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A method for h9c2 cardiomyocyte culture medium to induce iPSCs directed myocardial differentiation

A technology for cardiomyocytes and cell culture, applied in the field of medicine, can solve different problems

Active Publication Date: 2019-09-17
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few reports on the induction of stem cell differentiation by H9C2 cardiomyocyte culture medium, and different types of cells have different induction effects on the same stimulus

Method used

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  • A method for h9c2 cardiomyocyte culture medium to induce iPSCs directed myocardial differentiation
  • A method for h9c2 cardiomyocyte culture medium to induce iPSCs directed myocardial differentiation
  • A method for h9c2 cardiomyocyte culture medium to induce iPSCs directed myocardial differentiation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Preparation of iPSCs

[0020] 1) Preparation of trophoblast layer (MEF): Add 0.1% (volume fraction) gelatin into a T25 culture flask, put it in a cell culture incubator at 37°C for 20 minutes, then suck it off, add 5-6 mL of MEF culture medium preheated at 37°C At the same time, the mouse embryonic fibroblasts (MEF) (purchased from the cell bank of the Chinese Academy of Sciences in Shanghai) were quickly taken out from the liquid nitrogen, placed in a 37°C water bath to melt quickly, and immediately wiped and frozen with 75% alcohol by volume fraction Transfer the cell suspension in the cryopreservation tube to a 15mL centrifuge tube containing MEF culture medium, centrifuge at 1000rpm for 5min, discard the supernatant and resuspend it, add it to a T25 culture bottle, and place it in a CO 2 In a constant temperature incubator, iPSCs can be added to the feeder layer after 24 hours of culture;

[0021] 2) Cultivation and passage of iPSCs: The trophoblast obtai...

Embodiment 2H9

[0022] Example 2 H9C2 cardiomyocyte culture medium induces iPSCs differentiation

[0023] H9C2 cardiomyocytes were resuspended in DMEM medium containing 15% FBS, 50 U / mL penicillin, and 10 μg / mL streptomycin, and inoculated at an appropriate cell density in T25 culture flasks for culture, and passaged for 1-2 days. Aspirate the culture medium before subculture for 3 days, and filter it through a 0.22 μm filter membrane to obtain the induction culture medium, that is, the H9C2 cardiomyocyte culture medium, and set aside.

[0024] Take iPSCs in the logarithmic phase of growth, and use 100×10 6 cells / mL iPSCs hanging drop cultured for 48 hours, inoculated in a six-well plate pretreated with 0.1% gelatin for 1 hour, and the pre-prepared conventional iPSCs culture medium without LIF and H9C2 cardiomyocyte culture medium were mixed at a ratio of 1:0, 1:1. , 1:2 ratio mixed to prepare 15% fetal bovine serum differentiation medium to culture iPSCs. Conventional untreated iPSCs serve...

Embodiment 3

[0026] Example 3 Inverted Microscope and Cellular Immunofluorescence Detection of Multilineage Differentiation Potential Gene Oct-4-EGFP and α-sarcomeric actinin (α-sarcomeric actinin, α-actin) expression

[0027] After induction culture for 14 days, wash with PBS for 3 times, fix with 4% paraformaldehyde, wash with PBS, block with 5% BSA, add rabbit anti-mouse monoclonal primary antibody α-actin (1:500) dropwise, spend 4 nights in PBS Rinse, add PE-labeled goat anti-rabbit IgG (1:50) dropwise, incubate at 37°C in the dark for 30 minutes, rinse with PBS and counterstain the cell nucleus with DAPI for 10 minutes, and fix the slide with resin glue. Observed under a fluorescence microscope, 10 fields of view were observed in each well, a total of 30 fields of view.

[0028] Changes in the expression of EGFP in induced pluripotent stem cells, such as figure 1 As shown, the results show that: iPSCs cell culture medium and H9C2 cardiomyocyte culture medium induce their differentiat...

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Abstract

The invention discloses a method for inducing directional myocardiac differentiation of iPSCs (induced Pluripotent Stem Cells) with a H9C2 myocardial cell culture solution, and belongs to the technical field of medicine. The method comprises the following steps: mixing a conventional iPSCs culture solution from which LIF is removed with the H9C2 myocardial cell culture solution; culturing the iPSCs. The method proves that the H9C2 myocardial cell culture solution can effectively induce directional myocardiac differentiation of the iPSCs. Fluorescence quenching of a multi-directional differentiation potential gene OCT-4 is facilitated. As proved by a cell immunofluorescence experiment, the expression of alpha-actinin can be enhanced. As proved by a qPCR result, the expressions of mRNAs (messenger Ribonucleic Acids) such as beta-MHC, GATA4, Mef2C and NCX-1 are increased remarkably.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a method for inducing iPSCs directional myocardial differentiation with H9C2 cardiomyocyte culture medium. Background technique [0002] Myocardial infarction (MI) is one of the most common diseases that cause death, and once the cardiomyocytes are damaged, they lack the ability to regenerate. Inducing and culturing cardiomyocytes in vitro for heart transplantation has become an important research focus for the treatment of cardiovascular diseases. Induced pluripotent stem cells (iPSCs) have the potential to differentiate into a variety of cells. Although current studies have confirmed that iPSCs can differentiate cardiomyocytes in vitro, their natural differentiation efficiency is not high. The differentiation of IPSCs into cardiomyocytes is regulated by many factors, especially changes in the extracellular microenvironment. At present, there are few reports on th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10
Inventor 郭军江灿
Owner JINAN UNIVERSITY
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