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Induced differentiation method for efficiently shaping entoderm cells

A technique for finalizing endoderm and inducing differentiation, applied to embryonic cells, biochemical equipment and methods, germ cells, etc., can solve the problems of high differentiation cost, incomplete differentiation, and low differentiation efficiency, and achieve the effect of reducing costs

Pending Publication Date: 2021-03-26
SHANTOU UNIV MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] The purpose of the present invention is to solve the problems of high differentiation cost, low differentiation quality, incomplete differentiation, low differentiation efficiency and insufficient purity of definitive endoderm cells obtained after differentiation in existing differentiation methods, by analyzing the key signals in the DE differentiation process Pathways, optimize the ratio of different small molecules and cytokines, so that hESCs can efficiently differentiate to DE while blocking their differentiation to other lineages, so as to establish an efficient method for induction of definitive endoderm cells, and then obtain more mature and endoderm cells. hepatoid cells

Method used

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  • Induced differentiation method for efficiently shaping entoderm cells
  • Induced differentiation method for efficiently shaping entoderm cells
  • Induced differentiation method for efficiently shaping entoderm cells

Examples

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Effect test

Embodiment 1

[0059] Taking induced differentiated liver sample cells as an example, a method of inducing differentiation of highly active constitutive endoderm cells, including the following steps:

[0060] A, DAY0, adding A (Activin A), C (CHIR999021), T (Torin 2) in stem cells for inducing differentiation, in the first stage medium, and the concentration of A is 20 ng / ml, C is 3 μm, T is 15 nm;

[0061] B, DAY1, the addition of A and D (LDN193189 / DM3189) induced by 20 ng / ml, D of 250 nm, and 2 days in the first stage medium completed 2 days. Stage.

[0062] C, DAY3, after completing the stages of the steering endoderm cells, into induce the hepatic procedure stage, add dimethyl sulfoxide (DMSO) induction differentiation, and completed the induced hepatic procedure stage in the second stage medium, DMSO concentration is 1 %;

[0063] D, DAY8, completing the induced hepatic progenitor cell stage, into induced hepatic cell stage, adding hepatocyte growth factor (HGF), antimotonin M, sugar...

Embodiment 2

[0071] Taking induced differentiated liver sample cells as an example, a method of inducing differentiation of highly active constitutive endoderm cells, including the following steps:

[0072] A, DAY0, adding A (Activin A), C (LY2090314), T (RAPAMYCIN) in stem cells to cultivate in the first stage medium, and a concentration of A is 100 ng / ml, C is 10 μm, T 1000 nm;

[0073] B, DAY1, the addition of A and D (K02288) induced by 10 ng / mL, D of 1000 nm, and 2 days in the first stage medium completed 2 days in the first phase of the cell.

[0074]C, DAY3, after completing the stages of the steering endoderm cells, into induce the hepatic procedure stage, add dimethyl sulfoxide (DMSO) induction differentiation, and completed the induced hepatic procedure stage in the second stage medium, DMSO concentration is 1 %;

[0075] D, DAY8, completing the induced hepatic progenitor cell stage, into induced hepatic cell stage, adding hepatocyte growth factor (HGF), antimotonin M, sugar cort...

Embodiment 3

[0081] Taking induced differentiated liver sample cells as an example, a method of inducing differentiation of highly active constitutive endoderm cells, including the following steps:

[0082] A, DAY0, adding A (Activin A), C (SAR502250), T (mtor inhibitor-3) in stem cells to induce differentiation, in the first stage medium, and the concentration of A is 5 ng / ml, C is 1μm, T is 10 nm;

[0083] B, DAY1, the addition of A and D (LDN193189) induced by 5 ng / mL, D of 10 nm, and completed 2 days in the first stage medium for 2 days to complete the stereotype endoderm cell stage.

[0084] C, DAY3, after completing the stages of the steering endoderm cells, into induce the hepatic procedure stage, add dimethyl sulfoxide (DMSO) induction differentiation, and completed the induced hepatic procedure stage in the second stage medium, DMSO concentration is 1 %;

[0085] D, DAY8, completing the induced hepatic progenitor cell stage, into induced hepatic cell stage, adding hepatocyte growt...

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Abstract

The invention belongs to the biotechnology field, and particularly relates to an induced differentiation method for efficiently shaping entoderm cells. The induced differentiation method comprises thefollowing steps of: A: adding TGF-[beta] activator, GSK-3 inhibitor and mTOR inhibitor into stem cells to carry out induced differentiation, and carrying out culture in a first-stage culture medium;B: adding the TGF-[beta] activator and a BMP type I receptor inhibitor to carry out the induced differentiation, and finishing a stage of shaping the entoderm cells in the first-stage culture medium.By use of the induced differentiation method disclosed by the invention, the problems that an existing differentiation method has a high differentiation cost, low differentiation quality, incomplete differentiation, low differentiation efficiency, unpurified shaped entoderm cells obtained after differentiation and the like can be solved.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and more particularly to a method of inducing differentiation of highly active steering endoderm cells. Background technique [0002] Human primary liver cell (PHH) transplant is one of an important way to effectively treat end-stage liver disease and hereditary liver metabolic diseases. At the same time, due to the important place for the liver is an important place of drug metabolism, human primary liver cells are also a gold standard for detecting drug metabolism and toxicity. PHH demand has a large demand but is limited due to limited source of donor, which is greatly restricted in clinical and scientific applications. In addition, hepatocytes are highly differentiated cells that are easy to divide and lose their hepatocyte function under in vitro culture conditions. Therefore, finding effective and adequate PHH alternatives have significant practical significance. There are currently many researchers...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073C12N5/0735C12N5/071
CPCC12N5/0603C12N5/067C12N2506/02C12N2501/15C12N2501/405C12N2501/155C12N2500/62C12N2501/237C12N2501/18C12N2501/39C12N2500/25C12N2500/38Y02A50/30
Inventor 孙平楠周小玲周奇钟志乾
Owner SHANTOU UNIV MEDICAL COLLEGE
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