JPWO2022168908A5 - - Google Patents
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- JPWO2022168908A5 JPWO2022168908A5 JP2022579599A JP2022579599A JPWO2022168908A5 JP WO2022168908 A5 JPWO2022168908 A5 JP WO2022168908A5 JP 2022579599 A JP2022579599 A JP 2022579599A JP 2022579599 A JP2022579599 A JP 2022579599A JP WO2022168908 A5 JPWO2022168908 A5 JP WO2022168908A5
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- pluripotent stem
- growth factor
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- 210000004027 cell Anatomy 0.000 claims 28
- 239000003112 inhibitor Substances 0.000 claims 20
- 238000000034 method Methods 0.000 claims 18
- 230000000968 intestinal effect Effects 0.000 claims 17
- 210000001778 pluripotent stem cell Anatomy 0.000 claims 11
- 239000012190 activator Substances 0.000 claims 10
- 238000012258 culturing Methods 0.000 claims 9
- 102000009024 Epidermal Growth Factor Human genes 0.000 claims 8
- 101800003838 Epidermal growth factor Proteins 0.000 claims 8
- 229940116977 epidermal growth factor Drugs 0.000 claims 8
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 claims 8
- 108091005735 TGF-beta receptors Proteins 0.000 claims 6
- 102000016715 Transforming Growth Factor beta Receptors Human genes 0.000 claims 6
- 239000012071 phase Substances 0.000 claims 6
- 239000007791 liquid phase Substances 0.000 claims 5
- 230000006698 induction Effects 0.000 claims 4
- 238000004519 manufacturing process Methods 0.000 claims 4
- 239000003814 drug Substances 0.000 claims 3
- 229940079593 drug Drugs 0.000 claims 3
- 239000011435 rock Substances 0.000 claims 3
- 239000000126 substance Substances 0.000 claims 3
- AQGNHMOJWBZFQQ-UHFFFAOYSA-N CT 99021 Chemical group CC1=CNC(C=2C(=NC(NCCNC=3N=CC(=CC=3)C#N)=NC=2)C=2C(=CC(Cl)=CC=2)Cl)=N1 AQGNHMOJWBZFQQ-UHFFFAOYSA-N 0.000 claims 2
- 108010078791 Carrier Proteins Proteins 0.000 claims 2
- 230000007067 DNA methylation Effects 0.000 claims 2
- 206010013710 Drug interaction Diseases 0.000 claims 2
- 102100031480 Dual specificity mitogen-activated protein kinase kinase 1 Human genes 0.000 claims 2
- 101710146526 Dual specificity mitogen-activated protein kinase kinase 1 Proteins 0.000 claims 2
- 108090000790 Enzymes Proteins 0.000 claims 2
- 102000004190 Enzymes Human genes 0.000 claims 2
- 102000018233 Fibroblast Growth Factor Human genes 0.000 claims 2
- 108050007372 Fibroblast Growth Factor Proteins 0.000 claims 2
- 102000019058 Glycogen Synthase Kinase 3 beta Human genes 0.000 claims 2
- 108010051975 Glycogen Synthase Kinase 3 beta Proteins 0.000 claims 2
- 229940124647 MEK inhibitor Drugs 0.000 claims 2
- 238000010521 absorption reaction Methods 0.000 claims 2
- 229940126864 fibroblast growth factor Drugs 0.000 claims 2
- OHCQJHSOBUTRHG-KGGHGJDLSA-N forskolin group Chemical group C(C)(=O)O[C@H]1[C@H]([C@@H]2[C@@]([C@]3(C(C[C@](O[C@]13C)(C)C=C)=O)O)([C@H](CCC2(C)C)O)C)O OHCQJHSOBUTRHG-KGGHGJDLSA-N 0.000 claims 2
- 210000004263 induced pluripotent stem cell Anatomy 0.000 claims 2
- 208000028774 intestinal disease Diseases 0.000 claims 2
- 239000012528 membrane Substances 0.000 claims 2
- 230000004060 metabolic process Effects 0.000 claims 2
- 210000002220 organoid Anatomy 0.000 claims 2
- 230000035699 permeability Effects 0.000 claims 2
- 230000001988 toxicity Effects 0.000 claims 2
- 231100000419 toxicity Toxicity 0.000 claims 2
- JCSGFHVFHSKIJH-UHFFFAOYSA-N 3-(2,4-dichlorophenyl)-4-(1-methyl-3-indolyl)pyrrole-2,5-dione Chemical compound C12=CC=CC=C2N(C)C=C1C(C(NC1=O)=O)=C1C1=CC=C(Cl)C=C1Cl JCSGFHVFHSKIJH-UHFFFAOYSA-N 0.000 claims 1
- VPVLEBIVXZSOMQ-UHFFFAOYSA-N 3-[[6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]oxy]phenol Chemical compound NC1=CC=CC(C=2NC3=NC=NC(OC=4C=C(O)C=CC=4)=C3C=2)=C1 VPVLEBIVXZSOMQ-UHFFFAOYSA-N 0.000 claims 1
- XAUDJQYHKZQPEU-KVQBGUIXSA-N 5-aza-2'-deoxycytidine Chemical compound O=C1N=C(N)N=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 XAUDJQYHKZQPEU-KVQBGUIXSA-N 0.000 claims 1
- DVKQVRZMKBDMDH-UUOKFMHZSA-N 8-Br-cAMP Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=CN=C2N)=C2N=C1Br DVKQVRZMKBDMDH-UUOKFMHZSA-N 0.000 claims 1
- MDZCSIDIPDZWKL-UHFFFAOYSA-N CHIR-98014 Chemical compound C1=C([N+]([O-])=O)C(N)=NC(NCCNC=2N=C(C(=CN=2)N2C=NC=C2)C=2C(=CC(Cl)=CC=2)Cl)=C1 MDZCSIDIPDZWKL-UHFFFAOYSA-N 0.000 claims 1
- DWJXYEABWRJFSP-XOBRGWDASA-N DAPT Chemical group N([C@@H](C)C(=O)N[C@H](C(=O)OC(C)(C)C)C=1C=CC=CC=1)C(=O)CC1=CC(F)=CC(F)=C1 DWJXYEABWRJFSP-XOBRGWDASA-N 0.000 claims 1
- SUZLHDUTVMZSEV-UHFFFAOYSA-N Deoxycoleonol Natural products C12C(=O)CC(C)(C=C)OC2(C)C(OC(=O)C)C(O)C2C1(C)C(O)CCC2(C)C SUZLHDUTVMZSEV-UHFFFAOYSA-N 0.000 claims 1
- 102100028412 Fibroblast growth factor 10 Human genes 0.000 claims 1
- 102100035290 Fibroblast growth factor 13 Human genes 0.000 claims 1
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims 1
- 102100028072 Fibroblast growth factor 4 Human genes 0.000 claims 1
- 101000917237 Homo sapiens Fibroblast growth factor 10 Proteins 0.000 claims 1
- 101001060274 Homo sapiens Fibroblast growth factor 4 Proteins 0.000 claims 1
- 102100022762 R-spondin-1 Human genes 0.000 claims 1
- 101710110302 R-spondin-1 Proteins 0.000 claims 1
- OHCQJHSOBUTRHG-UHFFFAOYSA-N colforsin Natural products OC12C(=O)CC(C)(C=C)OC1(C)C(OC(=O)C)C(O)C1C2(C)C(O)CCC1(C)C OHCQJHSOBUTRHG-UHFFFAOYSA-N 0.000 claims 1
- 238000011977 dual antiplatelet therapy Methods 0.000 claims 1
- 210000001671 embryonic stem cell Anatomy 0.000 claims 1
- 210000001035 gastrointestinal tract Anatomy 0.000 claims 1
- 230000001939 inductive effect Effects 0.000 claims 1
- 239000007788 liquid Substances 0.000 claims 1
- 102000045246 noggin Human genes 0.000 claims 1
- 108700007229 noggin Proteins 0.000 claims 1
- 230000007170 pathology Effects 0.000 claims 1
Claims (21)
（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、Ｗｎｔシグナル活性化剤、および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気相液相培養を行う工程。A method for producing intestinal cells from pluripotent stem cells, comprising the following steps (1) to (6), wherein the gas-phase liquid phase culture in step (6) is carried out for 22 to 30 days, and the intestinal tract is produced. A method in which the transepithelial electrical resistance (TEER) of the cells is 150 Ω·cm 2 or less.
(1) Step of differentiating pluripotent stem cells into endoderm-like cells;
(2) 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) a step of differentiating the spheroids formed in step (4) to form intestinal organoids, the step comprising culturing in the presence of an epidermal growth factor, a BMP inhibitor, and a Wnt signal activator; and (6) Cells constituting the intestinal organoid formed in step (5) are treated in the presence of epidermal growth factor, cAMP signal activator, TGFβ receptor inhibitor, Wnt signal activator, and Notch signal inhibitor. A step of performing gas phase liquid phase culture in the absence of a Notch signal inhibitor after performing gas phase liquid phase culture.
（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 following steps (1), (2), (4) and (5) are included, and a part of the culture in step (4) and step (5), step (5), or step (5) is a gas phase liquid. Method for producing intestinal cells from pluripotent stem cells in phase culture:
(1) Step of differentiating pluripotent stem cells into endoderm-like cells;
(2) 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 an epidermal growth factor, a MEK1/2 inhibitor, a DNA methylation inhibitor, a TGFβ receptor inhibitor, and a cAMP signal activator.
（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；The method according to claim 9, further comprising step (3) between step (2) and step (4).
(3) a step of culturing the intestinal stem cell-like cells obtained in step (2) in the presence of an epidermal growth factor and a ROCK inhibitor;
（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 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; and (15) culturing the cells after step (14) with epidermal growth factor , a MEK1/2 inhibitor, a DNA methylation inhibitor, a TGFβ receptor inhibitor, and a cAMP signal activator.
（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。The method according to claim 18 or 19, comprising the following steps (i) and (ii):
(i) contacting the intestinal cells according to 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.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2021016645 | 2021-02-04 | ||
| PCT/JP2022/004220 WO2022168908A1 (en) | 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 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPWO2022168908A1 JPWO2022168908A1 (en) | 2022-08-11 |
| JPWO2022168908A5 true JPWO2022168908A5 (en) | 2024-01-10 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2022579599A Pending JPWO2022168908A1 (en) | 2021-02-04 | 2022-02-03 |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JPWO2022168908A1 (en) |
| WO (1) | WO2022168908A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024063533A1 (en) * | 2022-09-21 | 2024-03-28 | 한국생명공학연구원 | High-performance mature intestinal organoid regenerative therapeutic agent derived from pluripotent stem cells |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9464275B2 (en) * | 2008-08-21 | 2016-10-11 | The Board Of Trustees Of The Leland Stanford Junior University | Ex vivo culture, proliferation and expansion of intestinal epithelium |
| JP7367992B2 (en) * | 2018-11-02 | 2023-10-24 | 公立大学法人名古屋市立大学 | Method for producing pluripotent stem cell-derived intestinal organoids |
-
2022
- 2022-02-03 WO PCT/JP2022/004220 patent/WO2022168908A1/en active Application Filing
- 2022-02-03 JP JP2022579599A patent/JPWO2022168908A1/ja active Pending
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