Method for producing intestinal organoid derived from pluripotent stem cells

A technology of pluripotent stem cells and a production method, which is applied in the field of making intestinal organoids from pluripotent stem cells, and can solve the problem of no pharmacokinetics and the like

Pending Publication Date: 2020-01-14
NAGOYA CITY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, embryonic stem cells (ES cells) and induced pluripotent stem cells (induced pluripotent stem cells: iPS cells) which have the same multi-differentiation potential and almost unlimited proliferation ability as ES cells are also expected to be used in drug discovery research. cell) differentiation-induced intestinal organoids are immature, and pharmacokinetic and functional analyzes have hardly been performed

Method used

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  • Method for producing intestinal organoid derived from pluripotent stem cells
  • Method for producing intestinal organoid derived from pluripotent stem cells
  • Method for producing intestinal organoid derived from pluripotent stem cells

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Experimental program
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Embodiment

[0152]

[0153] In order to create a novel method for producing functional intestinal organoids, the following research was conducted.

[0154] 1. Method

[0155] (1) cells

[0156]Human iPS cells (iPS-51: Windy) were transformed with octamer-binding protein 3 / 4 (OCT3 / 4), sex-determining region Y-box protein 2 (SOX2), kruppel-like factor 4 ( KLF4), v-myc myeloma virus oncogene homologue (avian) (c-MYC) was introduced into human fetal lung fibroblast MRC-5, and then cloned human ES cell-like colonies, obtained by the National Provided by Dr. Akihiro Umezawa, Center for Developmental Medical Research. Cynomolgus monkey iPS cells were introduced into fibroblasts cultured from cynomolgus monkey skin tissue with pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK, and pCXLE-hUL as episomal vectors by electroporation. Monkey ES cell-like colonies were cloned and established by the present inventors. As feeder cells, mouse fetal fibroblasts (MEFs) were used.

[0157] (2) culture medium

[0158]...

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Abstract

The present invention addresses the problem of producing a functional intestinal organoid from pluripotent stem cells. An intestinal organoid is produced from pluripotent stem cells through the following steps: (1) a step for differentiating the 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; (3) a step for culturing the intestinal stem cell-like cells obtained in step (2) to form a spheroid; and (4) a step for differentiating the spheroid formed in step (3) to form an intestinalorganoid, wherein culturing is carried out in the presence of, in addition to an epidermal growth factor, a BMP inhibitor and a Wnt signal activator, a MEK1 / 2 inhibitor, a DNA methylation inhibitor, aTGF[beta] receptor inhibitor and a [gamma]-secretase inhibitor.

Description

technical field [0001] The present invention relates to a method for making intestinal organoids from pluripotent stem cells and its use. This application claims priority based on Japanese Patent Application No. 2017-093418 for which it applied on May 9, 2017, The content of this patent application is used in this specification by reference. Background technique [0002] The small intestine is a very important organ in the study of the pharmacokinetics of orally administered pharmaceuticals. In the stage of non-clinical trials, in order to comprehensively evaluate the pharmacokinetics (absorption, excretion, metabolism) in the human small intestine, it is desired to use primary small intestinal epithelial cells, but it is difficult to obtain them. In recent years, three-dimensional tissue structures (organoids) that mimic intestinal tissue have attracted attention as a new type of in vitro evaluation system. However, embryonic stem cells (ES cells) and induced pluripotent ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10A61L27/38C12N5/071C12Q1/02
CPCA61L27/38C12N5/0679C12N2513/00C12N2506/45C12N2501/727C12N2501/15C12N2501/155C12N2501/734C12N2501/11C12N2501/415C12N2533/90C12N2501/16C07K14/50G01N33/5088C12N5/0697G01N33/5082A61L27/3834A61L27/3882C12N2501/115C12N2501/119C12N2503/02C12N2533/70A61L27/3895C12N2501/998C12N2501/999C12N2503/04
Inventor 松永民秀岩尾岳洋小野里太智小川勇
Owner NAGOYA CITY UNIVERSITY
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