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INDUCTION OF DIFFERENTIATION OF INDUCED PLURIPOTENT STEM CELLS INTO INTESTINAL EPITHELIAL CELLS UPDATE_EN

Foreign code F170009236
File No. (S2016-0443-N0)
Posted date Sep 21, 2017
Country WIPO
International application number 2017JP008616
International publication number WO 2017154795
Date of international filing Mar 3, 2017
Date of international publication Sep 14, 2017
Priority data
  • P2016-044088 (Mar 8, 2016) JP
Title INDUCTION OF DIFFERENTIATION OF INDUCED PLURIPOTENT STEM CELLS INTO INTESTINAL EPITHELIAL CELLS UPDATE_EN
Abstract This invention addresses the problem of providing: a novel method that allows, in a simple manner, preparation of cells that exhibit a function similar to that of intestinal epithelial cells of a living body; and an application therefor. Induced pluripotent stem cells are induced to differentiate into intestinal epithelial cells by: (1) a step for differentiating the induced pluripotent stem cells into endoderm-like cells; (2) a step for differentiating the endoderm-like cells obtained in step (1) into intestinal stem cell-like cells; and (3) a step for differentiating the intestinal stem cell-like cells obtained in step (2) into intestinal epithelial cell-like cells, the step including culturing in the presence of an MEK1 inhibitor, a DNA methylation inhibitor, a TGFβ receptor inhibitor, and EGF, and in a condition in which cAMP is fed to the cells.
Scope of claims [claim1]
1. The process (1)-(3) below is included, the artificial versatile characteristic trunk cell to the intestinal tract epithelium cell the method where it differentiates induces:
(1) the process which the endotoderm way differentiates the artificial versatile characteristic trunk cell to the cell;
(2) to be obtained with process in order (1) endotoderm, the process which the intestinal tract trunk cell way differentiates the cell to the cell;
(3) to be obtained with process in order (2) intestinal tract trunk cell, being the process which the intestinal tract epithelium cell way differentiates the cell to the cell, the MEK1 inhibiter, the DNA methylation inhibiter, the TGF.beta. receptor inhibiter and under the existing of EGF, at the same time cAMP the process which includes the culture under the condition to the cell for being supplied.
[claim2]
2. Process (3) period of the aforementioned culture is between 7 days between -40 day, in claim 1 method of statement.
[claim3]
3. Process (3), includes each culture process of A-C below, in claim 1 method of statement,
Culture process A: (A-1) It follows to the MEK1 inhibiter, the DNA methylation inhibiter, the TGF.beta. receptor inhibiter and the culture and the said culture under the existing of EGF, (a-2) the MEK1 inhibiter, the DNA methylation inhibiter, the TGF.beta. receptor inhibiter and under the existing of EGF, at the same time cAMP includes the culture under the condition for being supplied to the cell,
Culture process B: (B-1) The MEK1 inhibiter, the DNA methylation inhibiter, the TGF.beta. receptor inhibiter and under the existing of EGF, at the same time cAMP for being supplied follows to the culture and the said culture under the condition to the cell, (b-2) the MEK1 inhibiter, the DNA methylation inhibiter and the TGF.beta. receptor inhibiter, culture under the existing of EGF and the cAMP disassembly enzyme inhibiter is included,
Culture process C: (C-1) The MEK1 inhibiter, the DNA methylation inhibiter, the TGF.beta. receptor inhibiter and under the existing of EGF, at the same time cAMP includes the culture under the condition for being supplied to the cell.
[claim4]
4. (A-1) Period of culture is between 1 days between -5 day, (a-2) period of culture is between 3 days between -15 day,
(B-1) Period of culture is between 3 days between -15 day, (b-2) period of culture is between 3 days between -15 day,
(C-1) Period of culture is between 3 days between -15 day, in claim 3 method of statement.
[claim5]
5. (A-2) Culture, (b-1) culture, and (c-1) culture, is done under the conditions also the cAMP disassembly enzyme inhibiter existing in addition to MEK1 inhibiter, DNA methylation inhibiter, TGF.beta. receptor inhibiter and EGF, in claim 3 or 4 method of statement.
[claim6]
6. Culture process A, (a-2) follows to culture, (a-3) implication the MEK1 inhibiter, the DNA methylation inhibiter, the TGF.beta. receptor inhibiter and the culture under the existing of EGF,
Culture process B, (b-2) follows to culture, (b-3) implication the MEK1 inhibiter, the DNA methylation inhibiter, the TGF.beta. receptor inhibiter and the culture under the existing of EGF,
Culture process C, (c-1) follows to culture, (c-2) the MEK1 inhibiter, the DNA methylation inhibiter, the TGF.beta. receptor inhibiter and culture under the existing of EGF are included, either of the claim 2-5 in one section method of statement.
[claim7]
7. (A-3) Culture, (b-3) culture, and (c-2) period of culture is between 1 days between -10 day, in claim 6 method of statement.
[claim8]
8. CAMP for being supplied the condition, is that 8-Br-cAMP exists in the nutrient medium to the cell, either of the claim 1-7 in one section method of statement.
[claim9]
9. The CAMP disassembly enzyme inhibiter is IBMX, either of the claim 1-8 in one section method of statement.
[claim10]
10. The MEK1 inhibiter is PD98059, the DNA methylation inhibiter 5 - aza - 2' - is deoxy cytidine, the TGF.beta. receptor inhibiter is A-83-01, either of the claim 1-9 in one section method of statement.
[claim11]
11. In process akuchibin A is used (1) as the differentiation elicitor, either of the claim 1-10 in one section method of statement.
[claim12]
12. In process FGF2 is used (2) as the differentiation elicitor, either of the claim 1-11 in one section method of statement.
[claim13]
13. The artificial versatile characteristic trunk cell is the human artificial versatile characteristic trunk cell, either of the claim 1-12 in one section method of statement.
[claim14]
14. Either of the claim 1-13 way in one section it is obtained with method of statement intestinal tract epithelium cell, the cell.
[claim15]
15. In claim 14 intestinal tract epithelium cell as in statement the cell was used, internal movement of the suffering inspection substance or the method of appraising virulence.
[claim16]
16. The aforementioned internal movement, is metabolism, absorption, excretion, medicine interaction, induction of the medicine metabolism enzyme or induction of the medicine trance porter, in claim 15 method of statement.
[claim17]
17. Process (i)-(iii) below is included, in claim 15 or 16 method of statement:
(i) In claim 14 intestinal tract epithelium cell as in statement the process which prepares the cell layer which is formed in the cell;
(ii) The process which makes the suffering inspection substance the aforementioned cell layer contact;
(iii) The process which the suffering inspection substance which transmitted the aforementioned cell layer measures, absorptivity or membrane permeability of the suffering inspection substance, medicine interaction, induction of the medicine metabolism enzyme and induction of the medicine trance porter, or appraises virulence.
[claim18]
18. It includes process (i) and (ii) below, in claim 15 or 16 method of statement:
(i) In claim 14 intestinal tract epithelium cell as in statement the process which makes the suffering inspection substance the cell contact;
(ii) Metabolism or absorption, medicine interaction of the suffering inspection substance, induction of the medicine metabolism enzyme and induction of the medicine trance porter, or the process which it measures & appraises poisonous.
[claim19]
19. The method process below (a) and (b) of including, appraising the alimentary canal mucous membrane obstacle action of the suffering inspection substance:
(A) In claim 14 intestinal tract epithelium cell as in statement the process which makes the suffering inspection substance the cell contact;
(B) The aforementioned intestinal tract epithelium cell way being the process which detects the revelation of the mucin 2 in the cell, decides the alimentary canal mucous membrane obstacle action of the suffering inspection substance on the basis of the detection result, being able to recognize the revelation decrease of mucin 2, the process which becomes index of the thing where the suffering inspection substance has alimentary canal mucous membrane obstacle action.
[claim20]
20. Process below (A) and (B) is included, the method alimentary canal mucous membrane protection action of the suffering inspection substance being appraised:
(A) in claim 14 intestinal tract epithelium cell as in statement the process which makes the suffering inspection substance the cell contact;
(B) the aforementioned intestinal tract epithelium cell way being the process which detects the revelation of the mucin 2 in the cell, decides the alimentary canal mucous membrane protection action of the suffering inspection substance on the basis of the detection result, being able to recognize the revelation rise of the mucin 2 by the aforementioned substance, the process which becomes index of the thing where the suffering inspection substance has alimentary canal mucous membrane protection action.
[claim21]
21. In claim 14 intestinal tract epithelium cell as in statement the cell is included, the cell formulation.
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • NAGOYA CITY UNIVERSITY
  • Inventor
  • IWAO TAKAHIRO
  • KABEYA TOMOKI
  • MATSUNAGA TAMIHIDE
IPC(International Patent Classification)
Specified countries (WO2017154795)
National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DJ DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JP KE KG KH KN KP KR KW KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG
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