Preparation method and application of human induced hepatic-specified differentiation stem cells

A technology for inducing differentiation and stem cells, applied in artificially induced pluripotent cells, biochemical equipment and methods, hepatocytes, etc., can solve the problems of weak regeneration ability, long differentiation cycle, insufficient differentiation maturity, etc., to reduce costs , the effect of reducing differentiation time, enhancing regenerative capacity and maturity

Pending Publication Date: 2021-09-17
丁建强
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the diagnosis and treatment methods for these liver diseases have made great progress in recent years, there is still a lack of effective treatment methods for end-stage liver disease-cirrhosis. Liver transplantation is currently the only clinical treatment method, but there is a lack of sufficient and suitable methods. Allogeneic donor livers a long-standing problem for patients with advanced liver disease requiring transplantation
[0003] Cell transplantation is a popular technology developed in recent years, especially stem cell transplantation, due to the advantages of wide source of stem cells, in vitro expansion and industrial production, etc. Differentiation (hepatic-specified differentiation) still has the disadvantages of long differentiation cycle, high cost, insufficient differentiation maturity and weak regenerative ability, etc., which need to be overcome and solved.

Method used

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  • Preparation method and application of human induced hepatic-specified differentiation stem cells
  • Preparation method and application of human induced hepatic-specified differentiation stem cells
  • Preparation method and application of human induced hepatic-specified differentiation stem cells

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1. Preparation method of human-derived induced liver-differentiated stem cells

[0026] Such as figure 1 As shown, this embodiment provides a method for preparing human induced liver-differentiated stem cells, which includes the following steps:

[0027] Step 1. Stem cell culture

[0028] Coating of cell culture plate: Use a pipette to draw 6ml of basal medium (DMEM / F-12) into a 15ml falcon test tube, take out 0.25ml of matrigel (Corning, NY, USA) from the refrigerator, and put the matrigel Mix well in 0.5ml basal culture medium, dissolve and mix with the remaining basal culture medium; add 1ml mixed basal culture medium to each well of the 6-well plate, and rotate the six-well plate to make the gel evenly cover the plate on a clean bench for 1 hour. Aspirate off the gel solution and wash once with DMEM / F-12 to obtain a coated cell culture plate.

[0029] Add 1 million human iPS stem cells to 2ml mTeSR TM 1 medium, mix well. Then culture in a matrigel-coat...

Embodiment 2

[0035] Example 2. Transplantation of Human-derived Induced Liver to Differentiated Stem Cells

[0036] In this example, 35 male AATD transgenic mice (6-8 weeks old) were selected for cell transplantation experiment.

[0037] Among them, the experimental group transplanted 1 million hepatically differentiated stem cells prepared in Example 1 into the liver of transgenic mice by intramedullary injection in the spleen. Among them, AATD transgenic mice were produced with the assistance of Saiye (Suzhou) Biotechnology Company.

[0038] The blank control was injected with normal saline, and one week before transplantation, the mice were provided with drinking water containing 1 μg / g / day tacrolimus FK506 (Sigma-Aldrich, St. Louis, MO, USA) to resist rejection.

[0039] test results:

[0040] Such as figure 2 As shown, 1 month after the transplantation, human serum albumin (human serum albumin, HSA) immunofluorescent staining of transgenic mouse liver pathological sections showed 50...

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Abstract

The invention discloses a preparation method and application of human induced hepatic-specified differentiation stem cell. The method comprises the following steps of inoculating human induced pluripotent stem cells (hiPSC) on a six-hole cell culture dish coated with matrigel, and culturing by using an mTeSRTM1 stem cell culture solution; when the growth and cloning of the stem cells are obvious, adding activin A into the stem cell culture solution to treat the human iPS stem cells to carry out induced differentiation of endoderm so as to obtain endoderm cells; and culturing the endoderm cells in an RPMI + B27 culture medium containing bone morphogenetic protein 4 (BMP-4) and fibroblast growth factor-2 (FGF-2), so that the endoderm cell is directionally differentiated, and the hepatic-specified differentiation stem cell is obtained. According to the stem cell hepatic-specified differentiation transplantation method, the hepatic-specified differentiation time is shortened, the regeneration capacity and maturity of the stem cells in the liver of the alpha-1 antitrypsin deficiency transgenic mice are enhanced, the cost is reduced, and the technology has wide prospects in clinical application.

Description

technical field [0001] The invention relates to the technical field of biopharmaceuticals, in particular to a preparation method and application of human-derived induced liver differentiation stem cells. Background technique [0002] Acute and chronic liver diseases caused by various reasons are still one of the main factors affecting people's health, including various viral hepatitis, fatty liver, autoimmune hepatitis and hereditary liver disease. With the improvement of diagnostic technology and understanding of diseases in recent years, more and more liver diseases such as autoimmune hepatitis and genetic liver diseases are no longer rare. At the same time, with the improvement of people's living standards, the intake of alcohol and sugar shows a significant upward trend, and alcoholic liver disease / non-alcoholic liver disease / fatty liver has become a public health problem that cannot be ignored. These liver diseases can develop into liver fibrosis, liver cirrhosis and e...

Claims

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Application Information

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IPC IPC(8): C12N5/10A61K35/545A61P1/16
CPCC12N5/0696C12N5/0672A61K35/545A61P1/16C12N2510/00C12N2501/16C12N2501/155C12N2501/115
Inventor 丁建强
Owner 丁建强
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