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Directional differentiation system and method for liver stem cells

A technology for directed differentiation and hepatic stem cells, applied in biochemical equipment and methods, artificial cell constructs, non-embryonic pluripotent stem cells, etc., which can solve the problems of inability to maintain the differentiation system, long-term in vitro culture, directed differentiation and expansion of hepatic stem cells liver stem cells

Active Publication Date: 2017-04-19
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

) At present, it is still not possible to stably maintain the differentiation system at a specific intermediate developmental stage, such as the tissue stem cell stage
At the same time, there is currently no effective method to induce the differentiation of human pluripotent stem cells into hepatic stem cells that can be stably expanded.
[0005] Therefore, there is still a lack of effective means to expand hepatic stem cells in this field, especially the inability to achieve long-term in vitro culture and directed differentiation of hepatic stem cells under chemically defined culture conditions.

Method used

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  • Directional differentiation system and method for liver stem cells
  • Directional differentiation system and method for liver stem cells
  • Directional differentiation system and method for liver stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Expansion of mouse embryonic liver stem cells

[0055] Take mouse embryos at 12.5 days pregnant, take out the embryonic liver, digest with Accutase (Invitrogen), and inoculate the single cell suspension on 0.5% Matrigel (BD Bioscience) or 20 μg / ml Laminin (Invitrogen) for coating 6-well culture plate, medium is DMEM / F12 (Invitrogen Company), contains insulin-transferrin-sodium selenite mixed solution (Invitrogen Company), 3 μ M CHIR99021 (Tocris Company), 2 μ M E-616452 (Sigma-Aldrich Company ), 5 μM lysophosphatidic acid (Sigma-Aldrich), 0.5 μM sphingosine-1-phosphate (Sigma-Aldrich), 20 ng / mL epidermal growth factor (R&D Systems) and 50 μg / mL human recombinant white protein (R&D Systems). Cells were subcultured with Accutase enzyme when grown to 70% confluency.

[0056] This culture condition can selectively expand embryonic liver stem cells, such as figure 1 As shown, the presence of vascular endothelial cells and fibroblasts could no longer be detected...

Embodiment 2

[0060] Example 2: Expansion of liver stem cells obtained by differentiation of human pluripotent stem cells

[0061] Human pluripotent stem cells H1 and Hues9 cells (Wicell Company) were cultured in 6-well culture plates coated with 0.5% Matrigel, and the medium was DMEM / F12, containing 1% N2 (Invitrogen Company), 1% B27 (Invitrogen Company) and 20ng / ml bFGF (Invitrogen). When the cells grow to 50% confluence, replace the medium with DMEM / F12, B27 (insulin-free), and 100ng / ml Activin A (R&D Systems) for four days to induce cell differentiation into endoderm cells; then replace the medium with DMEM / F12 , containing 1% N2, 1% B27, 5 μM SB431542 (Sigma-Aldrich Company) and 10ng / ml BMP4 (Invitrogen Company) were cultured for 2 days to induce cells to further differentiate into the ventral foregut; then replace the medium with DMEM / F12, Containing 1% N2, 1% B27, 10ng / ml bFGF and 10ng / ml BMP4 cultured for 5 days to induce cells to differentiate into hepatoblasts. Replace the mediu...

Embodiment 3

[0064] Example 3: Inducing hepatic stem cells to differentiate into mature hepatocytes

[0065] Inoculate mouse or human hepatic stem cells cultured in vitro into 6-well culture plates coated with 0.5% Matrigel (10000 cells / well). The medium is DMEM / F12, containing insulin-transferrin-sodium selenite mixture, 20ng / mL hepatocyte growth factor, 20ng / mL Oncostatin M, 100nM dexamethasone, 1μM γ-secretase inhibitor Compound E and 2 μM of transforming growth factor beta receptor inhibitor E-616452.

[0066] After two weeks of in vitro culture, the expression of mature hepatocyte marker molecules was significantly up-regulated; under this induction condition, hepatic stem cells can differentiate into mature hepatocytes expressing and secreting ALB in a high proportion; the differentiated cells have a low density of uptake of acetylation The ability of lipoproteins, while taking up fluorescein-labeled bile acid and secreting bile acid into the intercellular bile duct; differentiated ...

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Abstract

The invention relates to the technical field of biomedical engineering, particularly to a directional differentiation system and method for liver stem cells for long term invitro culture of liver stem cells. The directional differentiation system comprises an expansion culture medium with explicit chemical compositions, which is used for invitro culture of mouse or human liver stem cells, and a differentiation culture medium with explicit chemical compositions, which is used for changing the mouse or human liver stem cells to be mature liver cells through induced differentiation. According to the system and method provided by the invention, liver stem cells from mouse fetal liver tissue or hepatic progenitor cells obtained through human pluripotent stem cell differentiation are selectively expanded, the liver stem cells are cultivated for more than 20 generations under the condition, and stable liver stem cell molecular phenotype can be maintained; further, the cultured mouse or human liver stem cells are changed through induced differentiation of the mouse or human liver stem cells to be mature liver cells with functions of secreting albumin, metabolizing urea and the like.

Description

[0001] The present invention is a divisional application with the filing date of November 12, 2014, the application number: CN201410635815.7, and the title of the invention is "a system and method for long-term in vitro culture and directed differentiation of hepatic stem cells". technical field [0002] The present invention relates to the technical field of biomedical engineering, in particular to a system and method for long-term in vitro culture and directional differentiation of hepatic stem cells, including an expansion medium with defined chemical composition, which is used for in vitro culture of mouse or human hepatic stem cells; and a chemically defined differentiation medium for inducing the differentiation of mouse or human hepatic stem cells into mature hepatocytes. Background technique [0003] In my country, more than 300,000 patients die from liver disease every year, but the annual liver transplantation can only meet the needs of 5% of patients with liver dis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074C12N5/071
CPCC12N5/067C12N2501/12C12N2501/15C12N2501/237C12N2501/39C12N2501/734
Inventor 李文林吕林洁张木子孙平新
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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