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Method for inducing differentiation of multipotent stem cells into myocardial cells by biogenic amine and application

A technology of pluripotent stem cells and pluripotent stem cells, applied in the field of biomedicine, can solve problems such as no research reports, achieve the effects of enhancing survival rate, facilitating storage, and shortening the time of differentiation

Active Publication Date: 2020-05-22
ZHONGSHAN HOSPITAL FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Histamine is involved in the process of hematopoietic cell generation and nerve cell differentiation, but there is no relevant research report on the role and mechanism of histamine in the differentiation of cardiac stem cells and heart development

Method used

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  • Method for inducing differentiation of multipotent stem cells into myocardial cells by biogenic amine and application
  • Method for inducing differentiation of multipotent stem cells into myocardial cells by biogenic amine and application
  • Method for inducing differentiation of multipotent stem cells into myocardial cells by biogenic amine and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] To detect the critical time window and concentration of histamine to promote the differentiation of iPS into cardiomyocytes.

[0058] Histamine is histamine dihydrochloride.

[0059] Add an appropriate amount of mTESR medium to the culture dish coated with matigel gel, add Y reagent (Y27632 s1049 selleck, ROCK inhibitor 10uM) at a ratio of 1 / 1000, add digested or recovered ips cells to it, mix gently, and observe under the microscope Observe (the cell mass should not be too small or too large) and put it in a 37°C cell culture incubator. Remove the culture medium after 2 hours, add mTESR medium without reagent Y, and subculture once every 4 days, depending on the state of the cells. Differentiation begins when the cell density reaches 80-90%. Remove the medium in the culture dish and wash it again with D-PBS. RPMI1640+B27minus insulin (GIBCO, A18956-01) culture medium was added with 8umol / L CHIR (GSK3β inhibitor s2924selleck), and differentiated for 48 hours. Then r...

Embodiment 2

[0063] Detection of downstream pathways by which histamine promotes the differentiation of iPS into cardiomyocytes.

[0064] According to the above method, on the tenth day of ips differentiation to myocardium, the cells were collected to extract protein and RNA, the expression of histamine receptor 1 and 2 was detected, the expression and phosphorylation of ERK1 / 2, the classic downstream pathway of histamine, were detected, and the ERK1 / 2 Expression and phosphorylation of classical downstream target molecule STAT3.

[0065] According to the above method, on the third day to the fifth day of the differentiation of ips into the myocardium, histamine receptor inhibitor 1, ERK1 / 2 phosphorylation inhibitor, and STAT3 phosphorylation inhibitor were added respectively at the same time as histamine, and the tenth day The beating area, frequency, and expression of cardiomyocyte markers a-MHC and cTNT were analyzed, and the proportion of a-MHC and cTNT double-positive cardiomyocytes w...

Embodiment 3

[0068] To detect the repair effect of histamine-induced differentiation of cardiomyocytes on myocardial infarction.

[0069] BALB / C male mice were anesthetized by isoflurane inhalation, intubated with a 22g venous catheter, then fully anesthetized with 1-2% isoflurane gas, and mechanically ventilated with a positive pressure ventilator. A small left chest incision was made to expose the heart, and the LAD coronary artery was ligated with 8-0 silk thread; histamine or PBS was added on the third to fifth day of ips differentiation to the myocardium, and after the tenth day of cell differentiation, the cells were digested and centrifuged to collect the cells with red dye ( Dil, excitation / emission = 644 / 665 nm, Sigma-Aldrich) for labeling. Then 30 μL of cell suspension (2x10 5 ) into the marginal zone of the infarcted myocardium, and each mouse was injected at three sites. To reduce immune rejection in MI mice, Cyspin was provided after injecting cells into MI mice. Two weeks ...

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Abstract

The invention provides a method for promoting and inducing differentiation and maturation of multipotent stem cells into myocardial cells by using biogenic amine and application. According to the method, histamine is added into an in-vitro culture for inducing differentiation of the multipotent stem cells into the myocardial cells at a fixed time point so as to induce differentiation and maturation of the multipotent stem cells into the myocardial cells. The invention also provides a method for detecting the time that the histamine promotes ips to differentiate into the myocardial cells, and the concentration of the myocardial cells. The method is mature, simple to operate and environmentally friendly. The cells obtained by the method have characteristic cell surface markers and morphological markers of the myocardial cells, can spontaneously and periodically contract, and are suitable for various applications such as heart disease research, drug screening, cell treatment and the like.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method and application for obtaining human cardiac muscle precursor cells and mature cardiac muscle cell lines. Background technique [0002] At present, there are two main methods for inducing myocardial differentiation. One is induced pluripotent stem cells (iPSCs) monolayer cell culture method, that is, iPSCs are cultured in a petri dish covered with Matrigel. Molecular compounds to modulate key signaling pathways in cardiac differentiation to obtain beating cardiomyocytes. The other is to form a structure containing multiple types of cells similar to an embryonic structure, that is, the embryoid body method (EBs). The EBs method induces myocardial differentiation. After iPSCs are purified, they are cultured in suspension for several days to induce the formation of EBs, and then transferred to Adherent cultures were performed on gelatin-coated dishes. There are many types of cel...

Claims

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Application Information

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IPC IPC(8): C12N5/077C12Q1/02A61P9/10
CPCC12N5/0657G01N33/5014G01N33/5044A61P9/10C12N2501/82C12N2506/45
Inventor 杨向东丁素玲朱小伟
Owner ZHONGSHAN HOSPITAL FUDAN UNIV
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