosis viral oncogene homolog（avian）（c-MYC）を、パントロピックレトロウイルスベクターを用いて導入後、ヒトES細胞様コロニーをクローン化したものであり、国立成育医療研究センター研究所の梅澤明弘博士よりご供与いただいた。フィーダー細胞はマウス胎仔線維芽細胞（MEF）を使用した。 <Example A>
<Method>
(1) Cells Human induced pluripotent stem cells (hiPSC) (iPS-51: Windy) are human fetal lung fibroblast MRC-5, octamer binding protein 3/4 (OCT3/4), sex determining region Y-box 2 (SOX2), kruppel-like factor 4 (KLF4), v-myc myelocytomatosis viral oncogene homolog (avian) (c-MYC) were introduced using a pantropic retroviral vector, and human ES cell-like colonies were cloned. It was provided by Dr. Akihiro Umezawa of the Research Institute of the National Center for Child Health and Development. Mouse embryonic fibroblasts (MEF) were used as feeder cells.
（２）培地
　MEFの培養には10%ウシ胎仔血清（FBS）、2 mmol/L L-グルタミン（L-Glu）、1%非必須アミノ酸（NEAA）、100 units/mLペニシリンG、100 μg/mLストレプトマイシンを含むダルベッコ改変イーグル培地（DMEM）を用いた。MEFの剥離液には0.05%トリプシン-エチレンジアミン四酢酸（EDTA）を、MEFの保存液にはセルバンカー1を用いた。ヒトiPSCの維持培養には20%ノックアウト血清代替物（KSR）、0.8% NEAA、2 mmol/L L-Glu、0.1 mmol/L 2-メルカプトエタノール（2-MeE）、5 ng/mL線維芽細胞増殖因子（FGF）2を含むDMEM Ham’s F-12（DMEM/F12）を用いた。ヒトiPSCの剥離液には1 mg/mLコラゲナーゼIV、0.25%トリプシン、20% KSR、1 mmol/L塩化カルシウムを含むダルベッコリン酸緩衝生理食塩水（PBS）を用いた。ヒトiPSCの保存液には霊長類ES/iPSC用凍結保存液を用いた。 (2) Medium For culture of MEFs, 10% fetal bovine serum (FBS), 2 mmol/L L-glutamine (L-Glu), 1% non-essential amino acids (NEAA), 100 units/mL penicillin G, 100 μg/ Dulbecco's Modified Eagle Medium (DMEM) containing mL streptomycin was used. 0.05% trypsin-ethylenediaminetetraacetic acid (EDTA) was used as the MEF stripping solution, and Cellbanker 1 was used as the MEF preserving solution. 20% knockout serum replacement (KSR), 0.8% NEAA, 2 mmol/L L-Glu, 0.1 mmol/L 2-mercaptoethanol (2-MeE), 5 ng/mL fibroblasts for human iPSC maintenance culture DMEM Ham's F-12 (DMEM/F12) containing growth factor (FGF)2 was used. Dulbecco's phosphate-buffered saline (PBS) containing 1 mg/mL collagenase IV, 0.25% trypsin, 20% KSR, and 1 mmol/L calcium chloride was used as a detachment solution for human iPSCs. Primate ES/iPSC cryopreservation medium was used as the preservation medium for human iPSCs.
（３）ヒトiPSCの培養
　hiPSCはマイトマイシンC処理を施したMEF（6×105 cells/100 mmディッシュ）上に播種し、5% CO2/95% air条件下CO2インキュベーター中37℃にて培養した。ヒトiPSCの継代は、3~5日培養後、1:2~1:3のスプリット比で行った。ヒトiPSCは解凍48時間後に培地を交換し、それ以降は毎日交換した。 (3) Culture of human iPSCs hiPSCs were seeded on mitomycin C-treated MEFs (6×10 5 cells/100 mm dish) and placed at 37°C in a CO 2 incubator under 5% CO 2 /95% air conditions. cultured. Human iPSCs were passaged at a split ratio of 1:2 to 1:3 after 3 to 5 days of culture. For human iPSCs, the medium was changed 48 hours after thawing, and changed every day thereafter.
（４）hiPSCの腸管オルガノイドへの分化誘導
　hiPSC由来腸管オルガノイド（HIOs）の分化誘導は、継代時にhiPSC用培地にて30倍に希釈したマトリゲル（成長因子除去）にてコートした培養ディッシュに播種し、35 ng/mL FGF2を含むStemSure(R) hPSC 培地にて培養し、未分化コロニーの占める割合が約80%になった状態で開始した。100 ng/mLアクチビンA、100 units/mLペニシリンG、100 μg/mLストレプトマイシン、2 mmol/L L-Gluを含むロズウェルパーク記念研究所（RPMI）培地で1日間、0.2% FBS、100 ng/mLアクチビンA、100 units/mLペニシリンG、100 μg/mLストレプトマイシン、2 mmol/L L-Gluを含むRPMI培地で1日間、2% FBS、100 ng/mLアクチビンA、100 units/mLペニシリンG、100 μg/mLストレプトマイシン、2 mmol/L L-Gluを含むRPMI培地で1日間培養することで内胚葉に分化させた。その後、ヒトiPSCでは、2% FBS、500 ng/mL FGF4、3 μmol/L CHIR99021、100 units/mLペニシリンG、100 μg/mLストレプトマイシンを含むRPMI+グルタマックス培地で4日間培養することで腸管幹細胞へ分化させた。FGF4、CHIR99021処理後、Y-27632（Rho結合キナーゼ阻害剤）を10 μmol/Lとなるように添加し、5% CO2/95% air条件下CO2インキュベーター中37℃にて60分間処理した細胞を0.05%トリプシン-EDTAにて剥離し、40 μmナイロンメッシュのセルストレーナーにて細胞塊を砕き、PBS（-）にて0.16 μg/mLに希釈した iMatrix 511をコーティングした100 mm dish 上に2.0×106細胞を播種した。細胞は、1% グルタマックス、2% FBS、100 units/mLペニシリンG、100 μg/mLストレプトマイシン、100 ng/mL上皮成長因子（EGF）、30 ng/mL FGF2、0.5 μmol/L A-83-01、3 μmol/L CHIR99021、10 μmol/L Y-27632を含むAdvanced-DMEM/F12で培養した。その後、3日毎に、最大2回、細胞をアキュターゼにて剥離し、PBS（-）にて0.16 μg/mLに希釈した iMatrix 511をコーティングした100 mm dish上に2.0×106細胞を播種した。細胞は、1% グルタマックス、2% FBS、100 units/mLペニシリンG、100 μg/mLストレプトマイシン、100 ng/mL上皮成長因子（EGF）、30 ng/mL FGF2、0.5 μmol/L A-83-01、3 μmol/L CHIR99021、10 μmol/L Y-27632を含むAdvanced-DMEM/F12で培養した。2あるいは3日後、同様に剥離した後、3.0×106細胞から6.0×106細胞を100 mm EZSPHERE(R)上に播種した。その後、1% グルタマックス、2% B27 supplement、1% N2 supplement、100 units/mLペニシリンG、100 μg/mLストレプトマイシン、100 ng/mL EGF、100 ng/mL Noggin、200 ng/mL R-spondin-1、10 μmol/L Y-27632を含むAdvanced-DMEM/F12で3日間培養後、1% グルタマックス、2% B27 supplement、1% N2 supplement、100 units/mLペニシリンG、100 μg/mLストレプトマイシン、100 ng/mL EGF、100 ng/mL Noggin、200 ng/mL R-spondin-1、成長因子を除去したマトリゲル3%を含むAdvanced-DMEM/F12で9日間から12日間超低接着100 mm dish上で浮遊培養することでHIOsへ分化させた。 (4) Differentiation induction of hiPSCs into intestinal organoids HiPSC-derived intestinal organoids (HIOs) were seeded on a culture dish coated with Matrigel (growth factor removed) diluted 30-fold with hiPSC medium at the time of passaging. Then, the cells were cultured in StemSure(R) hPSC medium containing 35 ng/mL FGF2, and started when the ratio of undifferentiated colonies reached about 80%. 1 day in Roswell Park Memorial Institute (RPMI) medium containing 100 ng/mL activin A, 100 units/mL penicillin G, 100 μg/mL streptomycin, 2 mmol/L L-Glu, 0.2% FBS, 100 ng/mL RPMI medium containing activin A, 100 units/mL penicillin G, 100 μg/mL streptomycin, 2 mmol/L L-Glu for 1 day, 2% FBS, 100 ng/mL activin A, 100 units/mL penicillin G, 100 They were differentiated into endoderm by culturing in RPMI medium containing μg/mL streptomycin and 2 mmol/L L-Glu for 1 day. After that, human iPSCs were cultured in RPMI + glutamax medium containing 2% FBS, 500 ng/mL FGF4, 3 µmol/L CHIR99021, 100 units/mL penicillin G, and 100 µg/mL streptomycin for 4 days to transform them into intestinal stem cells. differentiated. After FGF4 and CHIR99021 treatment, Y-27632 (Rho-binding kinase inhibitor) was added to 10 μmol/L and treated for 60 minutes at 37°C in a CO 2 incubator under 5% CO 2 /95% air conditions. Cells were detached with 0.05% trypsin-EDTA, cell clumps were crushed with a 40 μm nylon mesh cell strainer, diluted to 0.16 μg/mL with PBS (-) and placed on a 100 mm dish coated with iMatrix 511. ×10 6 cells were seeded. Cells were treated with 1% glutamax, 2% FBS, 100 units/mL penicillin G, 100 μg/mL streptomycin, 100 ng/mL epidermal growth factor (EGF), 30 ng/mL FGF2, 0.5 μmol/L A-83- 01, 3 μmol/L CHIR99021 and 10 μmol/L Y-27632 were cultured in Advanced-DMEM/F12. Thereafter, the cells were detached with Accutase up to twice every 3 days, and 2.0×10 6 cells were seeded on a 100 mm dish coated with iMatrix 511 diluted to 0.16 μg/mL with PBS(-). Cells were treated with 1% glutamax, 2% FBS, 100 units/mL penicillin G, 100 μg/mL streptomycin, 100 ng/mL epidermal growth factor (EGF), 30 ng/mL FGF2, 0.5 μmol/L A-83- 01, 3 μmol/L CHIR99021 and 10 μmol/L Y-27632 were cultured in Advanced-DMEM/F12. Two or three days later, after similarly detaching, 3.0×10 6 to 6.0×10 6 cells were seeded on a 100 mm EZSPHERE (R). Then add 1% glutamax, 2% B27 supplement, 1% N2 supplement, 100 units/mL penicillin G, 100 μg/mL streptomycin, 100 ng/mL EGF, 100 ng/mL Noggin, 200 ng/mL R-spondin- 1. After 3 days of culture in Advanced-DMEM/F12 containing 10 μmol/L Y-27632, add 1% glutamax, 2% B27 supplement, 1% N2 supplement, 100 units/mL penicillin G, 100 μg/mL streptomycin, 100 ng/mL EGF, 100 ng/mL Noggin, 200 ng/mL R-spondin-1, Advanced-DMEM/F12 containing 3% growth factor-free matrigel for 9 to 12 days on a 100 mm dish with ultra-low adhesion They were differentiated into HIOs by floating culture at .
（５）HIOs由来腸管細胞の二次元培養
　HIOs培養終了24時間前に10 μM Y-27632を処理した。HIOsを遠沈管に回収し、D-PBS（-）で二回洗浄した。160×gで5分間遠心後、上清を吸引除去した。0.5 nM EDTAを2 mL添加し懸濁した。5分間室温で静置した後、D-PBS（-）で洗浄した。160×gで5分間遠心後、上清を吸引除去した。0.1% trypsin/EDTAを2 mLにて懸濁し、10-20分間37°Cで静置した。ピペッティングにてシングルセル化した後、10% FBS含有Advanced-DMEM/F12培地で遠沈管に回収した。40 μm nylon-mesh cell strainerにて細胞を濾過し、160×gで5分間遠心した。STEMCELLBANKER等の細胞保存液に懸濁し、緩慢凍結法にて凍結保存した。解凍した後に10 μMのY-27632を添加したMedium(7)で懸濁し、0.5 μg/cm2のiMatrix-511 silkもしくは3% マトリゲルにてコーティングした細胞培養用24-well transwell インサート（ポアサイズ0.4 μm）に播種した。2,3日毎にMedium(7)で培地交換を行い、7-22日間培養を行った。培養の際、様々な化合物を添加し、最適な条件を探索した。 (5) Two-Dimensional Culture of HIOs-Derived Intestinal Cells Twenty-four hours before the end of HIOs culture, 10 μM Y-27632 was applied. HIOs were collected in a centrifuge tube and washed twice with D-PBS(-). After centrifugation at 160 xg for 5 minutes, the supernatant was removed by aspiration. 2 mL of 0.5 nM EDTA was added and suspended. After standing at room temperature for 5 minutes, it was washed with D-PBS(-). After centrifugation at 160 xg for 5 minutes, the supernatant was removed by aspiration. 0.1% trypsin/EDTA was suspended in 2 mL and allowed to stand at 37°C for 10-20 minutes. After single cells were formed by pipetting, they were collected in a centrifuge tube with Advanced-DMEM/F12 medium containing 10% FBS. Cells were filtered with a 40 μm nylon-mesh cell strainer and centrifuged at 160×g for 5 minutes. The cells were suspended in a cell preservation solution such as STEMCELLBANKER and cryopreserved by the slow freezing method. 24 - well transwell insert for cell culture (pore size 0.4 μm ). The medium was replaced with Medium (7) every 2 or 3 days, and cultured for 7 to 22 days. During the culture, various compounds were added to search for optimal conditions.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
（６）総リボ核酸（RNA）抽出
　総RNAは分化誘導終了後、Agencourt RNAdvence Tissueの添付マニュアルに従い抽出した。 (6) Total ribonucleic acid (RNA) extraction Total RNA was extracted according to the attached manual of Agencourt RNAdvence Tissue after completion of differentiation induction.
（７）逆転写反応
　相補的DNA（cDNA）の合成は、ReverTra Ace qPCR RT Master Mixを使用し、添付マニュアルに従い行った。 (7) Reverse Transcription Reaction Synthesis of complementary DNA (cDNA) was performed using ReverTra Ace qPCR RT Master Mix according to the attached manual.
（８）Real-Time RT-PCR法
　Real-Time RT-PCRはKAPA SYBR Fast qPCR Kitを用い、cDNAを鋳型にして、反応は添付マニュアルに従い行った。結果は内在性コントロールとしてヒポキサンチンホスホリボシルトランスフェラーゼ（HPRT）を用いて補正した。  (8) Real-Time RT-PCR Method For Real-Time RT-PCR, KAPA SYBR Fast qPCR Kit was used, cDNA was used as a template, and the reaction was carried out according to the attached manual. Results were corrected using hypoxanthine phosphoribosyltransferase (HPRT) as an endogenous control.
（９）ヘマトキシリン－エオシン（HE）染色
　分化誘導終了後、腸管オルガノイドを4％パラホルムアルデヒドにて固定し、OCTコンパウンドにて凍結包埋した。厚さ10 μmの凍結切片を作製後、スライドガラスに貼り付け、マイヤーヘマトキシリン及びエオシンアルコールを使用して染色した。 (9) Hematoxylin-Eosin (HE) Staining After completion of induction of differentiation, the intestinal organoids were fixed with 4% paraformaldehyde and cryo-embedded with OCT compound. Cryosections with a thickness of 10 μm were prepared, attached to glass slides, and stained with Mayer's hematoxylin and eosin alcohol.
（１０）免疫蛍光染色
　分化誘導終了後、腸管オルガノイドを4％パラホルムアルデヒドにて固定し、OCTコンパウンドにて凍結包埋した。厚さ10 μmの凍結切片を作製後、スライドガラスに貼り付け、抗原の賦活化を行った。5% FBS溶液にて30分間ブロッキングし、一次抗体（Table1）を4℃で1晩反応させた。その後、スライドガラスを洗浄し、二次抗体を室温で1時間反応させ、核染色として4′, 6-ジアミジノ-2-フェニルインドール（DAPI）を用いた。封入作業を行い、Zeiss LSM510共焦点レーザー顕微鏡を用いて、蛍光を観察した。 (10) Immunofluorescence Staining After completion of induction of differentiation, the intestinal organoids were fixed with 4% paraformaldehyde and cryo-embedded with OCT compound. After preparing frozen sections with a thickness of 10 μm, they were attached to glass slides for antigen activation. After blocking with a 5% FBS solution for 30 minutes, the primary antibody (Table 1) was allowed to react overnight at 4°C. After that, the slide glass was washed, reacted with a secondary antibody at room temperature for 1 hour, and 4',6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. Encapsulation was performed and fluorescence was observed using a Zeiss LSM510 confocal laser scanning microscope.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
＜結果＞
（１）HIOs由来腸管細胞の二次元培養条件の検討
　二次元培養10日目において液相条件である通常の培養方法では陰窩-絨毛様の立体構造の形成は認められなかったが、気相液相界面培養において陰窩-絨毛様構造の形成が認められた。また、立体構造の形成にはcyclic adenosine monophosphate（cAMP）シグナル活性化剤であるForskolinと形質転換増殖因子（TGF）β1阻害剤であるA-83-01が必須であることが示唆された（図１Ａ）。Forskolinとは異なる経路でcAMPを活性化する8-Br-cAMPもしくはIBMXでも立体構造の形成が可能であることが判明した（図１）。 <Results>
(1) Examination of two-dimensional culture conditions for HIOs-derived intestinal cells On day 10 of two-dimensional culture, the formation of a crypt-villus-like three-dimensional structure was not observed in the usual liquid phase culture method, but the gas phase Formation of crypt-villus-like structures was observed in liquid-phase interface culture. It was also suggested that Forskolin, a cyclic adenosine monophosphate (cAMP) signal activator, and A-83-01, a transforming growth factor (TGF) β1 inhibitor, are essential for the formation of the three-dimensional structure (Fig. 1A). It was found that 8-Br-cAMP or IBMX, which activates cAMP through a pathway different from that of Forskolin, can also form a three-dimensional structure (Fig. 1).
　さらに、培養10日目以降も立体構造を維持したまま培養可能かを検討するため、WntシグナルおよびNotchシグナルを制御しながら培養を継続して細胞の観察を行った。結果として、Wntシグナルを活性化するCHIR99021は添加し続け、Notchシグナル阻害剤であるDAPTは添加を途中で中断することにより少なくとも22日間は立体構造を維持したままの培養が可能であることが判明した（図２）。 Furthermore, in order to examine whether it is possible to culture while maintaining the three-dimensional structure after the 10th day of culture, we continued culturing while controlling the Wnt and Notch signals and observed the cells. As a result, it was found that by continuing to add CHIR99021, which activates Wnt signaling, and interrupting the addition of DAPT, a Notch signaling inhibitor, it was possible to maintain the three-dimensional structure for at least 22 days. (Fig. 2).
（２）HIOs由来腸管細胞の陰窩-絨毛様構造の形成
　培養10日目に光干渉式断層撮像システム（Cell3iMager Estier）により撮像した画像およびH&E染色の画像から、陰窩-絨毛様の立体構造が確認された（図３Ａ、Ｂ、Ｄ）。Z方向にスタックした結果、高密度で絨毛様の突起が存在していることが判明した（図３Ｃ）。また、白い部分が連なっていることから、指状のものだけでなく、十二指腸付近でよくみられる葉状の構造の存在も示唆された。 (2) Formation of crypt-villus-like structures of HIOs-derived intestinal cells Crypt-villus-like three-dimensional structures were identified from images taken with an optical coherence tomography system (Cell3iMager Estier) on day 10 of culture and H&E stained images. was confirmed (Figs. 3A, B, D). Stacking in the Z direction revealed the presence of high density, villus-like processes (Fig. 3C). In addition, the presence of not only finger-like structures but also leaf-like structures commonly seen near the duodenum was suggested because of the continuous white parts.
（３）作製したHIOs由来腸管細胞の性質および機能の解析
　気相液相界面培養にて作製したHIOs由来腸管細胞は液相培養と比較して経上皮電気抵抗が低値に抑えられ安定していた（図４Ａ）。生体腸管組織のTEERは80 Ω・cm2から150 Ω・cm2程度であり、非常に近い値となった。RT-qPCRにより、腸管上皮マーカーであるvillin、杯細胞マーカーであるMUC2、腸管幹細胞マーカーであるLGR5、Paneth細胞マーカーであるLYZ等の腸管関連遺伝子の発現量が生体腸管組織よりも高いことが判明した。また、腸管で主要な代謝酵素であるCYP3A4やその発現を誘導する核内受容体のPXR、ペプチドトランスポーター1（PEPT1）の遺伝子も生体腸管組織と同程度発現していた（図４Ｂ）。Rifampicinおよび活性型ビタミンD3（VD3）を用いた、PXRおよびVDRを介したCYP3A4遺伝子の誘導試験では、顕著な発現量の増加が確認された（図４Ｃ）。多剤排出トランスポーターであるMDR1を介したRhodamine123の輸送試験では排出方向優位な輸送が認められ、阻害剤ベラパミルによるEfflux ratio（ER）の顕著な低下が認められた（図４Ｄ）。 (3) Analysis of the properties and functions of the HIOs-derived intestinal cells produced HIOs-derived intestinal cells produced by air-liquid interface culture have a lower transepithelial electrical resistance and are more stable than those produced by liquid-phase culture. (Fig. 4A). The TEER of living intestinal tissue ranged from 80 Ω·cm 2 to 150 Ω·cm 2 , which were very close values. RT-qPCR revealed that the expression levels of gut-related genes such as intestinal epithelial marker villin, goblet cell marker MUC2, intestinal stem cell marker LGR5, and Paneth cell marker LYZ were higher than in living intestinal tissue. did. In addition, the genes of CYP3A4, a major metabolic enzyme in the intestinal tract, the nuclear receptor PXR that induces its expression, and peptide transporter 1 (PEPT1) were also expressed at the same level as in intestinal tissue (Fig. 4B). An induction test of the CYP3A4 gene via PXR and VDR using rifampicin and activated vitamin D 3 (VD3) confirmed a significant increase in the expression level (Fig. 4C). A transport study of Rhodamine123 via MDR1, a multidrug efflux transporter, showed preferential transport in the efflux direction, and the inhibitor verapamil significantly reduced the Efflux ratio (ER) (Fig. 4D).
（４）HIOs由来腸管細胞の長期培養
　経上皮電気抵抗値は培養10日目から22日目まで150 Ω・cm2で安定しており、バリア機能の維持が示唆された。腸管関連遺伝子および薬物トランスポーター遺伝子も22日目まで生体腸管組織以上の発現量を維持していた（図５Ａ、Ｂ）。また、tuft細胞マーカーであるDCLK1とM細胞マーカーであるGP2も生体腸管組織以上の遺伝子発現を維持していた。SPIBもGP2と同じくM細胞マーカーだが、その発現量は低いことが判明した（図５Ｃ）。 (4) Long-term culture of HIOs-derived intestinal cells Transepithelial electrical resistance was stable at 150 Ω·cm 2 from day 10 to day 22 of culture, suggesting maintenance of barrier function. Intestinal tract-related genes and drug transporter genes also maintained expression levels higher than those in living intestinal tissue until day 22 (FIGS. 5A and 5B). In addition, DCLK1, a tuft cell marker, and GP2, an M cell marker, also maintained gene expression higher than that of living intestinal tissue. SPIB is also an M cell marker like GP2, but its expression level was found to be low (Fig. 5C).
（５）HIOs由来腸管細胞のタンパク質発現解析
　免疫蛍光染色により、腸管上皮細胞の頂端膜に発現するvillinが陰窩－絨毛様構造の表面に沿って局在していることが明らかになった。また、杯細胞およびムコ多糖マーカーのMUC2、内分泌細胞マーカーのchromogranin A（CGA）も発現も確認された。免疫系細胞であるtuft細胞およびM細胞マーカーであるGirdinおよびGlycoprotein 2（GP2）もタンパク質レベルで発現していることが判明した。 (5) Protein expression analysis of HIOs-derived intestinal cells Immunofluorescent staining revealed that villin expressed in the apical membrane of intestinal epithelial cells was localized along the surface of crypt-villus-like structures. The expression of goblet cell and mucopolysaccharide marker MUC2 and endocrine cell marker chromogranin A (CGA) was also confirmed. An immune system cell, tuft cells, and M cell markers, Girdin and Glycoprotein 2 (GP2), were also found to be expressed at the protein level.
＜まとめ＞
　以上の結果より、実施例Ａでは、HIOsを二次元培養に展開し、陰窩-絨毛様の立体構造を有する腸管細胞を作製することに成功した。また、この立体構造形成には気相液相界面培養が必要であり、cAMPシグナル、TGF-βシグナル、Wntシグナル、Notchシグナルが関与していることが示唆された。特にWntシグナルやNocthシグナルに関与する低分子化合物であるCHIR99021とDAPTは添加する時期も重要であることが示唆された。作製した腸管細胞は従来の液相培養と比較して経上皮電気抵抗値が生体腸管組織に近似しており、過剰なバリア機能の発達を抑えられることが示唆された。各種遺伝子発現も生体償還組織と同程度以上で安定しており、薬物動態学的機能を有していることも明らかになった。M細胞やtuft細胞といった吸収上皮細胞以外の存在も遺伝子およびタンパク質レベルで確認され、腸管免疫等の評価系としての利用も期待できる。Wntシグナルを制御することで少なくとも22日間状態を維持したまま培養が可能であることも判明した。さらに、本細胞はHIOsから二次元培養系に展開する前段階で凍結保存することも可能になっており、利便性の面で非常に有用である。 <Summary>
From the above results, in Example A, HIOs were developed in two-dimensional culture, and intestinal cells having a crypt-villus-like three-dimensional structure were successfully produced. In addition, it was suggested that gas-liquid interface culture is necessary for this conformation, and that cAMP, TGF-β, Wnt and Notch signals are involved. In particular, it was suggested that the timing of addition of CHIR99021 and DAPT, which are low-molecular-weight compounds involved in Wnt and Nocth signals, is also important. The transepithelial electrical resistance of the prepared intestinal cells was similar to that of living intestinal tissue compared to conventional liquid-phase culture, suggesting that the development of excessive barrier function could be suppressed. Various gene expressions were also stable at the same level or higher than that of biological reimbursement tissue, and it was also clarified that it has a pharmacokinetic function. The presence of cells other than absorptive epithelial cells such as M cells and tuft cells was also confirmed at the gene and protein level, and it is expected to be used as an evaluation system for intestinal immunity and the like. It was also found that by controlling the Wnt signal, it was possible to maintain the culture for at least 22 days. Furthermore, the cells can be cryopreserved in the pre-stage of expanding from HIOs to a two-dimensional culture system, which is very useful in terms of convenience.
＜実施例Ｂ＞
＜方法＞
（１）細胞
　ヒトiPSCとしてWindy株を使用した。両細胞株は国立成育医療研究センター研究所の梅澤明弘博士よりご供与いただいた。iPSC(オンフィーダー培養)の維持培養時のフィーダー細胞としてはMEFを使用した。 <Example B>
<Method>
(1) Cells Windy strain was used as human iPSCs. Both cell lines were donated by Dr. Akihiro Umezawa of the National Center for Child Health and Development. MEFs were used as feeder cells for maintenance culture of iPSCs (on-feeder culture).
（２）培地
オンフィーダー培養：
　MEFの培養には10%ウシ胎仔血清（FBS）、2 mmol/L L-グルタミン（L-Glu）、1%非必須アミノ酸（NEAA）、100 units/mLペニシリンG、100 μg/mLストレプトマイシンを含むダルベッコ改変イーグル培地（DMEM）を用いた。MEFの剥離液には0.05%トリプシン-エチレンジアミン四酢酸（EDTA）を、MEFの保存液にはセルバンカー1を用いた。ヒトiPSCの維持培養には20%ノックアウト血清代替物（KSR）、0.8% NEAA、2 mmol/L L-Glu、0.1 mmol/L 2-メルカプトエタノール（2-MeE）、5 ng/mL線維芽細胞増殖因子（FGF）2を含むDMEM Ham’s F-12（DMEM/F12）を用いた。ヒトiPSCの剥離液には1 mg/mLコラゲナーゼIV、0.25%トリプシン、20% KSR、1 mmol/L塩化カルシウムを含むダルベッコリン酸緩衝生理食塩水（PBS）を用いた。ヒトiPSCの保存液には霊長類ES/iPSC用凍結保存液を用いた。 (2) Medium-on-feeder culture:
MEF cultures contain 10% fetal bovine serum (FBS), 2 mmol/L L-glutamine (L-Glu), 1% non-essential amino acids (NEAA), 100 units/mL penicillin G, 100 μg/mL streptomycin Dulbecco's Modified Eagle Medium (DMEM) was used. 0.05% trypsin-ethylenediaminetetraacetic acid (EDTA) was used as the MEF stripping solution, and Cellbanker 1 was used as the MEF preserving solution. 20% knockout serum replacement (KSR), 0.8% NEAA, 2 mmol/L L-Glu, 0.1 mmol/L 2-mercaptoethanol (2-MeE), 5 ng/mL fibroblasts for human iPSC maintenance culture DMEM Ham's F-12 (DMEM/F12) containing growth factor (FGF)2 was used. Dulbecco's phosphate-buffered saline (PBS) containing 1 mg/mL collagenase IV, 0.25% trypsin, 20% KSR, and 1 mmol/L calcium chloride was used as a detachment solution for human iPSCs. Primate ES/iPSC cryopreservation medium was used as the preservation medium for human iPSCs.
フィーダーレス培養：
　培地はmTeSR1を用いた。ヒトiPSCの剥離液には0.5 mmol/L EDTAを用いた。ヒトiPSCの保存液にはStemSure(R)凍結保存溶液を用いた。 Feederless culture:
The medium used was mTeSR1. 0.5 mmol/L EDTA was used as a stripping solution for human iPSCs. StemSure (R) cryopreservation solution was used as the preservation solution for human iPSCs.
（３）ヒトiPSCの培養
オンフィーダー培養：
　hiPSCはマイトマイシンC処理を施したMEF（6×105 cells/100 mmディッシュ）上に播種し、5% CO2/95% air条件下CO2インキュベーター中37℃にて培養した。ヒトiPSCの継代は、3~5日培養後、1:2~1:3のスプリット比で行った。ヒトiPSCは解凍48時間後に培地を交換し、それ以降は毎日交換した。 (3) Culture of human iPSCs On-feeder culture:
hiPSCs were seeded on mitomycin C-treated MEFs (6×10 5 cells/100 mm dish) and cultured at 37° C. in a CO 2 incubator under 5% CO 2 /95% air conditions. Human iPSCs were passaged at a split ratio of 1:2 to 1:3 after 3 to 5 days of culture. For human iPSCs, the medium was changed 48 hours after thawing, and changed every day thereafter.
フィーダーレス培養：
　DMEM/F12にて成長因子を除去したマトリゲルを30倍希釈し、この希釈液で細胞培養用プレートをコートした。コートしたプレートを4℃下に10時間以上置いた。使用30分前に、室温まで戻した。ヒトiPSCをマトリゲルでコートしたプレートに播種し、5% CO2/95% air条件下CO2インキュベーター中37℃にて培養した。ヒトiPSCの継代は、3~5日培養後、1:6のスプリット比で行った。ヒトiPSCは解凍時にY-27632を10 μmol/Lとなるように添加し、24時間後に培地を交換し、それ以降は毎日交換した。 Feederless culture:
Matrigel from which growth factors were removed was diluted 30-fold with DMEM/F12, and a cell culture plate was coated with this diluted solution. The coated plate was placed at 4°C for 10 hours or longer. 30 minutes before use, it was brought back to room temperature. Human iPSCs were seeded on matrigel-coated plates and cultured at 37°C in a CO 2 incubator under 5% CO 2 /95% air conditions. Human iPSCs were passaged at a split ratio of 1:6 after 3-5 days of culture. When human iPSCs were thawed, Y-27632 was added to 10 μmol/L, the medium was changed 24 hours later, and thereafter changed every day.
（４）ヒトiPSCの腸管上皮細胞への分化
　オンフィーダー培養したヒトiPSCの腸管上皮細胞への分化は、ヒトiPSCが培養ディッシュに対し、未分化コロニーの占める割合が約70%になった状態で開始した。0.5% FBS、100 ng/mLアクチビンA、100 units/mLペニシリンG、100 μg/mLストレプトマイシンを含むロズウェルパーク記念研究所（RPMI）＋グルタマックス培地で2日間（分化開始後0日目～2日目）、2% FBS、100 ng/mLアクチビンA、100 units/mLペニシリンG、100 μg/mLストレプトマイシンを含むRPMI＋グルタマックス培地で1日間（分化開始後２日目～３日目）培養することで内胚葉に分化させた。その後、2% FBS、1%グルタマックス、250 ng/mL FGF2を含むDMEM/F12で4日間（分化開始後3日目～7日目）培養することで腸管幹細胞へ分化させた。FGF2処理後、Y-27632（Rho結合キナーゼ阻害剤）を10 μmol/Lとなるように添加し、5% CO2/95% air条件下CO2インキュベーター中37℃にて60分間処理した細胞をアクターゼにて剥離し、あらかじめヒトiPSC用培地にて30倍に希釈した、成長因子を除去したマトリゲルにてコートした細胞培養用24 well-insertに播種した。その後、2% FBS、2 mmol/Lグルタマックス、1% NEAA、2% B27サプリメント、1% N2サプリメント、100 units/mLペニシリンG、100 μg/mLストレプトマイシン、50 ng/mL上皮成長因子（EGF）、10 μmol/L Y-27632を含むDMEM/F12で1日間(分化開始後7日目～8日目）、2% FBS、2 mmol/Lグルタマックス、1% NEAA、2% B27サプリメント、1% N2サプリメント、100 units/mLペニシリンG、100 μg/mLストレプトマイシン、50 ng/mL EGF、30 μMフォルスコリンを含むDMEM/F12で6日間（分化開始後8日目～14日目）、2% FBS、2 mmol/Lグルタマックス、1% NEAA、2% B27サプリメント、1% N2サプリメント、100 units/mLペニシリンG、100 μg/mLストレプトマイシン、50 ng/mL EGF、30 μM フォルスコリン、20 μmol/L PD98059、5 μmol/L 5-アザ-2’-デオキシシチジン、0.5 μmol/L A-83-01を含むDMEM/F12で12日間（分化開始後14日目～26日目）培養し、この分化方法を標準プロトコルとし、腸管上皮細胞へ分化させた。5 μmol/L 5-アザ-2’-デオキシシチジンを12日間（分化開始後14日目～26日目）、0.5 μmol/L A-83-01で18日間（分化開始8日目～26日目）もしくは12日間（分化開始後14日目～26日目）、2 μmol/L CHIR-99021で18日間（分化開始8日目～26日目）培養することで腸管上皮細胞へ分化させた。InsertのApical側の培地を除去し、分化開始8日目もしくは14日目から分化終了時まで気相液相培養を行った。 (4) Differentiation of human iPSCs into intestinal epithelial cells Differentiation of on-feeder cultured human iPSCs into intestinal epithelial cells was performed when the proportion of undifferentiated colonies of human iPSCs in the culture dish was about 70%. started. 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 2 days (days 0-2 after initiation of differentiation) eyes), cultured in RPMI + glutamax medium containing 2% FBS, 100 ng/mL activin A, 100 units/mL penicillin G, and 100 μg/mL streptomycin for 1 day (2nd to 3rd day after initiation of differentiation) differentiated into endoderm. After that, they were differentiated into intestinal stem cells by culturing in DMEM/F12 containing 2% FBS, 1% glutamax, and 250 ng/mL FGF2 for 4 days (days 3 to 7 after initiation of differentiation). After FGF2 treatment, Y-27632 (Rho-binding kinase inhibitor) was added to 10 μmol/L, and the cells were treated for 60 minutes at 37°C in a CO 2 incubator under 5% CO 2 /95% air conditions. The cells were exfoliated with actase and seeded in a 24-well-insert for cell culture coated with matrigel from which growth factors had been removed, which had been diluted 30-fold with human iPSC medium in advance. followed by 2% FBS, 2 mmol/L glutamax, 1% NEAA, 2% B27 supplement, 1% N2 supplement, 100 units/mL penicillin G, 100 μg/mL streptomycin, 50 ng/mL epidermal growth factor (EGF) , 1 day in DMEM/F12 containing 10 μmol/L Y-27632 (days 7-8 after initiation of differentiation), 2% FBS, 2 mmol/L glutamax, 1% NEAA, 2% B27 supplement, 1 % N2 supplement, 100 units/mL penicillin G, 100 μg/mL streptomycin, 50 ng/mL EGF, 30 μM forskolin in DMEM/F12 for 6 days (days 8-14 after initiation of differentiation), 2% FBS, 2 mmol/L glutamax, 1% NEAA, 2% B27 supplement, 1% N2 supplement, 100 units/mL penicillin G, 100 μg/mL streptomycin, 50 ng/mL EGF, 30 μM forskolin, 20 μmol/ The cells were cultured in DMEM/F12 containing L PD98059, 5 μmol/L 5-aza-2'-deoxycytidine, and 0.5 μmol/L A-83-01 for 12 days (days 14 to 26 after initiation of differentiation). Differentiation into intestinal epithelial cells was performed using a standard protocol for the differentiation method. 5 μmol/L 5-aza-2'-deoxycytidine for 12 days (days 14-26 after initiation of differentiation), 0.5 μmol/L A-83-01 for 18 days (days 8-26 after initiation of differentiation) ) or for 12 days (days 14 to 26 after the initiation of differentiation) and 2 μmol/L CHIR-99021 for 18 days (days 8 to 26 after the initiation of differentiation) to differentiate into intestinal epithelial cells. . The medium on the apical side of the insert was removed, and gas phase liquid phase culture was performed from day 8 or day 14 of differentiation to the end of differentiation.
　フィーダーレス条件で培養したヒトiPSCの腸管上皮細胞への分化では、フィーダーレス条件で培養したヒトiPSCが培養ディッシュに対し、細胞のコンフルエンシーが約90%になった状態で開始した。0.2% B27サプリメント、100 ng/mLアクチビンA、50 nmol/L PI-103、2 μmol/L CHIR99021、100 units/mLペニシリンG、100 μg/mLストレプトマイシン、1% NEAAを含むRPMI培地で2日間（分化開始後0日目～1日目）、0.2% B27サプリメント、100 ng/mLアクチビンA、20 ng/mL bFGF、100 units/mLペニシリンG、100 μg/mLストレプトマイシン、1% NEAAを含むRPMI培地で１日間（分化開始後2日目～3日目）、0.2% B27サプリメント、100 ng/mLアクチビンA、20 ng/mL bFGF、100 units/mLペニシリンG、100 μg/mLストレプトマイシン、1% NEAAを含むRPMI培地： Advanced RPMI培地＝3:1の混合培地で４日間（分化開始後4日目～7日目）培養することで内胚葉に分化させた。内胚葉の腸管上皮細胞への分化は、ヒトiPSC由来内胚葉を用い、2% FBS、2%B27サプリメント、1%グルタマックス、250 ng/mL FGF2を含むDMEM/F12で4日間（分化開始後8日目～11日目）培養することで腸管幹細胞へ分化させた。FGF2処理後、Y-27632を10 μmol/Lとなるように添加し、5% CO2/95% air条件下CO2インキュベーター中37℃にて60分間処理した細胞をアクターゼにて剥離し、あらかじめDMEM/F12にて30倍に希釈した成長因子を除去したマトリゲルにてコートした細胞培養用24 well-insertに播種した。その後、4% FBS、2 mmol/Lグルタマックス、1% NEAA、2% B27サプリメント、1% N2サプリメント、1% Hepextend サプリメント、100 units/mLペニシリンG、100 μg/mLストレプトマイシン、50 ng/mL EGF、10 μmol/L Y-27632を含むDMEM/F12で1日間、4% FBS、2 mmol/Lグルタマックス、1% NEAA、2% B27サプリメント、1% N2サプリメント、100 units/mLペニシリンG、100 μg/mLストレプトマイシン、50 ng/mL EGF及び30 μmol/Lフォルスコリンを含むDMEM/F12で6日間（分化開始後12日目～18日目）、4% FBS、2 mmol/Lグルタマックス、1% NEAA、2% B27サプリメント、1% N2サプリメント、1% Hepextend サプリメント、100 units/mLペニシリンG、100 μg/mLストレプトマイシン、50 ng/mL EGF、20 μmol/L PD98059、5 μmol/L 5-アザ-2’-デオキシシチジン、0.5 μmol/L A-83-01、30 μmol/Lフォルスコリンを含むDMEM/F12で12日間（分化開始後19日目～30日目）培養することで腸管上皮細胞へ分化させた。insertのApical側の培地を除去し、分化開始12日目、18日目もしくは24日目から分化終了時まで気相培養を行った。 Differentiation of human iPSCs cultured under feederless conditions into intestinal epithelial cells started when human iPSCs cultured under feederless conditions reached approximately 90% confluency in the culture dish. RPMI medium containing 0.2% B27 supplement, 100 ng/mL activin A, 50 nmol/L PI-103, 2 μmol/L CHIR99021, 100 units/mL penicillin G, 100 μg/mL streptomycin, 1% NEAA for 2 days ( 0 to 1 days after initiation of differentiation), RPMI medium containing 0.2% B27 supplement, 100 ng/mL activin A, 20 ng/mL bFGF, 100 units/mL penicillin G, 100 μg/mL streptomycin, and 1% NEAA for 1 day (days 2-3 after initiation of differentiation), 0.2% B27 supplement, 100 ng/mL activin A, 20 ng/mL bFGF, 100 units/mL penicillin G, 100 μg/mL streptomycin, 1% NEAA RPMI medium containing: Advanced RPMI medium = 3:1 mixed medium was cultured for 4 days (days 4 to 7 after initiation of differentiation) to differentiate into endoderm. Differentiation of endoderm into intestinal epithelial cells was performed using human iPSC-derived endoderm in DMEM/F12 containing 2% FBS, 2% B27 supplement, 1% glutamax, and 250 ng/mL FGF2 for 4 days (after initiation of differentiation). Days 8 to 11) The cells were differentiated into intestinal stem cells by culturing. After FGF2 treatment, Y-27632 was added to 10 μmol/L, and the cells were treated for 60 minutes at 37°C in a CO 2 incubator under 5% CO 2 /95% air conditions. The cells were seeded in a 24-well-insert for cell culture coated with matrigel from which growth factors were removed and diluted 30-fold with DMEM/F12. Then 4% FBS, 2 mmol/L glutamax, 1% NEAA, 2% B27 supplement, 1% N2 supplement, 1% Hepextend supplement, 100 units/mL penicillin G, 100 μg/mL streptomycin, 50 ng/mL EGF , 1 day in DMEM/F12 containing 10 μmol/L Y-27632, 4% FBS, 2 mmol/L glutamax, 1% NEAA, 2% B27 supplement, 1% N2 supplement, 100 units/mL penicillin G, 100 DMEM/F12 containing μg/mL streptomycin, 50 ng/mL EGF and 30 μmol/L forskolin for 6 days (days 12-18 after initiation of differentiation), 4% FBS, 2 mmol/L glutamax, 1 % NEAA, 2% B27 supplement, 1% N2 supplement, 1% Hepextend supplement, 100 units/mL penicillin G, 100 μg/mL streptomycin, 50 ng/mL EGF, 20 μmol/L PD98059, 5 μmol/L 5-aza Intestinal epithelial cells were cultured in DMEM/F12 containing -2'-deoxycytidine, 0.5 μmol/L A-83-01, and 30 μmol/L forskolin for 12 days (days 19 to 30 after initiation of differentiation). differentiated into The medium on the apical side of the insert was removed, and gas phase culture was performed from day 12, 18, or 24 of differentiation to the end of differentiation.
（５）総リボ核酸（RNA）抽出
　総RNAはヒトiPSCの分化誘導終了後、Agencourt RNAdvance Tissue Kitの添付マニュアルに従い抽出した。 (5) Total Ribonucleic Acid (RNA) Extraction Total RNA was extracted according to the manual attached to the Agencourt RNAdvance Tissue Kit after completion of human iPSC differentiation induction.
（６）逆転写反応
　相補的DNA（cDNA）の合成は、ReverTra Ace qPCR RT Master Kitを使用し、添付マニュアルに従い行った。 (6) Reverse Transcription Reaction Synthesis of complementary DNA (cDNA) was performed using ReverTra Ace qPCR RT Master Kit according to the attached manual.
（７）Real-Time RT-PCR法
　Real-Time RT-PCRはKAPA SYBR Fast qPCR Kitを用い、cDNAを鋳型にして、反応は添付マニュアルに従い行った。結果は内在性コントロールとしてヒポキサンチン-グアニンホスホリボシルトランスフェラーゼ（HPRT）を用いて補正した。 (7) Real-Time RT-PCR Method For Real-Time RT-PCR, KAPA SYBR Fast qPCR Kit was used, cDNA was used as a template, and the reaction was carried out according to the attached manual. Results were corrected using hypoxanthine-guanine phosphoribosyltransferase (HPRT) as an endogenous control.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
＜結果・考察＞
（１）細胞数によるヒトiPSC由来腸管上皮細胞への分化効率の影響
　オンフィーダー培養したヒトiPSC由来腸管幹細胞をinsertに播種する時細胞数（1×105 cells / insertまたは1.5×105 cells / insert（グラフには、それぞれ１また1.５で表記された））及び分化開始８日目からの気相培養による腸管上皮細胞への分化の影響を検討した。分化段階において、経時的に細胞の経上皮電気抵抗（TEER）値を測定した結果、液相培養群はd23からTEER値が上昇する傾向があり、気相培養群ではd23からd26までTEERが安定した。いずれの群においても、d26にTEERは200 Ω・cm2以上であったことから、タイトジャンクションが形成されていると考えられた（図６）。さらに、分化終了後、各条件下で分化した腸管上皮細胞の遺伝子発現量を調べた。気相培養条件下で得られた腸管上皮細胞は、液相培養した腸管上皮細胞と比較し、Villin、CYP3A4及びSucrase-isomaltase発現量の変動が認められなかったものの、OLFM4、Pgpの発現量の上昇が確認できた。細胞数は分化した腸管上皮細胞の遺伝子発現レベルへ影響を及ぼさなかった（図７及び図８）。 <Results/Discussion>
(1) Effect of cell number on differentiation efficiency into human iPSC - derived intestinal epithelial cells The effects of differentiation into intestinal epithelial cells by insert (indicated by 1 and 1.5, respectively in the graph)) and gas-phase culture from day 8 of the initiation of differentiation were investigated. As a result of measuring the transepithelial electrical resistance (TEER) value of the cells over time at the differentiation stage, the TEER value tends to increase from d23 in the liquid phase culture group, while the TEER value is stable from d23 to d26 in the gas phase culture group. did. In all groups, the TEER was 200 Ω·cm 2 or more at d26, suggesting the formation of tight junctions (Fig. 6). Furthermore, after completion of differentiation, gene expression levels of differentiated intestinal epithelial cells under each condition were examined. Intestinal epithelial cells obtained under gas-phase culture conditions showed no change in the expression levels of Villin, CYP3A4, and Sucrase-isomaltase compared to liquid-phase cultured intestinal epithelial cells. A rise was confirmed. Cell number had no effect on the gene expression levels of differentiated intestinal epithelial cells (Figs. 7 and 8).
（２）腸管上皮細胞への分化に対する5-アザ-2'-デオキシシチジン、気相培養期間の検討
　細胞数1×105 cells/insert、1.5×105 cells/insertの違いによる腸管上皮細胞の分化誘導に与える影響が認められなかったため、1×105 cells/insertでオンフィーダー培養したヒトiPSC由来腸管幹細胞を腸管上皮細胞へ分化させた。分化過程において、5-アザ-2'-デオキシシチジンを分化開始14日目から分化終了時まで添加する標準プロトコール及び、5-アザ-2'-デオキシシチジンの非添加プロトコールを比較した。また、気相培養期間について、分化開始8日目から、分化開始14日目から分化終了まで2つの条件を検討した。分化終了時に明視野観察した細胞の形態より、気相培養条件下で5-アザ-2'-デオキシシチジンの非添加プロトコールにて培養した腸管上皮細胞は、絨毛様の構造を形成したことが確認された。また、分化開始8日目より気相培養に比べ、分化開始14日目より気相培養群では、陰窩－絨毛様構造が明瞭であることが明らかにした（図９）。分化した腸管上皮細胞の遺伝子レベルで評価した結果、BCRP及びMuc2について、いずれの群においてもヒト成人小腸より高い発現量レベルが認められた。5-アザ-2'-デオキシシチジン非添加かつ気相培養条件下で得られた腸管上皮細胞は、他の検討群より、Sucrase-isomaltaseの遺伝子発現量が顕著に上昇した。CYP3A4、Pgp及びOLFM4の発現量について、同条件下で5-アザ-2'-デオキシシチジン添加群は非添加群に比べて高く、最も高かった群は分化開始14日目より気相培養かつ5-アザ-2'-デオキシシチジン添加群であった（図１０及び図１１）。 (2) Examination of 5 - aza-2'-deoxycytidine and gas phase culture period for differentiation into intestinal epithelial cells Since no effect on differentiation induction was observed, human iPSC-derived intestinal stem cells cultured on-feeder at 1×10 5 cells/insert were differentiated into intestinal epithelial cells. In the course of differentiation, a standard protocol in which 5-aza-2'-deoxycytidine was added from day 14 of differentiation until the end of differentiation was compared with a protocol in which 5-aza-2'-deoxycytidine was not added. In addition, regarding the gas phase culture period, two conditions from the 8th day of differentiation to the 14th day of differentiation to the end of differentiation were examined. From the bright field observation of the cell morphology at the end of differentiation, it was confirmed that the intestinal epithelial cells cultured under the protocol without the addition of 5-aza-2'-deoxycytidine under gas phase culture conditions formed a villus-like structure. was done. In addition, it was clarified that the crypt-villus-like structure was clearer in the gas phase culture group from the 14th day on than the 8th day from the start of differentiation compared to the gas phase culture group (Fig. 9). As a result of evaluating differentiated intestinal epithelial cells at the gene level, BCRP and Muc2 were found to have higher expression levels than in human adult small intestine in all groups. Intestinal epithelial cells obtained under gas-phase culture conditions without the addition of 5-aza-2'-deoxycytidine showed significantly higher levels of sucrase-isomaltase gene expression than those of the other groups. The expression levels of CYP3A4, Pgp, and OLFM4 were higher in the 5-aza-2'-deoxycytidine-added group than in the non-added group under the same conditions. -aza-2'-deoxycytidine addition group (Figs. 10 and 11).
（３）A83-01添加時期の検討
　細胞数1×105 cells/insert、5-アザ-2'-デオキシシチジン添加、分化開始14日目気相培養条件下でオンフィーダー培養したヒトiPSC由来腸管上皮細胞の分化誘導に対するA83-01の添加時期による影響を検討した。分化開始14日目よりA83-01添加群に比較し、分化開始8日目よりA83-01添加群は細胞数の増加が認められた(図１２)。分化した腸管上皮細胞のタイトジャンクションを確認するため、TEER値を評価した。その結果、分化終了時に、液相培養群に比較し、気相培養群におけるTEER値は低いものの、いずれの群においても経上皮電気抵抗値は150Ω・cm2より高い値を示した(図１３)。また、Sucrase-isomaltase、CYP3A4、Muc2、Pgp及びOLFM4の遺伝子発現レベルについて、気相培養群は液相培養群より高かった。気相培養を行うことでBCRPの遺伝子発現量は低下したが、ヒト成人小腸より高い発現レベルを有した（図１４及び図１５）。 (3) Examination of A83-01 addition timing Cell number 1 × 10 5 cells/insert, 5-aza-2'-deoxycytidine addition, 14 days after initiation of differentiation Human iPSC-derived intestinal tract cultured on feeder under gas phase culture conditions The influence of the addition timing of A83-01 on induction of differentiation of epithelial cells was examined. Compared with the A83-01 addition group from the 14th day after the start of differentiation, the A83-01 addition group showed an increase in the number of cells from the 8th day after the start of differentiation (Fig. 12). TEER values were evaluated to confirm tight junctions of differentiated intestinal epithelial cells. As a result, at the end of differentiation, the TEER value in the gas phase culture group was lower than that in the liquid phase culture group, but the transepithelial electrical resistance value was higher than 150 Ω·cm 2 in both groups (Fig. 13). ). In addition, the gene expression levels of Sucrase-isomaltase, CYP3A4, Muc2, Pgp and OLFM4 were higher in the gas-phase culture group than in the liquid-phase culture group. Although the amount of gene expression of BCRP decreased by performing gas phase culture, it had a higher expression level than that of human adult small intestine (Figs. 14 and 15).
（４）GSK-3β阻害剤であるCHIR99021添加の有無による腸管上皮細胞への分化誘導について検討
　細胞数1×105 cells/insert、分化開始14日目より5-アザ-2'-デオキシシチジン、A83-01添加かつ気相培養条件下でオンフィーダー培養したヒトiPSC由来腸管上皮細胞の分化誘導に対するCHIR99021添加の有無による影響を検討した。分化終了時に、CHIR99021添加群では陰窩－絨毛様構造の形成が認められた（図１６）。CHIR99021添加群は非添加群に比べ、分化した細胞のLGR5遺伝子発現量は上昇した。Muc2、CYP3A4、CYP2C9、Villin、Pgp及びBCRPの遺伝子発現量について、気相培養群は液相培養群より高く発現し、CHIR99021非添加群は添加群より同程度またより高い発現量を有した（図１７～図１９）。 (4) Examination of induction of differentiation into intestinal epithelial cells with or without the addition of CHIR99021, a GSK - 3β inhibitor The influence of the presence or absence of addition of CHIR99021 on induction of differentiation of human iPSC-derived intestinal epithelial cells cultured on-feeder under gas phase culture conditions with the addition of A83-01 was examined. At the end of differentiation, formation of crypt-villus-like structures was observed in the CHIR99021-added group (Fig. 16). The LGR5 gene expression level of the differentiated cells increased in the CHIR99021-added group compared to the non-added group. Regarding gene expression levels of Muc2, CYP3A4, CYP2C9, Villin, Pgp, and BCRP, the gas phase culture group expressed higher than the liquid phase culture group, and the CHIR99021 non-addition group had the same or higher expression level than the addition group ( 17-19).
（５）気相培養期間によるフィーダーレスで培養したヒトiPSC由来腸管上皮細胞へ分化誘導に与える影響
　フィーダーレスで維持培養したヒトiPSC由来腸管上皮細胞を分化誘導した際に、分化開始12日目、18日目及び24日目より気相培養を行い、遺伝子レベルで薬物動態関連マーカー、腸管特異的なマーカーを調べた。CYP3A4、Muc2、Pgp、Villin及びSucrase-isomaltaseの発現量について、いずれの群においても、Caco-2より高い発現レベルを有した。また、気相培養期間は短いほど、各マーカー遺伝子の発現量は高くなる傾向が認められ、その中で、気相培養を分化開始24日目より行った群はMuc2以外のマーカー遺伝子発現量が最も高かった（図２０及び２１）。 (5) Effect of gas-phase culture period on induction of differentiation into feederless-cultured human iPSC-derived intestinal epithelial cells From the 18th and 24th days, gas-phase culture was performed, and pharmacokinetic-related markers and intestinal-specific markers were examined at the gene level. Regarding the expression levels of CYP3A4, Muc2, Pgp, Villin and Sucrase-isomaltase, all groups had higher expression levels than Caco-2. In addition, the shorter the gas phase culture period, the higher the expression level of each marker gene. highest (Figures 20 and 21).
＜結論＞
　以上の結果より、気相培養を行うことで、ヒトiPSC由来小腸上皮細胞は、5-アザ-2’-デオキシシチジンの非添加またCHIR99021添加条件下で、陰窩－絨毛様構造を持つことが可能になった。気相培養条件下で、分化した腸管上皮細胞はタイトジャンクションを形成できたことが明らかとなった。ヒトiPSCの維持培養法に関わらず、同様な分化条件下で、気相培養により、ヒトiPSC由来腸管上皮細胞は小腸上皮細胞マーカー、薬物トランスポーター及び薬物代謝酵素の遺伝子レベル発現を向上させることが示された。さらに、フィーダーレスで培養したヒトiPSC由来腸管上皮細胞の遺伝子レベルにて評価では、分化細胞の成熟前に6日間気相培養することで、各マーカー遺伝子の発現量の向上に寄与することが明らかとなった。したがって、気相培養はヒトiPSCから小腸上皮細胞への分化誘導に有用であることが示唆された。 <Conclusion>
From the above results, by performing gas phase culture, human iPSC-derived small intestinal epithelial cells can have a crypt-villus-like structure under conditions without addition of 5-aza-2'-deoxycytidine and addition of CHIR99021. became possible. It was found that differentiated intestinal epithelial cells could form tight junctions under gas phase culture conditions. Regardless of the maintenance culture method of human iPSCs, human iPSC-derived intestinal epithelial cells can enhance the gene-level expression of small intestinal epithelial cell markers, drug transporters, and drug-metabolizing enzymes by gas-phase culture under similar differentiation conditions. shown. Furthermore, at the gene level evaluation of human iPSC-derived intestinal epithelial cells cultured feederless, it is clear that gas phase culture for 6 days prior to the maturation of differentiated cells contributes to the improvement of the expression level of each marker gene. became. Therefore, it was suggested that gas-phase culture is useful for inducing the differentiation of human iPSCs into small intestinal epithelial cells.
＜実施例Ｃ＞
＜方法＞
（１）細胞
　ヒトiPS細胞由来腸管上皮細胞としてFUJIFILMより市販されているF-hiSIECTMを使用した。 <Example C>
<Method>
(1) Cells F-hiSIEC ™ commercially available from FUJIFILM was used as human iPS cell-derived intestinal epithelial cells.
（２）F-hiSIECTMの培養
　F-hiSIECTMを解凍してF-hiSIECTM Seeding Mediumに懸濁した後、あらかじめマトリゲルでコートした24ウェルセルカルチャーインサート上に播種し、5% CO2/95% air条件下CO2インキュベーター中37°Cにて培養した。翌日、培地を5-aza-2’-deoxycitidineを含むまたは含まないF-hiSIECTM Culture Mediumに交換し、その後は週に3回の頻度で培地交換を行った。3日間気相培養を行う際は、細胞播種後8日目から、7日間気相培養を行う際は、細胞播種後4日目から、apical側の培地を除去した。 (2) Cultivation of F-hiSIEC TM After thawing F-hiSIEC TM and suspending it in F-hiSIEC TM Seeding Medium, it was inoculated onto a 24-well cell culture insert pre-coated with Matrigel and placed in 5% CO 2 /95. It was cultured at 37°C in a CO 2 incubator under % air conditions. The next day, the medium was replaced with F-hiSIEC ™ Culture Medium containing or not containing 5-aza-2'-deoxycitidine, and thereafter the medium was changed three times a week. The medium on the apical side was removed from day 8 after cell seeding when gas phase culture was performed for 3 days, and from day 4 after cell seeding when gas phase culture was performed for 7 days.
（３）経上皮電気抵抗（TEER）値の測定
　細胞播種後4日目から培地交換のタイミングでTEER値の測定を行った。測定にはMillicell ERS-2を用いた。 (3) Measurement of transepithelial electrical resistance (TEER) value The TEER value was measured at the timing of medium exchange from day 4 after cell seeding. Millicell ERS-2 was used for the measurement.
（４）ミダゾラムの膜透過試験および代謝試験
　細胞播種後11日目に行った。Apical側の培地は10 mM MESおよび0.45 w/v% D-glucoseを含みpHが6.5になるように調製したHanks’ balanced salt solution（HBSS）（HBSS-MES）に交換し、basal側の培地は10 mM HEPESおよび0.45 w/v% D-glucoseを含みpHが7.4になるように調製したHBSS（HBSS-HEPES）に交換して60分間プレインキュベーションした。その後、apical側を10 μmol/Lミダゾラムを含むHBSS-MESに、basal側をHBSS-HEPESに置換することで透過試験を開始した。Apical側からは透過試験開始直後および60分後に、basal側からは透過試験開始30および60分後にサンプリングした。サンプリングした液中に含まれるミダゾラムおよび1’-ヒドロキシミダゾラムの量はLC-MS/MSにより定量した。また、ミダゾラムの見かけの膜透過係数（Papp）は以下の式により算出した。 (4) Midazolam Membrane Permeation Test and Metabolism Test Conducted 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 and adjusted to pH 6.5. The cells were replaced with HBSS (HBSS-HEPES) containing 10 mM HEPES and 0.45 w/v% D-glucose and adjusted to pH 7.4, and pre-incubated for 60 minutes. After that, the apical side was replaced with HBSS-MES containing 10 μmol/L midazolam, and the basal side was replaced with HBSS-HEPES to start the permeation test. Samples were taken from the apical side immediately after and 60 minutes after the start of the permeation test, and from the basal side at 30 and 60 minutes after the start of the permeation test. The amounts of midazolam and 1'-hydroxymidazolam contained in the sampled liquid were quantified by LC-MS/MS. In addition, the apparent membrane permeability coefficient (P app ) of midazolam was calculated by the following formula.
Figure JPOXMLDOC01-appb-C000004
Figure JPOXMLDOC01-appb-C000004
ここで、dQ/dtは単位時間あたりに透過した化合物の量、Aはセルカルチャーインサートの表面積、C0はドナーチャンバー中の化合物の初期濃度である。 where dQ/dt is the amount of compound permeated per unit time, A is the surface area of the cell culture insert, and C0 is the initial concentration of the compound in the donor chamber.
（５）Lucifer yellowの膜透過試験
　細胞播種後11日目に行った。Apical側の培地はHBSS-MESに、basal側の培地はHBSS-HEPESに交換して60分間プレインキュベーションした。その後、apical側を110 μmol/L lucifer yellowを含むHBSS-MESに、basal側をHBSS-HEPESに置換することで透過試験を開始した。Apical側からは透過試験開始直後に、basal側からは透過試験開始60分後にサンプリングした。サンプリングした液中に含まれるlucifer yellowの量は蛍光プレートリーダーにより定量した。測定の際の励起波長は430 nm、蛍光波長は535 nmとした。なお、lucifer yellowのPappは上記と同様の式を用いて算出した。 (5) Membrane permeation test of Lucifer yellow Conducted 11 days after cell seeding. The medium on the apical side was replaced with HBSS-MES, and the medium on the basal side was replaced with HBSS-HEPES, followed by preincubation for 60 minutes. After that, the apical side was replaced with HBSS-MES containing 110 μmol/L lucifer yellow, and the basal side was replaced with HBSS-HEPES to start the permeation test. The apical side was sampled immediately after the start of the permeation test, and the basal side was sampled 60 minutes after the start of the permeation test. The amount of lucifer yellow contained in the sampled liquid was quantified using a fluorescence plate reader. The excitation wavelength for the measurement was 430 nm, and the fluorescence wavelength was 535 nm. The P app of lucifer yellow was calculated using the same formula as above.
（６）総リボ核酸（RNA）抽出
　細胞播種後11日目にRNeasy Mini Kitの添付マニュアルに従い総RNAを抽出した。 (6) Total ribonucleic acid (RNA) extraction Total RNA was extracted 11 days after seeding the cells according to the manual attached to the RNeasy Mini Kit.
（７）逆転写反応
　相補的DNA（cDNA）の合成は、ReverTra Ace qPCR RT Master Mix with gDNA Remover Kitを使用し、添付マニュアルに従い行った。 (7) Reverse Transcription Reaction Synthesis of complementary DNA (cDNA) was performed using ReverTra Ace qPCR RT Master Mix with gDNA Remover Kit according to the attached manual.
（８）Real-Time RT-PCR法
　Real-Time RT-PCRはKAPA SYBR Fast qPCR Kitを用い、cDNAを鋳型にして、反応は添付マニュアルに従い行った。結果は内在性コントロールとしてヒポキサンチン-グアニンホスホリボシルトランスフェラーゼ（HPRT）を用いて補正した。
Figure JPOXMLDOC01-appb-T000005
 (8) Real-Time RT-PCR Method For Real-Time RT-PCR, KAPA SYBR Fast qPCR Kit was used, cDNA was used as a template, and the reaction was carried out according to the attached manual. Results were corrected using hypoxanthine-guanine phosphoribosyltransferase (HPRT) as an endogenous control.
Figure JPOXMLDOC01-appb-T000005
＜結果＞
（１）経上皮電気抵抗（TEER）値の測定
　F-hiSIECTMの培養は２回行った。１回目のF-hiSIECTMの培養実験におけるTEER値の測定の結果を図２２に示す。２回目のF-hiSIECTMの培養実験におけるTEER値の測定の結果を図３０に示す。5-aza-2’-deoxycitidineを添加しない群では、TEER値の低下が認められた。 <Results>
(1) Measurement of transepithelial electrical resistance (TEER) value F-hiSIEC ™ was cultured twice. FIG. 22 shows the results of TEER measurement in the first F-hiSIEC ™ culture experiment. FIG. 30 shows the results of TEER measurement in the second F-hiSIEC ™ culture experiment. A decrease in the TEER value was observed in the group to which 5-aza-2'-deoxycitidine was not added.
（２）ミダゾラムの膜透過試験および代謝試験
　１回目のF-hiSIECTMの培養実験におけるミダゾラムの膜透過試験および代謝試験の結果を図２３～図２６に示す。図２４においては、気相培養した群では見かけの透過係数の減少が認められた。図２６においては、気相培養群では、代謝物の生成量が多く、5-aza-2’-deoxycitidineを添加しない群は、5-aza-2’-deoxycitidineを添加した群に比べ、代謝物の生成量が増加した。 (2) Membrane Permeation Test and Metabolic Test of Midazolam The results of the membrane permeation test and metabolic test of midazolam in the first F-hiSIEC ™ culture experiment are shown in FIGS. 23 to 26. FIG. In FIG. 24, a decrease in apparent permeability coefficient was observed in the group subjected to gas phase culture. In FIG. 26, in the gas phase culture group, the amount of metabolites produced was large, and the group without addition of 5-aza-2'-deoxycitidine produced more metabolites than the group with addition of 5-aza-2'-deoxycitidine. increased the production of
　２回目のF-hiSIECTMの培養実験におけるミダゾラムの代謝試験の結果を図３６に示す。図３６においては、5-aza-2’-deoxycitidineを添加しない群ではCYP3A4の代謝活性の上昇が認められた。 FIG. 36 shows the results of the midazolam metabolism test in the second F-hiSIEC ™ culture experiment. In FIG. 36, an increase in CYP3A4 metabolic activity was observed in the group to which 5-aza-2'-deoxycitidine was not added.
（３）Lucifer yellowの膜透過試験
　２回目のF-hiSIECTMの培養実験におけるLucifer yellowの膜透過試験の結果を図３１に示す。図３１においては、5-aza-2’-deoxycitidineを添加しない群ではLucifer yellowの見かけの膜透過係数の低下（バリア機能の上昇）が認められた。 (3) Lucifer yellow Membrane Permeation Test The results of the Lucifer yellow membrane permeation test in the second F-hiSIEC ™ culture experiment are shown in FIG. In FIG. 31, a decrease in the apparent membrane permeability coefficient of Lucifer yellow (increase in barrier function) was observed in the group to which 5-aza-2'-deoxycitidine was not added.
（４）遺伝子発現の解析
　１回目のF-hiSIECTMの培養実験における遺伝子発現の解析結果を図２７～図２９に示す。
　２回目のF-hiSIECTMの培養実験における遺伝子発現の解析結果を図３２～図３５に示す。 (4) Gene Expression Analysis Results of gene expression analysis in the first F-hiSIEC ™ culture experiment are shown in FIGS.
Gene expression analysis results in the second F-hiSIEC ™ culture experiment are shown in FIGS.
＜結論＞
　5-aza-2’-deoxycitidineを添加しない条件下で、TEER値の低下と、腸管上皮関連マーカーの遺伝子発現量および薬物代謝酵素CYP3A4のの上昇が認められた。さらに、気相培養することで、CYP3A4の代謝活性が上昇し、midazolamの見かけの膜透過係数が低下した。
　よって、5-aza-2’-deoxycitidine非添加及び気相培養は、F-hiSIECの分化誘導に有用であることが示された。 <Conclusion>
In the absence of 5-aza-2'-deoxycitidine, the TEER value decreased, and the gene expression levels of intestinal epithelium-related markers and the drug-metabolizing enzyme CYP3A4 increased. Furthermore, gas-phase culture increased the metabolic activity of CYP3A4 and decreased the apparent membrane permeability coefficient of midazolam.
Therefore, it was shown that 5-aza-2'-deoxycitidine-free and gas-phase culture is useful for inducing differentiation of F-hiSIEC.

Claims (21)

  1. 以下の工程（１）～（６）を含む、多能性幹細胞から腸管細胞を作製する方法：
    （１）多能性幹細胞を内胚葉様細胞へと分化させる工程；
    （２）工程（１）で得られた内胚葉様細胞を腸管幹細胞様細胞へと分化させる工程；
    （３）工程（２）で得られた腸管幹細胞様細胞を、上皮成長因子、線維芽細胞増殖因子、TGFβ受容体阻害剤、GSK-3β阻害剤及びROCK阻害剤の存在下で培養する工程；
    （４）工程（３）後の細胞を培養し、スフェロイドを形成させる工程；
    （５）工程（４）で形成されたスフェロイドを分化させ、腸管オルガノイドを形成させる工程であって、上皮成長因子、BMP阻害剤及びWntシグナル活性化剤の存在下での培養を含む工程；及び
    （６）工程（５）で形成された腸管オルガノイドを構成する細胞を、上皮成長因子、cAMPシグナル活性化因子、TGFβ受容体阻害剤、及びWntシグナル活性化剤の存在下で気相液相培養する工程。     A method for producing intestinal cells from pluripotent stem cells, comprising the following steps (1) to (6):
    (1) differentiating pluripotent stem cells into endoderm-like cells;
    (2) a step of differentiating the endoderm-like cells obtained in step (1) into intestinal stem cell-like cells;
    (3) culturing the intestinal stem cell-like cells obtained in step (2) in the presence of epidermal growth factor, fibroblast growth factor, TGFβ receptor inhibitor, GSK-3β inhibitor and ROCK inhibitor;
    (4) culturing the cells after step (3) to form spheroids;
    (5) differentiating the spheroids formed in step (4) to form intestinal organoids, comprising culturing in the presence of an epidermal growth factor, a BMP inhibitor and a Wnt signal activator; and (6) Cells composing the intestinal organoids formed in step (5) are cultured in gas-phase liquid phase in the presence of epidermal growth factor, cAMP signal activator, TGFβ receptor inhibitor, and Wnt signal activator. process to do.
  2. 工程（３）において、線維芽細胞増殖因子がFGF2、FGF4又はFGF10であり、TGFβ受容体阻害剤がA-83-01であり、GSK-3β阻害剤がCHIR99021、SB216763、CHIR98014、TWS119、Tideglusib、SB415286、BIO、AZD2858、AZD1080、AR-A014418、TDZD-8、LY2090314、IM-12、Indirubin、Bikinin又は1-Azakenpaulloneであり、ROCK阻害剤がY-27632である、請求項１に記載の方法。 In step (3), the fibroblast growth factor is FGF2, FGF4 or FGF10, the TGFβ receptor inhibitor is A-83-01, the GSK-3β inhibitor is CHIR99021, SB216763, CHIR98014, TWS119, Tideglusib, 2. The method of claim 1, wherein the SB415286, BIO, AZD2858, AZD1080, AR-A014418, TDZD-8, LY2090314, IM-12, Indirubin, Bikinin or 1-Azakenpaullone and the ROCK inhibitor is Y-27632.
  3. 工程（５）において、BMP阻害剤がNogginであり、Wntシグナル活性化剤がR-spondin-1である、請求項１又は２に記載の方法。 3. The method according to claim 1 or 2, wherein in step (5), the BMP inhibitor is Noggin and the Wnt signal activator is R-spondin-1.
  4. 工程（６）において、cAMPシグナル活性化因子がフォルスコリン、8-Br-cAMP又はIBMXであり。TGFβ受容体阻害剤がA-83-01であり、Wntシグナル活性化剤がCHIR99021である、請求項１～３のいずれか一項に記載の方法。 In step (6), the cAMP signal activator is forskolin, 8-Br-cAMP or IBMX. The method according to any one of claims 1 to 3, wherein the TGFβ receptor inhibitor is A-83-01 and the Wnt signal activator is CHIR99021.
  5. 工程（６）の気相液相培養を、上皮成長因子、cAMPシグナル活性化因子、TGFβ受容体阻害剤、及びWntシグナル活性化剤に加え、Notchシグナル阻害剤の存在下で行う、請求項１～４のいずれか一項に記載の方法。 Claim 1, wherein the gas-liquid phase culture in step (6) is performed in the presence of an epidermal growth factor, a cAMP signal activator, a TGFβ receptor inhibitor, and a Wnt signal activator, as well as a Notch signal inhibitor. 5. The method according to any one of -4.
  6. Notchシグナル阻害剤が、DAPTである、請求項５に記載の方法。 6. The method of claim 5, wherein the Notch signaling inhibitor is DAPT.
  7. 工程（６）の気相液相培養を、Notchシグナル阻害剤の存在下で行った後に、Notchシグナル阻害剤の非存在下で行う、請求項５又は６に記載の方法。 7. The method according to claim 5 or 6, wherein the gas-liquid phase culture in step (6) is performed in the presence of a Notch signal inhibitor and then in the absence of a Notch signal inhibitor.
  8. 工程（６）の気相液相培養を、４日～３０日間行う、請求項１～７のいずれか一項に記載の方法。 The method according to any one of claims 1 to 7, wherein the gas phase liquid phase culture in step (6) is performed for 4 to 30 days.
  9. 以下の工程（１）、（２）、（４）および（５）を含み、工程（４）及び工程（５）、工程（５）、又は工程（５）の培養の一部が気相液相培養である、多能性幹細胞から腸管細胞を作製する方法：
    （１）多能性幹細胞を内胚葉様細胞へと分化させる工程；
    （２）工程（１）で得られた内胚葉様細胞を腸管幹細胞様細胞へと分化させる工程；
    （４）工程（２）後の細胞を、上皮成長因子及びcAMPシグナル活性化因子の存在下で培養する工程；
    （５）工程（４）後の細胞を、上皮成長因子、MEK1/2阻害剤、DNAメチル化阻害剤、TGFβ受容体阻害剤、及びcAMPシグナル活性化因子の存在下で培養する工程。     comprising the following steps (1), (2), (4) and (5), wherein steps (4) and (5), step (5), or a portion of the culture of step (5) is a gas phase liquid A method of generating intestinal cells from pluripotent stem cells in phase culture:
    (1) differentiating pluripotent stem cells into endoderm-like cells;
    (2) a step of differentiating the endoderm-like cells obtained in step (1) into intestinal stem cell-like cells;
    (4) culturing the cells after step (2) in the presence of epidermal growth factor and cAMP signal activator;
    (5) A step of culturing the cells after step (4) in the presence of epidermal growth factor, MEK1/2 inhibitor, DNA methylation inhibitor, TGFβ receptor inhibitor, and cAMP signal activator.
  10. 工程（２）と工程（４）の間に、工程（３）をさらに含む、請求項９に記載の方法。
    （３）工程（２）で得られた腸管幹細胞様細胞を、上皮成長因子、及びROCK阻害剤の存在下で培養する工程；     10. The method of claim 9, further comprising step (3) between steps (2) and (4).
    (3) culturing the intestinal stem cell-like cells obtained in step (2) in the presence of epidermal growth factor and a ROCK inhibitor;
  11. 多能性幹細胞から作製された腸管細胞を、5-アザ-2'-デオキシシチジンを含まない培地で培養することを含む、腸管細胞を作製する方法。 A method of producing intestinal cells, comprising culturing intestinal cells produced from pluripotent stem cells in a medium that does not contain 5-aza-2'-deoxycytidine.
  12. 5-アザ-2'-デオキシシチジンを含まない培地での培養の少なくとも一部が、気相液相培養である、請求項１１に記載の方法。 12. The method of claim 11, wherein at least part of the culture in the medium not containing 5-aza-2'-deoxycytidine is gas phase liquid phase culture.
  13. 多能性幹細胞から作製された腸管細胞が、以下の工程（１１）～（１４）を含む方法により得られた細胞である、請求項１１又は１２に記載の方法。
    （１１）多能性幹細胞を内胚葉様細胞へと分化させる工程；
    （１２）工程（１１）で得られた内胚葉様細胞を腸管幹細胞様細胞へと分化させる工程；
    （１４）工程（１２）で得られた腸管幹細胞様細胞を、上皮成長因子及びcAMPシグナル活性化因子の存在下で培養する工程；および
    （１５）工程（１４）後の細胞を、上皮成長因子、MEK1/2阻害剤、DNAメチル化阻害剤、TGFβ受容体阻害剤、及びcAMPシグナル活性化因子の存在下で培養する工程。     The method according to claim 11 or 12, wherein the intestinal cells produced from pluripotent stem cells are cells obtained by a method comprising the following steps (11) to (14).
    (11) differentiating the pluripotent stem cells into endoderm-like cells;
    (12) differentiating the endoderm-like cells obtained in step (11) into intestinal stem cell-like cells;
    (14) culturing the intestinal stem cell-like cells obtained in step (12) in the presence of epidermal growth factor and cAMP signal activator; , a MEK1/2 inhibitor, a DNA methylation inhibitor, a TGFβ receptor inhibitor, and a cAMP signal activator.
  14. 多能性幹細胞が人工多能性幹細胞又は胚性幹細胞である、請求項１～１３のいずれか一項に記載の方法。 The method of any one of claims 1-13, wherein the pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells.
  15. 多能性幹細胞がヒト人工多能性幹細胞である、請求項１～１４のいずれか一項に記載の方法。 The method of any one of claims 1-14, wherein the pluripotent stem cells are human induced pluripotent stem cells.
  16. 多能性幹細胞が、フィーダーレスで培養した多能性幹細胞である、請求項１～１５のいずれか一項に記載の方法。 The method according to any one of claims 1 to 15, wherein the pluripotent stem cells are feederless cultured pluripotent stem cells.
  17. 請求項１～１６のいずれか一項に記載の方法で得られた腸管細胞。 Intestinal cells obtained by the method according to any one of claims 1 to 16.
  18. 請求項１７に記載の腸管細胞を用いた、被検物質の体内動態又は毒性を評価する方法。 A method for evaluating pharmacokinetics or toxicity of a test substance using the intestinal cells according to claim 17 .
  19. 前記体内動態が、代謝、吸収性、膜透過性、薬物相互作用、薬物代謝酵素の誘導、又は薬物トランスポーターの誘導である、請求項１８に記載の方法。 19. The method of claim 18, wherein the pharmacokinetics is metabolism, absorption, membrane permeability, drug interactions, induction of drug-metabolizing enzymes, or induction of drug transporters.
  20. 以下の工程（ｉ）及び（ｉｉ）を含む、請求項１８又は１９に記載の方法：
    （ｉ）請求項１７に記載の腸管細胞に被検物質を接触させる工程；
    （ｉｉ）被検物質の代謝、吸収性、膜透過性、薬物相互作用、薬物代謝酵素の誘導、又は薬物トランスポーターの誘導、或いは毒性を評価する工程。     20. A method according to claim 18 or 19, comprising steps (i) and (ii) of:
    (i) contacting the intestinal cells of claim 17 with a test substance;
    (ii) evaluating the metabolism, absorption, membrane permeability, drug interaction, induction of drug-metabolizing enzymes, induction of drug transporters, or toxicity of the test substance;
  21. 請求項１～１６のいずれか一項に記載の方法で得られた腸管細胞に腸疾患の病態を誘導することを特徴とする、腸疾患モデルの作製方法。 A method for preparing an intestinal disease model, which comprises inducing an intestinal disease state in intestinal cells obtained by the method according to any one of claims 1 to 16.
PCT/JP2022/004220 2021-02-04 2022-02-03 Production method for intestinal tract cells derived from pluripotent stem cells and having crypt-villus-like structures, and use thereof WO2022168908A1 (en)

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