A number of genetic mutations is associated with cardiomyopathies. A mutation in the coding region of the phospholamban (PLN) gene (R14del) is identified in families with hereditary heart failure. Heterozygous patients exhibit left ventricular dilation and ventricular arrhythmias. Here we generate induced pluripotent stem cells (iPSCs) from a patient harbouring the PLN R14del mutation and differentiate them into cardiomyocytes (iPSC-CMs). We find that the PLN R14del mutation induces Ca2+ handling abnormalities, electrical instability, abnormal cytoplasmic distribution of PLN protein and increases expression of molecular markers of cardiac hypertrophy in iPSC-CMs. Gene correction using transcription activator-like effector nucleases (TALENs) ameliorates the R14del-associated disease phenotypes in iPSC-CMs. In addition, we show that knocking down the endogenous PLN and simultaneously expressing a codon-optimized PLN gene reverses the disease phenotype in vitro. Our findings offer novel strategies for targeting the pathogenic mutations associated with cardiomyopathies.

The generation of human ventricular cardiomyocytes from human embryonic stem cells and/or induced pluripotent stem cells could fulfill the demand for therapeutic applications and in vitro pharmacological research; however, the production of a homogeneous population of ventricular cardiomyocytes remains a major limitation. By combining small molecules and growth factors, we developed a fully chemically defined, directed differentiation system to generate ventricular-like cardiomyocytes (VCMs) from human embryonic stem cells and induced pluripotent stem cells with high efficiency and reproducibility. Molecular characterization revealed that the differentiation recapitulated the developmental steps of cardiovascular fate specification. Electrophysiological analyses further illustrated the generation of a highly enriched population of VCMs. These chemically induced VCMs exhibited the expected cardiac electrophysiological and calcium handling properties as well as the appropriate chronotropic responses to cardioactive compounds. In addition, using an integrated computational and experimental systems biology approach, we demonstrated that the modulation of the canonical Wnt pathway by the small molecule IWR-1 plays a key role in cardiomyocyte subtype specification. In summary, we developed a reproducible and efficient experimental platform that facilitates a chemical genetics-based interrogation of signaling pathways during cardiogenesis that bypasses the limitations of genetic approaches and provides a valuable source of ventricular cardiomyocytes for pharmacological screenings as well as cell replacement therapies. STEM CELLS

Self-renewable human pluripotent stem cells (hPSCs) serve as a potential unlimited ex vivo source of human cardiomyocytes (CMs) for cell-based disease modeling and therapies. Although recent advances in directed differentiation protocols have enabled more efficient derivation of hPSC-derived CMs with an efficiency of ∼50%-80% CMs and a final yield of ∼1-20 CMs per starting undifferentiated hPSC, these protocols are often not readily transferrable across lines without first optimizing multiple parameters. Further, the resultant populations are undefined for chamber specificity or heterogeneous containing mixtures of atrial, ventricular (V), and pacemaker derivatives. Here we report a highly cost-effective and reproducibly efficient system for deriving hPSC-ventricular cardiomyocytes (VCMs) from all five human embryonic stem cell (HES2, H7, and H9) and human induced PSC (hiPSC) (reprogrammed from human adult peripheral blood CD34(+) cells using nonintegrating episomal vectors) lines tested. Cardiogenic embryoid bodies could be formed by the sequential addition of BMP4, Rho kinase inhibitor, activin-A, and IWR-1. Spontaneously contracting clusters appeared as early as day 8. At day 16, up to 95% of cells were cTnT(+). Of which, 93%, 94%, 100%, 92%, and 92% of cardiac derivatives from HES2, H7, H9, and two iPSC lines, respectively, were VCMs as gauged by signature ventricular action potential and ionic currents (INa(+)/ICa,L(+)/IKr(+)/IKATP(+)); Ca(2+) transients showed positive chronotropic responses to β-adrenergic stimulation. Our simple, cost-effective protocol required the least amounts of reagents and time compared with others. While the purity and percentage of PSC-VCMs were comparable to a recently published protocol, the present yield and efficiency with a final output of up to 70 hPSC-VCMs per hPSC was up to 5-fold higher and without the need of performing line-specific optimization. These differences were discussed. The results may lead to mass production of hPSC-VCMs in bioreactors.