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Method for creating hepatocyte by human embryo stem cell external evoked differentiation

A technique for inducing differentiation of human embryonic stem cells, applied in the field of spheroid structure-embryoid body, which can solve the problems of unfavorable and sufficient contact, and achieve the effect of facilitating morphological observation

Inactive Publication Date: 2008-02-06
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the existing method of inducing differentiation is to directly inoculate EB as a sphere, which will cause the cells to stack, which is not conducive to full contact, and the morphology can only be observed through the cells that climb out of the periphery during operation. Therefore, the current Some methods of inducing differentiation still need to be improved

Method used

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  • Method for creating hepatocyte by human embryo stem cell external evoked differentiation
  • Method for creating hepatocyte by human embryo stem cell external evoked differentiation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1, the culture of human ES cell and the formation of EB

[0030] Human ES cell line H9 (purchased from Wicell Company) was inoculated on mouse embryonic fibroblast feeder layer (Kunming mice, serum, 0.25 trypsin-0.02% EDTA and DMEM medium (purchased from Hyclone Company) ( The mitotic activity was inactivated by gamma ray irradiation, seeded on a 0.1% gelatin-coated culture dish), and the culture medium was serum-free human ES cell culture medium: Knockout DMEM medium (purchased from Gibco Company), containing 20% ​​serum Substitute (purchased from Gibco), 0.1mM β-mercaptoethanol (purchased from Gibco), 1mM glutamine (purchased from Gibco), 4ng / ml basic fibroblast growth factor (bFGF, purchased from Chemicon) , 50IU / mL penicillin, 50IU / mL streptomycin, 1% non-essential amino acids (purchased from Gibco company). 37 ° C, 5% CO 2 cultured under the same conditions, and the culture medium was changed every day. The cells grew confluently for about a week, incu...

Embodiment 2

[0033] Example 2, EB digested into single cells

[0034] Under the microscope, use the mouth pipette technique to select EBs that have been cultured for 7 days and have a relatively uniform size, and place them in two bacteria dishes containing phosphate buffered saline (PBS) to wash twice, and then transfer (use the mouth pipette to transfer under the microscope) Digest in a bacteria dish pre-added with 0.25% trypsin (Amresco), 37°C, 5% CO 2 Incubate under the conditions for about 5 minutes until all single cells can be formed by gently pipetting, then add medium containing 10% serum (HYCLONE) to stop digestion for 1 minute, transfer the single cell suspension to a 10ml centrifuge tube with a dropper, 1000rpm , centrifuge for 5 minutes, and discard the supernatant. Example 3, Induced Differentiation of Single Cells to Hepatocytes

Embodiment 3

[0035] The pellet obtained by centrifugation in Example 2 was resuspended in the hepatocyte-inducing conditioned medium, and the components of the medium were IMDM medium (Sigma), containing 20% ​​fetal bovine serum (Gibco), 50 nM dexamethasone (Sigma), 0.0625 U / ml human insulin (Lilly France S.A.S). Then inoculated onto culture plates pre-coated with type I collagen. 37°C, 5% CO 2 Cultured under conditioned conditions, the culture medium was changed every two days, and the directional induction and differentiation effect of conditioned cultured was observed.

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Abstract

The present invention provides a method that embryo of human embryonic stem cells (HES) is directionally induced and divided into liver cells. First, the biological property of high degree of self-regeneration multiplication of HES cell is made use of, the collagenase IV digestion is adopted for transfer of culture and augmentation, thereby obtaining a plurality of stem cells; second, the HES cell is inoculated into a cell utensil with lower adsorbability to form a mature imitated embryonic plant (EB); third, the mature cystic EB is digested with 0.25 tryptic enzyme to 0.02 percent EDTA to a single cell, and then is inoculated to a tissue culture dish which is packed with I-shaped collagen in advance, solution culture is induced under the condition of dexamethasone and human trypsin, and the differentiation towards the liver cell direction is observed and judged. The prevent invention provides the method that the HES is efficiently divided into liver cells, at the same time, the method makes the follow-up judging work more simply and more efficient, and lay a foundation for the stem cellular transplantation or biologic artificial liver curing the disease such as serious hepatitis, hepatic failure and so on.

Description

technical field [0001] The present invention relates to a method of utilizing the biological characteristics of human embryonic stem cells (human embryonic stem cells, hES cells) to obtain a spheroid structure similar to early embryos - embryoid body (embryoid body, EB), combined with dexamethasone and human insulin I Collagen-type method to realize the technology of directing and efficiently inducing the differentiation of human embryonic stem cells into hepatocytes in vitro. Background technique [0002] End-stage liver disease caused by various reasons (viruses, drugs, tumors and genetic diseases, etc.) seriously threatens human health. At present, its treatment 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. This restricts the widespr...

Claims

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Application Information

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IPC IPC(8): C12N5/08C12N5/071C12N5/0735
Inventor 裴雪涛裴海云王韫芳杨印祥习佳飞施双双刘雨潇南雪陈琳白慈贤
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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