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Method for inducing differentiation of pluripotent stem cells into hepatocytes

A technology of pluripotent stem cells and hepatocytes, applied in the field of induction of differentiation from pluripotent stem cells to hepatocytes, can solve the problems of large batch differences, difficulty in correctly evaluating the toxicity of human liver cells, and supply limitations of liver cells

Pending Publication Date: 2020-07-17
TOKYO INST OF TECH +2
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since tests using experimental animals have "obstacles of interspecies differences", it is difficult to accurately evaluate the toxicity to human hepatocytes, and it is not preferable from the viewpoint of animal protection.
On the other hand, human primary cultured hepatocytes have problems such as limited supply, large lot-to-lot variability, or a decrease in the activity of drug-metabolizing enzymes in a short period of time from the start of culture.

Method used

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  • Method for inducing differentiation of pluripotent stem cells into hepatocytes
  • Method for inducing differentiation of pluripotent stem cells into hepatocytes
  • Method for inducing differentiation of pluripotent stem cells into hepatocytes

Examples

Experimental program
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Embodiment

[0105] Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples.

[0106] (A) Materials and Methods

[0107] (1) Human iPS cell line

[0108] The undifferentiated human iPS cell line ChiPS18 cells (Asplund et al., 2015, Stem Cell Rev. Reports) were treated with AK02 StemFit medium (Ajinomoto) on a cell culture dish (Invitrogen) previously coated with Synthemax (registered trademark) II. )maintain. To deplete methionine, ChiPS18 cells were cultured with StemFit complete medium (Ajinomoto) or KA01 medium lacking methionine (Ajinomoto).

[0109] (2) Preparation of Collagen Vitrigel membrane chamber

[0110] The collagen xerogel film was manufactured by Kanto Chemical Co., Ltd. (Tokyo, Japan). Briefly, Collagen Vitrigel membranes are as previously reported (Oshikata-Miyazaki and Takezawa, 2016, Cytotechnology 68, 1801-1811; Yamaguchi et al., 2013, Toxicol. Sci. 135, 347-355; Yamaguchi et ...

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Abstract

Provided is a method for preparing hepatocytes or cells that can be differentiated into hepatocytes from pluripotent stem cells for the purpose of preparing mature hepatocytes that are similar in manyrespects to primary cultured hepatocytes, wherein the method is characterized by including (1) a step for culturing pluripotent stem cells in a medium that contains an activator of activin receptor-like kinase-4,7, (2) a step for culturing the cells obtained in step (1) in a medium that contains an osteogenic factor and a fibroblast growth factor, (3) a step for culturing the cells obtained in step (2) in a medium that contains a hepatocyte growth factor receptor activator and an oncostatin M receptor activator, and (4) a step for culturing the cells obtained in step (3) and obtaining hepatocytes or cells that can be differentiated into hepatocytes, at least one of steps (2), (3), and (4) involving culturing the cells on a high-density collagen gel membrane.

Description

technical field [0001] The present invention relates to a method for producing hepatocytes or cells capable of differentiating into hepatocytes, hepatocytes or cells capable of differentiating into hepatocytes, and methods for evaluating properties of test substances using the hepatocytes. Background technique [0002] Many pharmaceutical agents have either altered efficacy or exhibited toxicity by being metabolized by the liver. Therefore, when a drug is developed, it is necessary to examine the toxicity of drug candidate substances to the liver. Currently, in vivo tests using experimental animals such as mice, in vitro tests using primary cultured human hepatocytes, etc. are performed for the evaluation of hepatotoxicity. However, since tests using experimental animals have "barriers to differences between species", it is difficult to accurately evaluate the toxicity to human hepatocytes, and it is not preferable from the viewpoint of animal protection. On the other hand...

Claims

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Application Information

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IPC IPC(8): C12N5/077C12N5/0735C12N5/10C12Q1/06
CPCC12N5/10C12Q1/06C12N5/067C12N2533/54C12N2501/119C12N2501/12C12N2501/155C12N2501/16C12N2501/237C12N2501/727C12Y207/11
Inventor 粂昭苑白木伸明山口宏之井上智彰中川俊人清川顺平
Owner TOKYO INST OF TECH
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