Abstract
BackgroundMost infarctions occur in the left anterior descending coronary artery and cause myocardium damage of the left ventricle. Although current pluripotent stem cells (PSCs) and directed cardiac differentiation techniques are able to generate fetal-like human cardiomyocytes, isolation of pure ventricular cardiomyocytes has been challenging. For repairing ventricular damage, we aimed to establish a highly efficient purification system to obtain homogeneous ventricular cardiomyocytes and prepare engineered human ventricular heart muscles in a dish.MethodsThe purification system used TALEN-mediated genomic editing techniques to insert the neomycin or EGFP selection marker directly after the myosin light chain 2 (MYL2) locus in human pluripotent stem cells. Purified early ventricular cardiomyocytes were estimated by immunofluorescence, fluorescence-activated cell sorting, quantitative PCR, microelectrode array, and patch clamp. In subsequent experiments, the mixture of mature MYL2-positive ventricular cardiomyocytes and mesenchymal cells were cocultured with decellularized natural heart matrix. Histological and electrophysiology analyses of the formed tissues were performed 2 weeks later.ResultsHuman ventricular cardiomyocytes were efficiently isolated based on the purification system using G418 or flow cytometry selection. When combined with the decellularized natural heart matrix as the scaffold, functional human ventricular heart muscles were prepared in a dish.ConclusionsThese engineered human ventricular muscles can be great tools for regenerative therapy of human ventricular damage as well as drug screening and ventricular-specific disease modeling in the future.
Highlights
Most infarctions occur in the left anterior descending coronary artery and cause myocardium damage of the left ventricle
Insertion of the neomycin or Enhanced green fluorescent protein (EGFP) selection marker after the myosin light chain 2 (MYL2) locus in Human embryonic stem cell (hESC) by Transcription activator-like effector nuclease (TALEN)-mediated homologous recombination To insert the neomycin/EGFP selection marker under the C-terminus of the MYL2 gene located on chromosome 12, we used TALEN-mediated genomic editing techniques in this study
Final DNA sequencing of the genomic sequence of the MYL2 allele confirmed the correct insertion of the neomycin or EGFP cassette (Additional file 3: Figure S2)
Summary
Most infarctions occur in the left anterior descending coronary artery and cause myocardium damage of the left ventricle. Current pluripotent stem cells (PSCs) and directed cardiac differentiation techniques are able to generate fetal-like human cardiomyocytes, isolation of pure ventricular cardiomyocytes has been challenging. Largescale production of human cardiomyocytes is achievable through directed differentiation of hPSCs to the cardiac lineage [4,5,6,7], which is able to provide abundant human cardiomyocytes for bioengineering human heart tissues and for cell therapy of myocardial damage. Cardiac differentiation of hPSCs results in multiple subtypes of cells, including ventricular-like, atrial-like, and sinus nodal-like cardiomyocytes as well as cells of other lineages. For the purpose of human heart tissue engineering or repairing ventricular muscle damage, using a homogeneous population of ventricular-specific myocytes would be more attractive
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