Aims Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have proven valuable for studies in drug discovery and safety, although limitations regarding their structural and... Show moreAims Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have proven valuable for studies in drug discovery and safety, although limitations regarding their structural and electrophysiological characteristics persist. In this study, we investigated the electrophysiological properties of Pluricyte (R) CMs, a commercially available hiPSC-CMs line with a ventricular phenotype, and assessed arrhythmia incidence by I-Kr block at the single-cell and 2D monolayer level.Methods and results Action potentials were measured at different pacing frequencies, using dynamic clamp. Through voltage-clamp experiments, we determined the properties of I-Na, I-Kr, and I-CaL. Intracellular Ca2+ measurements included Ca2+-transients at baseline and during caffeine perfusion. Effects of I-Kr block were assessed in single hiPSC-CMs and 2D monolayers (multi-electrode arrays). Action-potential duration (APD) and its rate dependence in Pluricyte (R) CMs were comparable to those reported for native human CMs. I-Na, I-Kr, and I-CaL revealed amplitudes, kinetics, and voltage dependence of activation/inactivation similar to other hiPSC-CM lines and, to some extent, to native CMs. Near-physiological Ca2+-induced Ca2+ release, response to caffeine and excitation-contraction coupling gain characterized the cellular Ca2+-handling. Dofetilide prolonged the APD and field-potential duration, and induced early afterdepolarizations. Beat-to-beat variability of repolarization duration increased significantly before the first arrhythmic events in single Pluricyte (R) CMs and 2D monolayers, and predicted pending arrhythmias better than action-potential prolongation.Conclusion Taking their ion-current characteristics and Ca2+ handling into account, Pluricyte (R) CMs are suitable for in vitro studies on action potentials and field potentials. Beat-to-beat variability of repolarization duration proved useful to evaluate the dynamics of repolarization instability and demonstrated its significance as proarrhythmic marker in hiPSC-CMs during I-Kr block. Show less
Unexpected induction of arrhythmias in the heart is still one of the major risks of new drugs despite recent improvements in cardiac safety assays. Here we address this in a novel emerging assay... Show moreUnexpected induction of arrhythmias in the heart is still one of the major risks of new drugs despite recent improvements in cardiac safety assays. Here we address this in a novel emerging assay system. Eleven reference compounds were administrated to spontaneously beating clusters of cardiomyocytes from human pluripotent stem cells (hPSC-CM) and the responses determined using multi-electrode arrays. Nine showed clear dose-dependence effects on field potential (FP) duration. Of these, the Ca(2+) channel blockers caused profound shortening of action potentials, whereas the classical hERG blockers, like dofetilide and d,l-sotalol, induced prolongation, as expected. Unexpectedly, two potent blockers of the slow component of the delayed rectifier potassium current (I(Ks)), HMR1556 and JNJ303, had only minor effects on the extracellular FP of wild-type hPSC-CM despite evidence of functional I(Ks) channels. These compounds were therefore re-evaluated under conditions that mimicked reduced "repolarization reserve," a parameter reflecting the capacity of cardiomyocytes to repolarize and a strong risk factor for the development of ventricular arrhythmias. Strikingly, in both pharmacological and genetic models of diminished repolarization reserve, HMR1556 and JNJ03 strongly increased the FP duration. These profound effects indicate that I(Ks) plays an important role in limiting action potential prolongation when repolarization reserve is attenuated. The findings have important clinical implications and indicate that enhanced sensitization to repolarization-prolonging compounds through pharmacotherapy or genetic predisposition should be taken into account when assessing drug safety. Show less
Dambrot, A.; Braam, S.R.; Freund, C.M.A.H.; Pas, S. van de; Oostwaard, D.W.V.; Atsma, D.E.; Mummery, C.L. 2012
Recent advances in pluripotent stem cell biology now make it possible to generate human cardiomyocytes in vitro from both healthy individuals and from patients with cardiac abnormalities. This... Show moreRecent advances in pluripotent stem cell biology now make it possible to generate human cardiomyocytes in vitro from both healthy individuals and from patients with cardiac abnormalities. This offers unprecedented opportunities to study cardiac disease development 'in a dish' and establish novel platforms for drug discovery, either to prevent disease progression or to reverse it. In this review paper, we discuss some of the genetic diseases that affect the heart and illustrate how these new paradigms could assist our understanding of cardiac pathogenesis and aid in drug discovery. In particular, we highlight the limitations of other commonly used model systems in predicting the consequences of drug exposure on the human heart. Show less
Recent advances in pluripotent stem cell biology now make it possible to generate human cardiomyocytes in vitro from both healthy individuals and from patients with cardiac abnormalities. This... Show moreRecent advances in pluripotent stem cell biology now make it possible to generate human cardiomyocytes in vitro from both healthy individuals and from patients with cardiac abnormalities. This offers unprecedented opportunities to study cardiac disease development 'in a dish' and establish novel platforms for drug discovery, either to prevent disease progression or to reverse it. In this review paper, we discuss some of the genetic diseases that affect the heart and illustrate how these new paradigms could assist our understanding of cardiac pathogenesis and aid in drug discovery. In particular, we highlight the limitations of other commonly used model systems in predicting the consequences of drug exposure on the human heart. Show less
Human embryonic stem cells (hESC) hold great potential as a model for human development, disease pathology, drug discovery and safety pharmacology. All these applications will depend on... Show moreHuman embryonic stem cells (hESC) hold great potential as a model for human development, disease pathology, drug discovery and safety pharmacology. All these applications will depend on comprehensive knowledge of their biology and control of their signaling mechanisms and fate choices. To begin to address this, we developed a standardized feeder-free hESC culture protocol. This system is optimized and tested for 12 independently derived stem cell lines, and optimal for clonal growth and efficient gene transfer without loss of pluripotency (Chapter 2,3). Using these protocols we created stem cells ubiquitously expressing EGFP, showed efficient SOX2 knockdown and created a fluorescent reporter stem cell line for the stem cell regulator OCT4. Next we used mass spectroscopy to investigate the plasma membrane of hESC (Chapter 4). We were able to show that these cells express a uniform epithelial plasma membrane profile and that VIMENTIN, normally associated with mesenchymal cells is also expressed. This expression turned out to be related to stress and associated with hardness of the tissue culture plastic substrate rather than differentiation. We continued to investigate the plasma membrane of hESC and decided to focus on integrins, the cell surface receptors that bind extracellular matrix proteins. Functional analyses of their function showed human embryonic stem cells have the capacity to bind to a wide range of extracellular matrix proteins via specific integrin receptors. We were able to show that recombinant vitronectin robustly supports the maintenance of hESC in an undifferentiated state in completely defined culture medium. Having validated a completely defined culture protocol we began to investigate differentiation mechanisms under defined conditions (Chapter 5). We used Stable Isotope Labeling in Cell Culture (SILAC) and quantitative phopspho-proteomics to investigate how human embryonic stem cells exit the pluripotent state. BMP4 was used to trigger differentiation and protein samples were analyzed at 30 min, 60 min and 240 min. We showed that approximately 50% of the 3067 identified phosphosites were regulated within 1 hr of differentiation induction, revealing a complex interplay of phosphorylation networks spanning different signaling pathways. Among the phosphorylated proteins was the pluripotency-associated protein SOX2, which was SUMOylated as a result of phosphorylation. Using the data to predict kinase-substrate relationships we reconstructed the hESC kinome, with CDK1/2 emerging as a key kinase in controlling self-renewal and lineage specification. Next we used gene targeting to create a fluorescent cardiac reporter cell line. EGFP was targeted into one allele of the NKX2-5 gene. EGFP fluorescence driven by the endogenous Nkx2-5 promoter faithfully reported cardiovascular lineage commitment of differentiating hESC under defined culture conditions. Using fluorescence activated cell sorting we showed that the early NKX2-5 positive cell population contained multipotent progenitor cells capable of directed differentiation to cardiomyocytes, endothelial and vascular smooth muscle cells (Chapter 7). Finally, we used the cardiomcyocytes from hESC to develop a system for cardiac safety pharmacology (Chapter 8). Recent withdrawals of prescription drugs from clinical use because of unexpected side effects on the heart have highlighted the need for more reliable cardiac safety pharmacology assays. In particular, blocking of the human Ether-a-go go Related Gene ion channel is associated with life-threatening arrhythmias, such as Torsade de Pointes. We demonstrated that, as predicted, patient serum levels of drugs and known responses on QT interval overlapped with field potential duration values derived from hESC-CM,. On this basis, we propose field potential duration prolongation as a directly applicable safety criterion for pre-clinical evaluation of new drugs in development. In conclusion, the availability of human cardiomyocytes from stem cell sources is now expected to accelerate cardiac drug discovery and safety pharmacology by offering more clinically relevant human culture models than presently available (Chapter 9,10). Show less
Braam, S.R.; Tertoolen, L.; Stolpe, A. van de; Meyer, T.; Passier, R.; Mummery, C.L. 2010
Recent withdrawals of prescription drugs from clinical use because of unexpected side effects on the heart have highlighted the need for more reliable cardiac safety pharmacology assays. Block of... Show moreRecent withdrawals of prescription drugs from clinical use because of unexpected side effects on the heart have highlighted the need for more reliable cardiac safety pharmacology assays. Block of the human Ether-a-go go Related Gene (hERG) ion channel in particular is associated with life-threatening arrhythmias, such as Torsade de Pointes (TdP). Here we investigated human cardiomyocytes derived from pluripotent (embryonic) stem cells (hESC) as a renewable, scalable, and reproducible system on which to base cardiac safety pharmacology assays. Analyses of extracellular field potentials in hESC-derived cardiomyocytes (hESC-CM) and generation of derivative field potential duration (FPD) values showed dose-dependent responses for 12 cardiac and noncardiac drugs. Serum levels in patients of drugs with known effects on QT interval overlapped with prolonged FPD values derived from hESC-CM, as predicted. We thus propose hESC-CM FPD prolongation as a safety criterion for preclinical evaluation of new drugs in development. This is the first study in which dose responses of such a wide range of compounds on hESC-CM have been generated and shown to be predictive of clinical effects. We propose that assays based on hESC-CM could complement or potentially replace some of the preclinical cardiac toxicity screening tests currently used for lead optimization and further development of new drugs. Show less
The absence of identified cell surface proteins and corresponding antibodies to most differentiated derivatives of human embryonic stem cells (hESCs) has largely limited selection of specific cell... Show moreThe absence of identified cell surface proteins and corresponding antibodies to most differentiated derivatives of human embryonic stem cells (hESCs) has largely limited selection of specific cell types from mixed cell populations to genetic approaches. Here, we describe the use of mass spectrometry (MS)-based proteomics on cell membrane proteins isolated from hESCs that were differentiated into cardiomyocytes to identify candidate proteins for this particular lineage. Quantitative MS distinguished cardiomyocyte-specific plasma membrane proteins that were highly enriched or detected only in cardiomyocytes derived from hESCs and human fetal hearts compared with a heterogeneous pool of hESC-derived differentiated cells. For several candidates, cardiomyocyte-specific expression and cell surface localization were verified by conventional antibody-based methodologies. Using an antibody against elastin microfibril interfacer 2 (EMILIN2), we demonstrate that cardiomyocytes can be sorted from live cell populations. Besides showing that MS-based membrane proteomics is a powerful tool to identify candidate proteins that allow purification of specific cell lineages from heterogeneous populations, this approach generated a plasma membrane proteome profile suggesting signaling pathways that control cell behavior. Show less
In recent years the differentiation efficiency of human embryonic stem cells (hESCs) to cardiomyocytes has improved considerably. In general, hESC-derived cardiomyocytes are formed in aggregates,... Show moreIn recent years the differentiation efficiency of human embryonic stem cells (hESCs) to cardiomyocytes has improved considerably. In general, hESC-derived cardiomyocytes are formed in aggregates, which require dissociation for follow-up experimental analyses and (clinical) applications. Here, we show that inhibition of the Rho-associated kinase (ROCK) by Y-27632 improved survival of dissociated hESC-derived differentiated cells. A maximum effect on cell survival was already observed within the first 24 hours. Hereafter, no further differences in the percentage of apoptotic and proliferating cells were observed with or without ROCK-inhibitor treatment. Improved survival was observed in both cardiomyocyte as well as non-cardiomyocyte cell populations. Viable cardiomyocytes were indicated by the appearance of beating, sarcomeric organization of cardiac-specific proteins, and fluorescence of a mitochondrion-selective dye. These results facilitate development of applications of hESC-derived cardiomyocytes in multiple research areas. Furthermore, these findings may be applied to other cell types differentiated from hESCs or other stem cells. Show less
