Engineering human ventricular heart muscles based on a highly efficient system for purification of human pluripotent stem cell-derived ventricular cardiomyocytes

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0651-x) contains supplementary material, which is available to authorized users.

Tissue-engineered human embryonic stem cell-containing cardiac patches: evaluating recellularization of decellularized matrix

“…of the CMs increased about 40% to 1.6 μm, which is at the upper range of the sarcomere size in human fetal cardiac myocytes (1-1.7 μm). [25][26][27][28] Li et al 19 also showed an increase in sarcomere length when PSC-derived CMs were cultured over dECM, although their reported values (1-2.5 μm) were above the physiologic range for human CMs in vivo, suggesting overstretch of the cells in their studies.…”

“…This finding supports previous data showing electrical coupling of CMs that were cultured on the surface of dECM. 18,19 Because hESC-CMs typically have an immature phenotype in vitro, 20,21 some investigators have seeded these cells on dECM and other biomaterials to induce maturation. [22][23][24] We reasoned that incorporation of the cells into the matrix would be superior to surface coating because of the greater probability of matrix-cell interactions.…”

“…This finding supports previous data showing electrical coupling of CMs that were cultured on the surface of dECM. 18 , 19 …”

Mending a broken heart: current strategies and limitations of cell-based therapy

“…On the other hand, a stable transgenic hPSC line harboring fluorescent reporter under the transcriptional control of human myosin light chain-2V promoter (MLC2V) [130][131][132] and chick ovalbumin upstream promoter transcription factor II (COUP-TFII) [133] were developed to isolate ventricular and atrial cardiomyocytes, respectively. Despite the robust and high efficiency in enriching specific subtypes after cardiac differentiation of PSCs, the use of virus-based vector, again, has raised safety issues including immunogenicity and insertional mutagenesis risk that hinder their application in future clinical treatment.…”

Subtype-specific cardiomyocytes for precision medicine: Where are we now?

“…First, a number of ventricular-specific reporter lines have been created to enrich the ventricular cardiomyocyte subtype (Table S1) [98]. In these reporter lines, a fluorescence protein is cloned into the endogenous MLC2v locus or driven by an MLC2v promoter for selection of ventricular-like cardiomyocytes [99,100]. This reporter can also be combined with a second cardiac marker such as NKX2.5 for double selection [101].…”