Pluripotent stem cell-hereditary cardiomyopathy cardiac muscle cell and preparation method thereof

A technology of pluripotent stem cells and cardiomyocytes, applied in the field of human pluripotent stem cells-hereditary cardiomyopathy cardiomyocytes and its preparation, can solve the problems of difficult long-term survival, scarce sources, and difficulty in obtaining hereditary cardiomyopathy cardiomyocytes, etc., to achieve Effects from a wide range of sources

Inactive Publication Date: 2015-11-11
FUDAN UNIV
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

[0005] It is very difficult to obtain the diseased human cardiomyocytes of inherited cardiomyopathy from the patient's heart, because the source is very rare and it is not easy to survive for a long time in vitro, and these diseased cardiomyocytes are already in the late stage of the disease, so there is not much research on the early mechanism of the disease. Multi-function
Although transgenic animal models of hereditary cardiomyopathy have also been established and have played an important role in the study of hereditary

Method used

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  • Pluripotent stem cell-hereditary cardiomyopathy cardiac muscle cell and preparation method thereof
  • Pluripotent stem cell-hereditary cardiomyopathy cardiac muscle cell and preparation method thereof
  • Pluripotent stem cell-hereditary cardiomyopathy cardiac muscle cell and preparation method thereof

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Effect test

Embodiment 1

[0078] 1) Construction of TALEN or CRISPR / CAS9 plasmids and homologous gene donor plasmids with corresponding mutations

[0079] On the basis of the backbone plasmid, construct TALEN or CRISPR / CAS9 plasmids targeting different mutation targets and homologous gene donor plasmids with corresponding mutations. For example, HCM (DE160, R403Q), DCM (DK210, E244D), RCM (R145W, I79N), LVNC (L301Q, R131W), ARVC (Q62K, D1373A), etc., TALEN and mutant homologous gene plasmid construction (such as figure 1 , figure 2 as shown in the legend);

[0080] 2) Activity detection for different target TALENs or CRISPR / CAS9 plasmids

[0081] Transfect 293T cells with TALEN or CRISPR / CAS9 plasmids, extract the genomic DNA of the surviving cells after Puromycin screening for PCR, and detect the activity by observing the set peak strength, T7E1 mismatch enzyme digestion efficiency and TA clone sequencing ratio (such as image 3 shown);

[0082] 3) Co-electrotransfect human pluripotent stem cells...

Embodiment 2

[0094] The contractile function and electrophysiological characteristics of the human pluripotent stem cells-hereditary cardiomyopathy cardiomyocytes obtained by the above method can be detected by multi-electrode microarray; further, different small molecule chemical drugs or Chinese herbal medicines are added to the cardiomyocyte culture environment Preparations, etc., can detect changes in their contractile function and electrophysiological function through a multi-electrode microarray system, predict the possible impact of the tested small molecule chemical drugs or Chinese herbal preparations on the function of human heart tissue, and screen new and effective therapeutic drugs; for example Figure 5 It shows that the present invention adds epinephrine, a catecholamine small molecule drug epinephrine, to the human embryonic stem cells-hypertrophic cardiomyopathy cardiomyocytes carrying the TNNT2DE160 mutation, and it is observed that epinephrine has a positive inotropic effe...

Embodiment 3

[0096] The cardiotoxicity of drugs is a key factor hindering the development of drugs. Drug cardiotoxicity screening based on transgenic cells and animals often has some false negative and false positive results, resulting in unnecessary death, and the cardiotoxic effect of drugs is more likely to occur in the presence of Patients with underlying heart disease. Through the above method, a series of human pluripotent stem cells-hereditary cardiomyopathy cardiomyocytes can be constructed, which contain all the ion channels on the surface of human normal cardiomyocytes, which can reflect the complex interactions between ion channels and more accurately predict the cardiotoxicity of drugs. Become a good platform for drug toxicity screening.

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Abstract

The invention belongs to the field of researching and application of biomedicine and particularly relates to human pluripotent stem cell-hereditary cardiomyopathy cardiac muscle cells and a preparation method thereof. The invention provides a human hereditary cardiomyopathy-pluripotent stem cell, which is constructed by means of TALEN or CRISPR/CAS9 genome editing technology. The human hereditary cardiomyopathy cardiac muscle cell can be combined with any scaffold materials to culture various in-vitro human hereditary cardiomyopathy cardiac muscle tissues. The human hereditary cardiomyopathy cardiac muscle cells in the invention have disease phenotypes and electrophysiology change being similar as the cardiac muscle cells of human hereditary cardiomyopathy patients. The human hereditary cardiomyopathy cardiac muscle cells are wide in sources and can be cultured in-vitro for a long time. The invention provides excellent tools and platforms for researching an effective new therapy means and researching an effective corresponding treatment medicine.

Description

technical field [0001] The invention belongs to the field of biomedical research and application, and specifically relates to human pluripotent stem cells-hereditary cardiomyopathy cardiomyocytes and preparation technology thereof. Background technique [0002] Human hereditary cardiomyopathy includes five kinds of cardiomyopathy: hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), restricted cardiomyopathy, left ventricular noncompaction cardiomyopathy (left ventricular noncompaction, LVNC) and arrhythmogenic right ventricular cardiomyopathy (ARVC) are major heart diseases caused by multiple single gene mutations. These single genes include genes expressing sarcomere proteins, cytoskeletal proteins, ion channel proteins, lamins, mitochondrial proteins such as myosin heavy chain MHC, myosin light chain MLC, troponin TNNT2, myosin Binding protein MBPC3, sodium ion channel protein SCN5a, potassium ion channel protein KCNH2, nuclear lamin lamin, etc. (see Morita H...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10
Inventor 孙宁詹永坤李宾
Owner FUDAN UNIV
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