Method for inducing human embryo stem cell for directional differentiation into hepatocyte and special culture medium

A technology of human embryonic stem cells and differentiation medium, applied in artificial cell constructs, biochemical equipment and methods, tissue culture, etc., can solve the problems of short survival period, limited treatment methods, difficult expansion, etc.

Active Publication Date: 2009-08-05
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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Problems solved by technology

At present, the treatment of such diseases mainly depends on orthotopic liver transplantation. However, a series of problems such as the extreme shortage of donors, death related to transplantation and surgery itself, life-long administration of immunosuppressants and the fatal complications caused by them are extremely serious. The earth limits the widespread development of this treatment
Hepat

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  • Method for inducing human embryo stem cell for directional differentiation into hepatocyte and special culture medium
  • Method for inducing human embryo stem cell for directional differentiation into hepatocyte and special culture medium
  • Method for inducing human embryo stem cell for directional differentiation into hepatocyte and special culture medium

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Embodiment 1

[0045] Example 1. Directed differentiation of human embryonic stem cells into hepatocytes in vitro

[0046] The following media were used in the examples:

[0047] Differentiation medium I (embryoid body medium): on the basis of DF-12 medium (Gibco), 20% (15-25% is acceptable, volume percentage concentration, V / V) serum substitute (SR, Gibco), 0.1 mM (0.05-0.15 mM is acceptable) β-mercaptoethanol (Gibco) and 1 mM (0.5-1.5 mM is acceptable) glutamine (Gibco), pH 7.2-7.6. To increase nutrition, 0.5-1.5% (volume percent concentration, V / V) of non-essential amino acids (HyClone) can be added to the differentiation medium I.

[0048] Differentiation medium II (definitive endoderm differentiation medium): on the basis of DF-12 medium, 1% (0.5-1.5% is acceptable, volume percentage concentration, V / V) fetal bovine serum (FBS) is added, 0.1mM (0.05-0.15mM is acceptable) β-mercaptoethanol, 1mM (0.5-1.5mM is acceptable) glutamine and 100ng / mL activin A (activin A), pH 7.2-7.6.

[0049...

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Abstract

The invention discloses a method for differentiating human embryonic stem cells into hepatic cells by in vitro inducement, and a special culture medium thereof. The in vitro inducement method comprises the following steps: 1). human embryonic stem cells are suspended by a differential medium I; cell suspension is filled in a low absorbability germ culture dish, and is cultured for one to three days in 5 percent CO2 at 37 DEG C to form an embryoid body which is cultured for two to four days in a differentiating culture medium under the same conditions and is differentiated into sizing endoblast cells; 2). the sizing endoblast cells differentiated by the step 1 are inoculated to human fetal liver stroma cell culture layers of expressing basic fibroblast growth factors and are added with a differentiating culture medium III; after the sizing endoblast cells are cultured for four to six days in 5 percent CO2 at 37 DEG C, the culture medium III is replaced by a differentiating culture medium IV; and the sizing endoblast cells are continuously cultured for five to twenty days under the same conditions so as to obtain liver cells. The invention has the advantages of simple operation, low cost, high differentiating efficiency, and the like, exerts important action in medical fields, and has wide application prospects.

Description

technical field [0001] The invention relates to a method for inducing differentiation of human embryonic stem cells and its special medium, in particular to a method for inducing human embryonic stem cells to differentiate into hepatocytes in vitro and its special medium. Background technique [0002] End-stage liver disease caused by various reasons (viruses, drugs, tumors and genetic diseases, etc.) seriously threatens human health. According to statistics, more than 110,000 patients die of end-stage liver failure each year nationwide. At present, the treatment of such diseases mainly depends on orthotopic liver transplantation. However, a series of problems such as the extreme shortage of donors, death related to transplantation and surgery itself, life-long administration of immunosuppressants and the fatal complications caused by them are extremely serious. The earth limits the widespread development of this treatment. Hepatocyte transplantation and bioartificial live...

Claims

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Application Information

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IPC IPC(8): C12N5/08C12N5/071
Inventor 裴雪涛裴海云王韫芳白宗良杨印祥习佳飞岳文南雪白慈贤
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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