A method for inducing differentiation of amniotic epithelial stem cells into functional liver cells and its application

A technology for inducing differentiation and stem cells, applied in cell dissociation methods, cell culture active agents, biochemical equipment and methods, etc., can solve the problems of difficult clinical practice of cells, tumorigenicity of stem cells, limited sources of bone marrow and umbilical cord blood, etc. Achieve the effect of enriching the source of cells, inhibiting the activity of transaminase, and restoring the level of albumin in the liver

Active Publication Date: 2021-07-20
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the limited sources of bone marrow and umbilical cord blood, the ethical issues and tumorigenicity of embryonic stem cells, and the tumorigenicity of induced pluripotent stem cells make it difficult for these cells to be used in clinical practice.

Method used

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  • A method for inducing differentiation of amniotic epithelial stem cells into functional liver cells and its application
  • A method for inducing differentiation of amniotic epithelial stem cells into functional liver cells and its application
  • A method for inducing differentiation of amniotic epithelial stem cells into functional liver cells and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1. Isolation, culture, expansion and GFP marker of amniotic membrane epithelial stem cells (HAESCs):

[0090] Under the premise of obtaining the consent of the newborn's family members, the fresh amniotic membrane was isolated. After 2 hours of antibiotic treatment, microbiological detection and safety detection of infectious disease pathogens were carried out. After the amniotic membrane was digested by trypsin / EDTA (0.25%) in a water bath at 37°C for 1 hour, stop solution was added. After centrifugation, the supernatant was removed, and the amnion epithelial stem cell culture medium was placed in 5% CO 2 , 95% humidity, and cultured in a 37°C incubator. Change the medium after 2-3 days to remove unattached cells, and then change the medium every 2 days. When the confluence of the cells reached 80%, trypsin / EDTA was used for passage. The amniotic epithelial stem cells of passage 3 were transfected with GFP using lentivirus. Immunofluorescence and flow cyto...

Embodiment 2

[0176] Example 2. In vivo and in vitro tumorigenicity detection of amniotic membrane epithelial stem cells

[0177] 1. In vivo and in vitro tumorigenicity detection of amniotic membrane epithelial stem cells

[0178]1) Take the third-generation amnion epithelial cells in good growth condition, which are characterized by vigorous growth, large cell bodies, clear nuclei, rich cytoplasm, and strong refraction under the microscope, and digest them with trypsin-EDTA (0.25% ) digestion, under the microscope, when the cells became a single round shape, the digestion was terminated with DMEM medium containing 10% (volume concentration) fetal bovine serum, centrifuged after gentle blowing, and the cell pellet was obtained, washed with PBS (PBS without calcium and magnesium ions) solution) and washed twice (the purpose is to wash away trypsin, fetal bovine serum and other substances);

[0179] 2) Inoculate the amnion epithelial stem cells in step 1) on soft agar, and after culturing fo...

Embodiment 3

[0187] Example 3. Induced differentiation of amniotic membrane epithelial stem cells into hepatocytes and its function detection

[0188] The specific operating procedures are as follows:

[0189] 1. Directed induction of amniotic epithelial stem cells to differentiate into hepatocytes in vitro

[0190] 1) Take the well-growing cells of the third generation and digest them with trypsin-EDTA digestion solution (0.25%). The DMEM medium was digested, centrifuged after gently pipetting to obtain cell pellets, and washed twice with PBS (PBS washing solution without calcium and magnesium ions) (the purpose is to wash away trypsin, fetal bovine serum and other substances);

[0191] 2) The above-mentioned cell pellets washed with PBS were inoculated into Matrigel pretreated 6-well plates for induction culture, and the seeding cell density was 3×10 5 cells / well, add 2ml of amniotic epithelial stem cell general medium to each well, at 37°C, 5% CO 2 , 95% saturated humidity incubator ...

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Abstract

The invention discloses a method for using human amniotic epithelial stem cells to induce differentiation into liver cells with biological functions, comprising the following steps: 1) isolation, cultivation, amplification, identification and GFP (Green Fluorescent Protein) marker of amniotic epithelial stem cells; 2) Differentiation in vitro: In the experiment, the 3rd to 7th passages of amniotic epithelial stem cells with GFP markers were used, and the system 1 containing induction medium Ⅰ, Ⅱ, and Ⅲ or the system containing induction medium Ⅳ, Ⅴ, and Ⅵ were selected. 2 to induce differentiation. Liver cell-like cells that can express liver cell-specific markers and have normal liver cell functions can be obtained by induction and differentiation of system 1 (induction time of 14-15 days) or system 2 (induction time of 22-23 days); 3) in vivo Transplantation: Transplantation of induced liver-like cells into acute liver injury model mice can significantly alleviate acute liver injury, restore liver function of mice and increase survival rate of mice.

Description

technical field [0001] The invention relates to a method for inducing differentiation of liver cells by using amniotic membrane epithelial stem cells. Background technique [0002] Orthotopic liver transplantation is the gold standard for the treatment of end-stage liver disease. However, the shortage of liver sources is the biggest bottleneck in clinical hepatocyte transplantation. Therefore, finding a suitable and abundant source of hepatocytes is a hotspot in the field of biology. In 1981, Evans et al. established the first mouse ES cell line and used it as a model to confirm that mouse ES cells could not only be induced to differentiate into hepatic progenitor cells in vitro, but also further differentiate into hepatocytes with biological functions. Animal experiments have shown that when hepatocytes differentiated in vitro are transplanted into liver injury model mice, the transplanted cells can fuse to the liver tissue and alleviate the liver injury of the mice, and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071
CPCC12N5/067C12N2500/24C12N2500/32C12N2500/44C12N2501/11C12N2501/115C12N2501/12C12N2501/33C12N2509/00
Inventor 辛洪波柳全文李婧嫄刘倩玉任康康
Owner NANCHANG UNIV
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