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Method for acquiring highly-purified conduction cell derived from multipotent stem cell on basis of HCN4 double reporter genes

A technology of pluripotent stem cells and dual reporter genes is applied in the field of obtaining highly purified conducting cells derived from pluripotent stem cells based on the HCN4 double reporter gene, which can solve the problems of low differentiation efficiency of conducting cells and efficient acquisition of conducting cells, so as to avoid adverse effects. , the effect of wide application prospects

Inactive Publication Date: 2019-07-09
XIAMEN UNIV
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  • Application Information

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Problems solved by technology

However, due to the low differentiation efficiency of conductive cells, there is no effective method to efficiently obtain conductive cells (Michael A.Laflamme and Charles E.Murry et al,.2011, Heart regeneration)

Method used

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  • Method for acquiring highly-purified conduction cell derived from multipotent stem cell on basis of HCN4 double reporter genes
  • Method for acquiring highly-purified conduction cell derived from multipotent stem cell on basis of HCN4 double reporter genes
  • Method for acquiring highly-purified conduction cell derived from multipotent stem cell on basis of HCN4 double reporter genes

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Embodiment Construction

[0062] The present invention will be further described below, but the implementation of the present invention is not limited thereto.

[0063] 1. Cultivation and identification of hiPSC-HCN4-GFP-puromycin ( figure 1 ) and hESC-HCN4-GFP-puromycin ( figure 2 ).

[0064] a) Culture in E8 medium on a Matrigel-coated petri dish, add 10 μM ROCK inhibitor, and change the E8 medium the next day;

[0065] b) When the density reaches 80% again, use Oct4 / SSEA4 to verify the dryness.

[0066] 2. Targeted induction of hiPSC-HCN4-GFP-puromycin ( image 3 ) and hESC-HCN4-GFP-puromycin ( Figure 4 ) differentiate into conducting cells

[0067] a) On the fourth day, when the cell growth density reached 80%-90%, the culture medium was replaced with a differentiation medium, which was the first day of differentiation, and CHIR (concentration 0.6 μL / mL), BMP4 (concentration 10 ng / mL), ACTA ( Concentration 6ng / mL) and bFGF (concentration 5ng / mL), maintain 72h;

[0068] b) On the third day ...

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Abstract

The invention discloses a method for acquiring a highly-purified conduction cell derived from a multipotent stem cell on the basis of HCN4 double reporter genes. The method includes: using puromycin to screen and differentiate cells; detecting conduction cell proportion according to GFP expression flow type. The multipotent stem cell with a double-reporter system and comprises an inducing multipotent stem cell and an embryonic stem cell. hPSC contains a fluorescent screening marker and an antibiotic screening marker and analyzes a method for verifying the conduction cell proportion through antibiotic screening and fluorescent sorting. The conduction cell comprises a sinus node, a front internodal tract, a rear internodal tract, an upper interatrial tract, an atrioventricular node, a Kent-His bundle, a left bundle branch, a right bundle branch and a Purkinje's fibrocyte. The conduction cell can be used for screening small molecules to promote conduction cell differentiation, studying asignal pathway in the process of promoting conduction cell differentiation, studying conduction cell drug toxicology and conducting clinical cell transplanting.

Description

technical field [0001] The present invention relates to the culture of hPSC and the differentiation of conduction cells, in particular to a method for obtaining highly purified conduction cells derived from pluripotent stem cells based on the HCN4 double reporter gene based on reporter cell line resistance screening and flow cytometry analysis. Background technique [0002] Arrhythmia is caused by the abnormal function of conduction cells such as the sinoatrial node, which leads to abnormal excitation, or the excitation of conduction cells occurs outside the sinoatrial node, and the conduction of the excitation is slow, blocked, or conducted through abnormal channels, that is, the origin and (or) of cardiac activity. Conduction disorder leads to abnormal frequency and (or) rhythm of heart beat, which can easily lead to sudden death, heart failure and stroke, which greatly affects people's daily life. The best clinical treatment is to install a pacemaker. However, chest surg...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02
CPCG01N33/5005
Inventor 徐秀琴林瑜刘靖
Owner XIAMEN UNIV
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