Method for inducing human embryo stem cell differentiation to liver cell and the special-purpose medium

A technology of human embryonic stem cells and differentiation medium, which is applied in the field of inducing human embryonic stem cells to differentiate into liver cells, and achieves the effects of high safety, broad application prospects and short cycle.

Active Publication Date: 2006-12-27
PEKING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, although the differentiated cells obtained in this way express the marker proteins of hepatocytes and have some functions, it is still questionable to what extent they are similar to normal hepatocytes

Method used

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  • Method for inducing human embryo stem cell differentiation to liver cell and the special-purpose medium
  • Method for inducing human embryo stem cell differentiation to liver cell and the special-purpose medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1. Inducing human embryonic stem cells to differentiate into liver cells

[0025] 1. Routine culture of human embryonic stem cells

[0026] Reagent:

[0027] PBS: weigh 8g NaCl, 0.2g KCl, 1.44g Na 2 HPO 4 and 0.24g KH 2 PO 4 , plus ddH 2 O was adjusted to 1000 mL, and the pH value of the solution was adjusted to 7.4 with HCl.

[0028] 200mM glutamine storage solution (200×): Weigh 0.292g glutamine, dissolve it in 10mL PBS, filter and sterilize, aliquot and store in a -70°C refrigerator.

[0029] 2M β-mercaptoethanol (20000×): Take 1mL of 14.3M β-mercaptoethanol, add 6.15mL PBS to dilute, and filter to sterilize.

[0030] Human embryonic stem cell medium (HESM): 20% serum replacement (Knock-out SerumReplacement, KSR), 1mM glutamine, 0.1mM β-mercaptoethanol, 1% non-essential amino acids (Non-essentialAminoAcids) (Gibco, USA) , 4ng / mL basic fibroblast growth factor (bFGF), with ddH 2 O was adjusted to 1000mL.

[0031] 0.5 mg / mL Dispase: Weigh 10 mg of Dis...

Embodiment 2

[0059] Embodiment 2, detection of differentiated cells

[0060] Reagent:

[0061] PBST: PBS solution containing 0.2% (volume percentage) Triton X100.

[0062] Blocking solution: PBST solution containing 2-3% goat serum (or horse serum).

[0063] Secondary antibody diluent (0.1% BSA solution): weigh 0.1 g of bovine serum albumin (BSA), and dissolve it in 100 mL of PBS.

[0064] 1. Detection of initial differentiation from definitive endoderm cells to hepatocytes

[0065] During the induction of differentiation, the cell morphology gradually changed to that of typical hepatocytes, see figure 1 Figures A-D in the figure (Figures A, B, C, and D are the cell morphology after 3, 4, 6, and 8 days of differentiation culture, respectively), while the cells in the control group that spontaneously differentiate without adding factors have completely different shapes. See figure 1 Figure E in.

[0066]The differentiation state of human embryonic stem cells induced by 3-day different...

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Abstract

The invention discloses a method of inducing the human embryo stem cell differentiation to liver cell and its special culture medium. The said culture medium comprises the following two culture mediums: differentiation medium I which is prepared by adding 80-120ng / activinA in RPMI 1640 culture medium and differentiation medium II which is prepared by adding 20-60ng / mL desmocyte growth factor and 15-25ng / mL bone formation protein in HCM medium. The culture medium is of positive components and high use safety; the revulsion is of short periodic time, high differentiation rate and stable safety, and has a wide application foreground.

Description

technical field [0001] The invention relates to a method for inducing differentiation of human embryonic stem cells and a special medium thereof, in particular to a method for inducing differentiation of human embryonic stem cells to liver cells and a special medium for the same. Background technique [0002] Stem cells are a type of cells with self-renewal ability and multi-lineage differentiation potential. Such cells can maintain the size or expansion of their own cell population through cell division, and can further differentiate into more mature cell types. According to the differentiation potential of stem cells, they can be divided into totipotent stem cells (such as embryonic stem cells, which can differentiate into all adult tissue cells, and even develop into complete individuals), pluripotent stem cells (with multi-directional differentiation potential, which can differentiate into cells other than Other types of tissue cells other than own tissue cells, such as...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08C12N5/0735
Inventor 邓宏魁丁明孝蔡军张晶
Owner PEKING UNIV
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