Application of hepatocyte model derived in vitro by stem cell

A technology of hepatocytes and stem cells, applied in the fields of developmental biology and pharmacology, can solve problems such as the application of stem cell-derived hepatocyte models, etc.

Inactive Publication Date: 2008-10-15
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The in vitro differentiation of mouse ES cells into hepatocytes truly reproduces the process of hepatocyte differentiation in vivo, and has been widely used in various studies as an alternative model. So far, no stem cell-derived hepatocyte model has been used in the study of energy metabolism in liver development.

Method used

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  • Application of hepatocyte model derived in vitro by stem cell
  • Application of hepatocyte model derived in vitro by stem cell
  • Application of hepatocyte model derived in vitro by stem cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1: The ES cell in vitro derivation liver cell model is constructed by the following steps

[0018] 1. Hanging drop culture

[0019] ES cells were digested with trypsin to make a cell density of 2×10 4 cells / ml of single-cell suspension, inoculated on the inner surface of the lid of a 10-cm diameter petri dish with 30 μl, added 10 ml of D-Hanks solution into the petri dish, and then carefully covered the lid with many small drops to make the lid The small drops on the surface were hung upside down to form hanging drops (each hanging drop contained about 600 cells), and cultured in an incubator for 5 days. The differentiation medium was high-glucose DMEM, containing 0.1 mmol·L -1 β-mercaptoethanol, non-essential amino acids and 20% fetal bovine serum without LIF factor.

[0020] 2. Adhesive culture

[0021] Transfer the embryoid body (embryoid body, EB) formed by hanging drop culture with a pipette under the microscope to a 24-well plate coated with gelatin, a...

Embodiment 2

[0024] Example 2: In vitro derivation of ES cells into liver cells Mitochondrial membrane potential changes

[0025] ES cells were collected, EBs were digested into single cells, EBs were adhered to the wall and differentiated into hepatocyte samples, added JC-1 dye and incubated for 15 minutes, washed three times with PBS, and observed under a fluorescent inverted microscope.

[0026] The results showed that almost all ES cells exhibited green fluorescence, suggesting that the mitochondrial membrane potential was low; EB digested cells showed red fluorescence, while almost all hepatocytes cultured by adherent differentiation showed red fluorescence, which suggested that with the Differentiation and maturation, the cell membrane potential gradually increased with the maturation of liver cell mitochondria. see figure 2 , where (A) a single ES cell, ×100; (B) a single cell after digested EB, ×100; (C) the 12th day of EB adherent culture differentiation, ×50; (D) the 12th day o...

Embodiment 3

[0027] Example 3: Expression of PPARs family genes when ES cells are derivatized into hepatocytes in vitro

[0028] Collect ES cells, EBs, and hepatocytes on days 6, 12, and 18 of adherent differentiation, and extract total mRNA according to the instructions of Trizol reagent. See Table 1 for primer sequences and experimental conditions:

[0029] Table 1. Primer sequences and PCR reaction conditions

[0030]

[0031] PCR products were electrophoresed on 1.5% agarose gel, observed and photographed with a gel imaging system.

[0032] see image 3 , the experimental results showed that PPAR-α, PPAR-β and PPAR-γ genes were expressed during the differentiation of ES cells into liver cells, and all of them had a certain degree of up-regulation trend.

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Abstract

The invention provides an application of mouse embryonic stem cell-derived in vitro hepatic cell model, which is mainly in screening cell differential agent with peroxisome proliferator-activated receptor as the target. The stem cell-derived in vitro hepatic cell model can also be used to explain an expression profile and functions of PPARs family related to the energy metabolism during liver development and to reveal the nature of a life phenomenon of embryonic liver development, even to construct a highly-efficient screening model with PPARs family as the target to regulate the primary screening and evaluation of the potency of energy supply to livers and develop a new energy metabolism inducer for improving the differentiation of stem cell to hepatic cells. The invention provides a new application of the embryonic stem cell-derived in vitro hepatic cell model which can be used as a surrogate model for use in research of energy metabolism during the liver development to explain the expression traits of PPARs related to the energy system development of liver mitochondrion, and can also be used to screening cell differential agents for stimulating the energy metabolism of liver cells.

Description

technical field [0001] The invention belongs to the field of developmental biology and pharmacology, and relates to the use of a model, in particular to the application of a stem cell in vitro derivation liver cell model in the study of energy metabolism in liver development, which can be used to use peroxisome proliferation-activated receptors as Target cell differentiation agent screening. Background technique [0002] The in vitro directed differentiation of mouse embryonic stem cells (ES cells) into hepatocytes can simulate the formation and development of hepatocytes in vivo, and is rich in a large amount of biological information dependent on liver development. In this system, the expression of many key genes in hepatocytes is higher than that of primary mature hepatocytes; it has the functions of albumin synthesis and ammonia degradation; its metabolic function is also stronger than that of primary hepatocytes, and the maintenance time is longer, so both structure and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06C12Q1/02C12N5/071
Inventor 朱丹雁楼宜嘉吴佳莹严洁萍沃燕波
Owner ZHEJIANG UNIV
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