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Method used for inducing differentiation of human pluripotent stem cells into hepatocytes using small molecular compound

A technology of human pluripotent stem cells and small molecule compounds, applied in the field of stem cell biology and regenerative medicine, can solve the problems of poor reproducibility, difficult induction, and unstable experimental system.

Active Publication Date: 2018-09-04
THE THIRD AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method has the following defects: the induction time is longer (17 days); the experimental system is unstable, and the cell damage is heavier (1% DMSO has heavier cytotoxicity); the experimental reproducibility is poor (the first stage is only through the regulation of Wnt signal The pathway is difficult to efficiently induce definitive endoderm differentiation; the second stage is difficult to induce definitive endoderm differentiation into hepatic lineage cells only through DMSO)

Method used

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  • Method used for inducing differentiation of human pluripotent stem cells into hepatocytes using small molecular compound
  • Method used for inducing differentiation of human pluripotent stem cells into hepatocytes using small molecular compound
  • Method used for inducing differentiation of human pluripotent stem cells into hepatocytes using small molecular compound

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0152] Example 1 Inducing Human Pluripotent Stem Cells to Differentiate into Hepatocytes

[0153] A method for inducing human pluripotent stem cells to differentiate into hepatocytes (the flowchart of the differentiation process is shown in figure 1 ), divided into the following three steps:

[0154] S1. Inducing Human Pluripotent Stem Cells to Differentiate into Definitive Endoderm

[0155] Method: (0.5% DMSO+human embryonic stem cell medium mTeSR) was pre-cultured for 24 hours, then cultured with (RPMI1640+insulin-free B-27 cell culture supplement+CHIR99021 (3μM)) for 24 hours, and then replaced with insulin-free B-27 RPMI1640 medium with 27 cell culture supplements, continued to culture for 24 hours, after a total of 72 hours of culture.

[0156] The specific plan is:

[0157] (1) Preparation for induction of differentiation 24 hours in advance: Coat a six-well plate with Matrigel and put it in a 37-degree incubator; the confluence of human pluripotent stem cells is 70-9...

Embodiment 2

[0169] (3) Continue culturing for 5 days, and replace fresh medium every day to promote the differentiation of hepatic progenitor cells into hepatocytes. Example 2 Optimization of Conditions for Inducing Human Pluripotent Stem Cells to Differentiate into Definitive Endoderm

[0170] 1. Effects of different basal media on stem cell pluripotency and endoderm differentiation

[0171] Comparison of the effects of two media on stem cell pluripotency and endoderm differentiation

[0172] Medium 1: Essential 8 TM Medium

[0173] Medium 2: 1640B27 medium, RPMI1640 medium containing insulin-free B-27 cell culture supplement, wherein the volume ratio of RPMI1640 medium to insulin-free B-27 cell culture supplement is 49:1.

[0174] 1. Operation method

[0175] (1) Embryonic stem cells H1 were inoculated in a 6-well plate 24 hours in advance, so that the cell fusion degree before induction reached 30-50%;

[0176] (2) When inducing differentiation, add 3 μM CHIR99021 to Essential8 an...

Embodiment 3

[0204] Example 3 Condition optimization for inducing the differentiation of defined endoderm into hepatic progenitor cells

[0205] In order to determine the effect of (A83-01+NaB+DMSO) in inducing the formation of hepatic progenitor cells, a traditional cytokine group (BMP4+FGF4) was designed as a comparison.

[0206] 1. Operation method

[0207] After the embryonic stem cells H1 were induced into defined endoderm according to the optimal scheme above, the medium was replaced with the hepatic cell induction medium: GlutaMAX, non-essential amino acids (NEAA), B-27 cell culture supplement, Fatty acid complex (Chemically Defined Lipid Concentrate) and serum replacement (Knockout TM Serum Replacement) with a volume of 95:1:1:1:1:1. Among them, the traditional cytokine group was supplemented with 10ng / ML of BMP4 and 10ng / ML of FGF4; the small molecule compound induction group was supplemented with 0.5μM A83-01, 200nM NaB and 0.5% DMSO. The induction time was 5 days, and fresh i...

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Abstract

The invention discloses a method used for inducing differentiation of human pluripotent stem cells into hepatocytes using a small molecular compound. The method comprises following steps: differentiation of human pluripotent stem cells into definitive endoderm cells is induced; differentiation of the definitive endoderm cells into hepatic progenitor cells is induced; and differentiation of the hepatic progenitor cells into mature hepatocytes is induced. Compared with the prior art, the method possesses following advantages: in the whole induced differentiation process, the small molecular compound, and culture mediums and additives with clear composition are adopted, using of cytokines and culture medium with unclear composition is avoided, the system is stable, and the repeatability is excellent; the cost of the small molecular compound is lower than that of cytokines, compared with conventional cytokine method, cost is reduced by 2 / 3, so that the whole induced differentiation processis lower in cost, and it is beneficial for further enlarged differentiation applications; the induced differentiation process takes 13 days, the time consumption is the shortest in whole small molecular compound induced differentiation schemes reported home and abroad; the concentration of adopted DMSO is low; and the cytotoxicity is low.

Description

technical field [0001] The invention relates to the fields of stem cell biology and regenerative medicine, in particular to a method for inducing differentiation of human pluripotent stem cells (including human iPS cells and human embryonic stem cells) into liver cells by using small molecule compounds. Background technique [0002] For various end-stage liver diseases, liver transplantation is by far the most effective treatment. However, the serious shortage of liver donors has become a global problem that limits its development. Therefore, it is urgent to find a suitable alternative therapy. Hepatocyte transplantation can alleviate this problem to some extent, but there are also problems such as lack of hepatocyte resources and difficulty in in vitro expansion. In recent years, with the vigorous development of stem cell and regenerative medicine research, a series of advances have brought new hope for humans to overcome the treatment problems of liver diseases. Human p...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/067C12N2501/30C12N2506/02C12N2506/45C12N2500/05C12N2500/30
Inventor 张琪杜聪奉源许燕邱东波
Owner THE THIRD AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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