Composition and application thereof

A composition and serum substitute technology, applied in the direction of microorganisms, animal cells, tissue culture, etc., can solve the problems of easy inactivation of growth factors, high cost of differentiation, and hindrance to the large-scale use of liver precursor cells

Active Publication Date: 2020-08-07
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although the use of growth factors to differentiate stem cells into hepatic precursors is well established, the extensive use of growth factors makes differentiation costly
In addition, growth factors are easily inactivated during long-term storage, which affects the reproducibility of experiments
The above reasons hinder the large-scale clinical use of stem cell-derived hepatic precursor cells

Method used

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  • Composition and application thereof
  • Composition and application thereof
  • Composition and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0118] Example 1 Acquisition of definitive endoderm cells

[0119] Day 0: When the cells grow to 50%-80% confluence, start the directed differentiation experiment, discard the mTeSR medium, wash twice with basal medium RPMI1640, add endoderm induction medium I, endoderm induction medium ⅠBased on RPMI1640, add B27 without insulin at a final concentration of 2%, CHIR99021 at 3 μM, IDE1 at 1 μM, and isoliquiritigenin at 2.5 μM;

[0120] Day 1-2: discard the medium, add endoderm induction medium II, endoderm induction medium II is based on RPMI1640, add B27 without insulin at a final concentration of 2%, 1 μM CHIR99021, 1 μM IDE1 , 2.5 μM isoliquiritigenin;

[0121] Day 3: discard the medium, add endoderm induction medium III, endoderm induction medium III is based on RPMI 1640, add B27 without insulin at a final concentration of 2%, 1 μM CHIR99021, 1 μM IDE1, 0.2 μM dorsomorphin 2HCl;

[0122] Day 4: Obtain definitive endoderm cells, which can be used for identification or su...

Embodiment 2

[0123] Example 2. Acquisition of liver precursor cells

[0124] Day 4-8: Repeat this step every day during the period, discard the medium, add liver precursor medium, the liver precursor medium is based on HepatoZYME-SFM, add B27 at a final concentration of 2%, 2.5 μM isoliquiritigenin , 2.5 μM 4-hydroxychalcone, 0-2 μM Smoothened Agonist HCl;

[0125] Day 9: Obtain liver precursor cells, which can be used for identification or subsequent differentiation;

[0126] Identification of marker genes and proteins: Use RT-PCR to detect genes specifically expressed in liver precursor cells, such as AFP, HNF4α, etc.; use flow cytometry to detect the protein AFP specifically expressed in liver precursor cells; use immunofluorescence to detect liver precursor cells Cell-specific expression of proteins AFP and HNF4α, the morphological changes of cells during differentiation see figure 1 , the results of hepatic progenitor cells (HPCs) induced by small molecule compounds are shown in Fig...

Embodiment 3

[0128] Example 3. Verification of differentiation potential of liver precursor cells

[0129] 1) Differentiation of liver precursor cells into hepatocytes

[0130] Day 9-13; Repeat this step every day during this period, discard the medium, and add liver induction medium Ⅰ. Liver induction medium Ⅰ is based on HepatoZYME-SFM, with a final concentration of 2% B27, 20ng / mL HGF, 20ng / mL KGF;

[0131] Day 14-18: Repeat this step every day during this period, discard the medium, and add liver induction medium II. Liver induction medium II is based on HepatoZYME-SFM, with a final concentration of 2% B27, 0.1 μM dexamethasone, and 10 ng / mL OSM;

[0132] Day 19: Obtain liver cells for identification;

[0133] Morphological changes of cells during differentiation figure 1 ;

[0134] Identification of marker genes and proteins: Use RT-PCR to detect genes specifically expressed in liver cells, such as ALB, AAT, etc.; use immunofluorescence to detect proteins ALB and AAT specifically...

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Abstract

The invention provides a composition and the application thereof. A culture medium prepared from the composition is used for inducing human pluripotent stem cells to be differentiated into liver precursor cells. According to the invention, active components are screened; optimizing component ratios, the method comprises the following steps: adding a GSK3-beta inhibitor, a Nodal activator, a BMP activator, a BMP inhibitor and a Hedgehog activator into differentiated cells; the method has the advantages of simple and efficient process and high positive cell content, reduces the cost required bycell differentiation, is used for related research and application of drug research and development and regenerative medical treatment, and has wide application prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a composition and its application, in particular to a composition and its use for preparing a cell culture medium for inducing human pluripotent stem cells to differentiate into liver precursor cells. Background technique [0002] The liver is the largest organ and gland in the human body, and it undertakes important endocrine and exocrine functions. At present, liver disease has a high morbidity and mortality rate. According to a report in 2014, there are more than 1.3 billion liver disease patients worldwide, and as many as 300 million liver disease patients in my country. Liver diseases include viral hepatitis, alcoholic steatohepatitis, and nonalcoholic steatohepatitis. End-stage liver disease (cirrhosis, liver cancer) is irreversible. And clinically, no effective drugs have been found to treat liver cirrhosis and liver cancer. The above reasons lead to a high mortality rate of...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0672C12N2506/45C12N2506/02C12N2500/30C12N2501/999C12N5/067C12N2501/727C12N2501/155C12N2501/41A61K35/407Y02A50/30C12N2500/05
Inventor李尹雄庄苑琦潘廷才陈彦
OwnerGUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI