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Method for inducing direct reprogramming of human fibroblasts into hepatic cells by small molecules

A technology of human fibroblasts and fibroblasts, applied in artificial cell constructs, animal cells, vertebrate cells, etc., can solve the problems of low differentiation efficiency, high cost, and incomplete differentiation of stem cells

Inactive Publication Date: 2019-03-22
TRANSCEND CYTOTHERAPY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing directed differentiation methods have one or more of the following defects: such as low differentiation efficiency, low transformation purity, lack of function of differentiated hepatocytes, possible immune rejection, and potential cancer risk due to incomplete differentiation of stem cells And the high cost, etc., cannot meet the clinical needs
However, transdifferentiation of human fibroblasts into hepatocytes using only small chemical molecules has not been reported

Method used

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  • Method for inducing direct reprogramming of human fibroblasts into hepatic cells by small molecules
  • Method for inducing direct reprogramming of human fibroblasts into hepatic cells by small molecules
  • Method for inducing direct reprogramming of human fibroblasts into hepatic cells by small molecules

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preparation example Construction

[0144] The preparation method of the composition of the present invention is determined according to the dosage form to be prepared and the route of administration. Those skilled in the art can use the conventional preparation method of the pharmaceutical composition after referring to the combination and proportioning provided by the present invention. Compositions of the present invention are prepared.

[0145] It should be understood that although in the specific embodiment, the inventor has listed several composition forms, those skilled in the art can also deduce from this that any other combination form of the present invention also has outstanding effects.

[0146] The inventors firstly confirmed that the small molecule composition of the present invention can be used to develop and prepare drugs or drug formulations for preventing, improving or treating liver fibrosis or chronic liver failure. When used to prevent, improve or treat liver fibrosis, or chronic liver fail...

Embodiment 1

[0181] Example 1. Small molecule composition for direct reprogramming (transdifferentiation) of human fibroblasts induced by small molecules into hepatocytes, and preparation of medium and reagents thereof

[0182] Design the following composition or medium, which can be prepared according to molar concentration or weight concentration:

[0183] 1. Formula of small molecule composition for fibroblast transdifferentiation liver cell

[0184] (1) Fibroblast transdifferentiation liver cell composition 1

[0185] GSK3β inhibitor CHIR-99021: final concentration 5uM;

[0186] TGFβ inhibitor SB431542: final concentration 2uM;

[0187] G9aHMTase inhibitor BIX01294: final concentration 3uM;

[0188] (2) Fibroblast transdifferentiation liver cell composition 2

[0189] GSK3β inhibitor CHIR-99021: final concentration 6uM;

[0190] TGFβ inhibitor SB431542: final concentration 0.5uM;

[0191] G9aHMTase inhibitor BIX01294: final concentration 5uM;

[0192] (3) Fibroblast transdiffere...

Embodiment 2

[0223] Example 2, Fibroblast Transdifferentiation Hepatocyte Medium 6 Induces Fibroblast Transdifferentiation Hepatocyte

[0224] 1. Fibroblast transdifferentiation hepatocyte culture

[0225] The fibroblasts were suspended in the basal cell medium DMEM containing 10% calf serum, plated, and cultured at 37°C.

[0226] After the fibroblasts adhered to the wall, the original medium was discarded and replaced with fibroblast-transdifferentiated liver cell medium 6, cultured at 37°C, and the medium was changed every 3 days; passage was performed once every 7 days.

[0227] 2. Subculture of fibroblast transdifferentiated hepatocytes

[0228] Subculture steps: Discard the original culture medium, wash once with PBS, add cell digestion solution to digest the cells, stop cell digestion at 37°C for 3 minutes, centrifuge, discard the supernatant, resuspend the cell pellet, and plate at a ratio of 1:2. Replace the fibroblast transdifferentiation liver cell medium 6 for culture, and cha...

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Abstract

The invention relates to a method for inducing direct reprogramming (transdifferentiation) of human fibroblasts into hepatic cells by small molecules. The invention discloses the method for inducing the direct reprogramming (transdifferentiation) of the human fibroblasts into the hepatic cells on the basis of a chemically induced direct cell reprogramming mechanism and a small-molecule compositionthereof, and the small-molecule composition can be developed and prepared into drugs or prodrugs used for treating hepatopathy. The invention further discloses a cell transdifferentiation medium anda reagent prepared from the small-molecule composition.

Description

technical field [0001] The invention belongs to the interdisciplinary fields of cell biology, stem cell biology (cell reprogramming), medicine and pharmacy; more specifically, the invention relates to a method for direct reprogramming (transdifferentiation) of human fibroblasts into hepatocytes induced by small molecules. Background technique [0002] China is a country with a large number of liver diseases. There are a large number of patients with hepatitis A, hepatitis B, fatty liver, alcoholic liver, liver cirrhosis (liver fibrosis), and acute and chronic liver failure caused by various liver diseases, chemical drugs, trauma, etc., The condition is critical and the case fatality rate is high. Liver transplantation is an effective method for the treatment of end-stage liver disease or liver failure. However, the lack of liver sources makes many patients, especially those with acute liver failure, lose the chance of treatment. Hepatocyte transplantation, bioartificial liv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/067C12N2501/15C12N2501/70C12N2501/72C12N2506/1307A01K2207/20A01K2227/105A01K2267/03A61P1/16C12N2501/065C12N2501/727C12N2501/999
Inventor 张培霖
Owner TRANSCEND CYTOTHERAPY CO LTD
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