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Method for inducing differentiation of pluripotent stem cells into intestinal epithelial cells

A technology of pluripotent stem cells and intestinal epithelial cells, which is applied in the field of induction of differentiation from pluripotent stem cells to intestinal epithelial cells, can solve problems such as difficult to accurately evaluate pharmacokinetics, observation of drug metabolism enzyme expression and enzyme induction, etc.

Active Publication Date: 2020-09-18
NAGOYA CITY UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the expression and enzyme induction of drug-metabolizing enzymes are hardly observed in Caco-2 cells, so it is difficult to accurately evaluate the pharmacokinetics in the small intestine
Therefore, in order to comprehensively evaluate drug metabolism and membrane permeability in the small intestine, primary small intestinal epithelial cells are expected to be used, but there are problems with function and supply, so it is difficult to be widely used for pharmacokinetic tests like primary hepatocytes system

Method used

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  • Method for inducing differentiation of pluripotent stem cells into intestinal epithelial cells
  • Method for inducing differentiation of pluripotent stem cells into intestinal epithelial cells
  • Method for inducing differentiation of pluripotent stem cells into intestinal epithelial cells

Examples

Experimental program
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Embodiment

[0156] Search for low-molecular-weight compounds useful for promoting differentiation / acquisition of function from human iPS cells to intestinal epithelial cells

[0157] The following studies were conducted for the purpose of establishing a method for easily and efficiently producing cells (enterocyte-like cells) exhibiting functions closer to living intestinal epithelial cells.

[0158]

[0159] 1. Method

[0160] (1) cells

[0161] As human iPS cells, Windy (iPS-51) and FF-1 strains were used. Windy is the use of a pan-retrotropic retroviral vector to combine octamer binding protein 3 / 4 (octamer binding protein 3 / 4) (OCT3 / 4), sex determining region Y-box protein 2 (sexdetermining region Y-box 2) (SOX2) , Krupp-like factor 4 (kruppel-like factor 4) (KLF4), V-Myc myelocytomatosis viral oncogenehomolog (avian) (c-MYC) introduction After human embryonic lung fibroblasts MRC-5, human ES cell-like colonies were cloned, provided by Dr. Mei Zeminghong of the National Medical R...

Embodiment 2

[0195] 1. Method

[0196] (1) Differentiation from human iPS cells to intestinal epithelial cells

[0197] Human iPS cells (FF-1) were treated with serum-free medium containing 100 ng / mL recombinant human activin A for 24 hours to initiate differentiation. Then, they were treated for 144 hours in a serum-free medium containing 100 ng / mL recombinant human activin A, 2.5 ng / mL BMP4, 10 ng / mL VEGF, and 5 ng / mL FGF2 to differentiate into endoderm. Then, differentiate into small intestine by culturing for 96 hours in AdvancedDMEM / F-12 containing 2% FBS, 1% GlutaMax, 100 units / mL penicillin G, 100 μg / mL streptomycin sulfate, 250 ng / mL FGF2 stem cell. The intestinal stem cell-like cells treated with recombinant human activin A and FGF2 were detached with Accutase, and seeded on GFR Matrigel diluted 30 times in human iPS medium. After inoculation, contain 2% FBS, 0.1mM NEAA, 2mM L-Glu, 100 units / mL penicillin G, 100μg / mL streptomycin sulfate, 2% B27 supplement, 1% N2supplement, 1% ...

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Abstract

The present invention addresses the problem of providing a novel means whereby cells showing a function more closely similar to the function of intestinal epithelial cells of a living body can be easily and efficiently prepared. According to the present invention, differentiation of pluripotent stem cells into intestinal epithelial cells is induced by: (1) a step for differentiating the pluripotent stem cells into intestinal stem cell-like cells; and (2) a step for differentiating the intestinal stem cell-like cells obtained in step (1) into intestinal epithelial cell-like cells with the combined use of an MEK1 inhibitor, a DNA methylation inhibitor, a TGF Beta receptor inhibitor, EGF and a cAMP activator.

Description

technical field [0001] The invention relates to a method for inducing the differentiation of pluripotent stem cells into intestinal epithelial cells and its application. Background technique [0002] Since many drug-metabolizing enzymes and drug transporters exist in the small intestine, it is very important as an organ involved in the first-pass effect of drugs like the liver. Therefore, in the development of pharmaceuticals with excellent pharmacokinetic properties, it is necessary to evaluate the membrane permeability and metabolism of small intestinal Chinese medicines from the early stage of pharmaceutical development. Currently, as a model system of the small intestine, Caco-2 cells derived from human colon cancer are commonly used. However, the expression pattern of drug transporters in Caco-2 cells differs from that in the human small intestine. Furthermore, expression and enzyme induction of drug-metabolizing enzymes are hardly observed in Caco-2 cells, so it is d...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N1/00C12N5/0735C12N5/10C12Q1/02
CPCC12N1/00C12N5/10A61K35/38A61P1/00C12N5/0679C12N5/0696C12N2501/01C12N2501/11C12N2501/115C12N2501/15C12N2501/727C12N2506/45G01N33/5073G01N33/5014C12Q1/68C12N2501/999C12Q2600/158
Inventor 松永民秀岩尾岳洋壁谷知树美马伸治宫下敏秀
Owner NAGOYA CITY UNIVERSITY
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