Method for inducing fetal hepatic stem cells to differentiate into mature hepatic cells in three-step sequential mode

A technology for inducing differentiation and stem cells, applied in the biological field, can solve the problems of low model efficiency and poor accuracy, achieve the effect of inhibiting the activity of transaminases and restoring the level of liver albumin

Pending Publication Date: 2019-01-22
刘卫辉
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, various induced differentiation models have

Method used

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  • Method for inducing fetal hepatic stem cells to differentiate into mature hepatic cells in three-step sequential mode
  • Method for inducing fetal hepatic stem cells to differentiate into mature hepatic cells in three-step sequential mode
  • Method for inducing fetal hepatic stem cells to differentiate into mature hepatic cells in three-step sequential mode

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] like figure 1 As shown, the present invention provides a three-step sequential method for inducing fetal liver stem cells to differentiate into mature hepatocytes, comprising the following steps:

[0047] (1) Obtaining and identification of fetal liver stem cells:

[0048] 1) Isolation, cultivation and expansion of fetal liver stem cells mainly include the following steps: a. Using Percoll discontinuous density gradient centrifugation to enrich rat fetal liver cells; b. Using jet lag attachment and jet lag digestion to culture fetal liver cells Purification; c, using Percoll continuous density gradient centrifugation to enrich rat fetal liver stem cells.

[0049] 2) Identification of fetal liver stem cells: the stem cells were identified by immunohistochemistry and immunofluorescence multiple antibody staining, wherein the stem cell marker used was CD133.

[0050] (2) Induced differentiation of fetal liver stem cells:

[0051] a) Collect normal liver tissue at low te...

Embodiment 2

[0057] like figure 1 As shown, the present invention provides a three-step sequential method for inducing fetal liver stem cells to differentiate into mature hepatocytes, comprising the following steps:

[0058] (1) Obtaining and identification of fetal liver stem cells:

[0059] 1) Separation, culture and expansion of fetal liver stem cells mainly include the following steps: a. Using Percoll discontinuous density gradient centrifugation to enrich rat fetal liver cells; b. Using jet lag attachment and jet lag digestion to culture fetal liver cells performing purification; c, using Percoll continuous density gradient centrifugation to enrich rat fetal liver stem cells.

[0060] 2) Identification of fetal liver stem cells: the stem cells were identified by immunohistochemistry and immunofluorescence multiple antibody staining, wherein the stem cell marker used was EpCAM.

[0061] (2) Induced differentiation of fetal liver stem cells:

[0062] a) Collect normal liver tissue a...

Embodiment 3

[0068] like figure 1 As shown, the present invention provides a three-step sequential method for inducing fetal liver stem cells to differentiate into mature hepatocytes, comprising the following steps:

[0069] (1) Obtaining and identification of fetal liver stem cells:

[0070] 1) Separation, culture and expansion of fetal liver stem cells mainly include the following steps: a. Using Percoll discontinuous density gradient centrifugation to enrich rat fetal liver cells; b. Using jet lag attachment and jet lag digestion to culture fetal liver cells performing purification; c, using Percoll continuous density gradient centrifugation to enrich rat fetal liver stem cells.

[0071] 2) Identification of fetal liver stem cells: the stem cells were identified by immunohistochemistry and immunofluorescence multiple antibody staining, wherein the stem cell marker used was CD49f.

[0072] (2) Induced differentiation of fetal liver stem cells:

[0073] a) Collect normal liver tissue a...

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Abstract

The invention discloses a method for inducing fetal hepatic stem cells to differentiate into mature hepatic cells in a three-step sequential mode, and belongs to the technical field of biology. The method comprises the following steps that (1) the fetal hepatic stem cells are obtained and identified, specifically, (a) the fetal hepatic steam cells are separated and amplified, and (b) the fetal hepatic stem cells are identified; (2) the fetal hepatic steam cells are induced to differentiate, specifically, (a) a tissue suspension prepared from normal hepatic tissue which is ground and homogenized is collected on dry ice at a low temperature, supernatant is collected after centrifuging and added into a Williams'E cell culture medium, and the fetal hepatic stem cells in the step (1) are cultivated to obtain hepatic progenitor cells, (b) the cells obtained in the step (a) are continuously cultivated through the Wiliams'E culture medium added with a Notch signal channel inhibitor to obtain hepatocyte-like cells, and (c) the cells obtained in the step (b) are cultivated through a rapid cell expansion culture medium added with HGF to obtain functional hepatic cells. Through the method, thefetal hepatic stem cells obtained early can be induced into the functional hepatic cells rapidly, accurately and efficiently.

Description

technical field [0001] The invention belongs to the technical field of biotechnology, and specifically relates to a three-step sequential method for inducing the differentiation of fetal liver stem cells into mature hepatocytes. Background technique [0002] Orthotopic liver transplantation is the gold standard for the treatment of end-stage liver disease. However, the shortage of liver sources has become the biggest bottleneck in the clinical application of hepatocyte transplantation, limiting its effective implementation. Therefore, finding a suitable and abundant source of hepatocytes is a hot spot in the field of biology. At the same time, a large number of studies have confirmed that cell therapy is one of the effective methods for the treatment of end-stage liver diseases (End-stage liver diseases, ESLDs), and the application prospect is very bright. Fetal liver stem / progenitor cells (FLSPCs) have the advantages of high survival rate, small proliferation and high saf...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/0735
CPCC12N5/067C12N5/0672C12N2501/12C12N2501/734
Inventor 刘卫辉王涛任丽娜冯亚星
Owner 刘卫辉
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