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Culture medium for inducing directional differentiation of human embryonic stem cells into liver-like tissue, inducing method and application

A technology of human embryonic stem cells and differentiation medium, which is applied in the field of medium for inducing human embryonic stem cells to differentiate into liver-like tissues, and can solve the problems of single liver cells, inability to further differentiate into tissue organoids, and complicated culture

Active Publication Date: 2018-10-02
TANGYI HLDG(SHENZHEN) LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the induced liver buds do not have intrahepatic bile ducts, and are not from the same kind of cells, and the culture is complicated
[0005] The patents with publication numbers CN1884494A, CN101497872A, CN101962630A and CN105385651A all disclose methods and / or special media for inducing human embryonic stem cells to differentiate into hepatocytes, but they are all induction differentiation schemes for a single germ layer, and only a single hepatocyte can be obtained , cannot be further differentiated into tissue organoids, and there are still major limitations in medical applications

Method used

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  • Culture medium for inducing directional differentiation of human embryonic stem cells into liver-like tissue, inducing method and application
  • Culture medium for inducing directional differentiation of human embryonic stem cells into liver-like tissue, inducing method and application
  • Culture medium for inducing directional differentiation of human embryonic stem cells into liver-like tissue, inducing method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Example 1 Medium for Inducing Directed Differentiation of Human Embryonic Stem Cells into Liver-like Tissues in Vitro

[0049] The culture medium for inducing the directed differentiation of human embryonic stem cells into liver-like tissue, including the following five mediums used in sequence:

[0050] (1) Differentiation medium I, directional endoderm differentiation medium A: based on RPIM1640 medium, 25% MTeSR™1, 100ng / mL Activin A (ActivinA), and 10ng / mL bone morphogenetic protein were added 4 (BMP4), 2% B27 (-insulin);

[0051] (2) Differentiation Medium II, i.e. Directed Endoderm Differentiation Medium B: Based on RPIM1640 medium, 25% MTeSR™1, 100ng / mL Activin A (ActivinA), 2% B27 (-insulin) were added ;

[0052] (3) Differentiation medium III, that is, liver-directed differentiation medium: on the basis of RPIM1640 medium, 25% MTeSR™1, 20ng / mL bone morphogenetic protein-2 (BMP2), 30ng / mL human fibroblast Cell growth factor-4 (FGF4), 2% insulin-free B27 (-ins...

Embodiment 2

[0055] Example 2 Induction of Directed Differentiation of Human Embryonic Stem Cells into Liver-like Tissues in Vitro

[0056] Using the method of the present invention to induce the directed differentiation of human embryonic stem cells (humanembryonicstemcells, hEScells) into liver-like tissues in vitro, the specific process includes the following steps:

[0057] 1. Obtainment of Committed Endoderm Cells

[0058] Day 1~2:

[0059] (1) Human embryonic stem cells were induced 2 to 3 days after H1 passage, and cells with good growth status were selected for differentiation experiments;

[0060] (2) Discard the human embryonic stem cell medium (MTeSR™1), and wash twice with DMEM / F12;

[0061] (3) Replace with differentiation medium I (endoderm induction medium A), at 37°C, 5% CO 2 conditions for two days.

[0062] Day 3-4:

[0063] (4) Discard yesterday's medium, replace with differentiation medium II (endoderm induction medium B), and culture for two days to obtain endoder...

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Abstract

The invention discloses a special culture medium for inducing directional differentiation of human embryonic stem cells into liver-like tissue in vitro, an inducing method and application, belonging to the technical field of culturing of stem cells. The special culture medium comprises a differentiation culture medium I, a differentiation culture medium II, a differentiation culture medium III, adifferentiation culture medium IV and a differentiation culture medium V which are successively used. The invention also discloses the application of the differentiation culture medium in induction ofdirectional differentiation of the human embryonic stem cells into the liver-like tissue in vitro, and a concrete method thereof. According to the invention, the liver-like tissue can be cultured byutilizing the culture medium and the method; a conventional scheme of differentiation induction of a single germ layer is changed; differentiation of multiple germ layers is realized; and the multiplegerm layers are further differentiated into a tissue-like organ rather than a single cell. The liver-like tissue prepared by using the method provided by the invention has potential clinical application value and can provide an ideal research platform for the fields of drug screening, liver development, etc.

Description

technical field [0001] The invention belongs to the technical field of culturing stem cells, and more specifically relates to a culture medium for inducing human embryonic stem cells to differentiate into liver-like tissues, an induction method and application. Background technique [0002] At present, there is no specific therapy and technical means for the clinical treatment of liver failure (such as hepatic necrosis, cirrhosis, etc.) and liver-related genetic diseases. Orthotopic liver transplantation is recognized as an effective treatment for advanced liver disease. However, serious shortage of donor liver sources and transplant rejection limit the widespread development of this treatment. Therefore, the rise of stem cell technology and its application is expected to become one of the most potential methods to solve advanced liver failure. [0003] Pluripotent stem cells, including embryonic stem cells (Embryonicstemcells, ESCs) and induced pluripotent stem cells (ind...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0671C12N2501/119C12N2501/117C12N2500/25C12N2500/76C12N2500/38C12N2500/36C12N2506/02C12N2501/999C12N2501/998C12N2501/50C12N2501/155C12N2501/16
Inventor 徐安龙吴芬芳吴迪任勇陈尚武
Owner TANGYI HLDG(SHENZHEN) LTD
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