Georgios Kosmidis

Functional maturation of human pluripotent stem cell derived cardiomyocytes in vitro--correlation between contraction force and electrophysiology.

Cardiomyocytes from human pluripotent stem cells (hPSC-CM) have many potential applications in disease modelling and drug target discovery but their phenotypic similarity to early fetal stages of cardiac development limits their applicability. In this study we compared contraction stresses of hPSC-CM to 2nd trimester human fetal derived cardiomyocytes (hFetal-CM) by imaging displacement of fluorescent beads by single contracting hPSC-CM, aligned by microcontact-printing on polyacrylamide gels. hPSC-CM showed distinctly lower contraction stress than cardiomyocytes isolated from hFetal-CM. To improve maturation of hPSC-CM in vitro we made use of commercial media optimized for cardiomyocyte maturation, which promoted significantly higher contraction stress in hPSC-compared with hFetal-CM. Accordingly, other features of cardiomyocyte maturation were observed, most strikingly increased upstroke velocities and action potential amplitudes, lower resting membrane potentials, improved sarcomeric organization and alterations in cardiac-specific gene expression. Performing contraction force and electrophysiology measurements on individual cardiomyocytes revealed strong correlations between an increase in contraction force and a rise of the upstroke velocity and action potential amplitude and with a decrease in the resting membrane potential. We showed that under standard differentiation conditions hPSC-CM display lower contractile force than primary hFetal-CM and identified conditions under which a commercially available culture medium could induce molecular, morphological and functional maturation of hPSC-CM in vitro. These results are an important contribution for full implementation of hPSC-CM in cardiac disease modelling and drug discovery.

Immaturity of human stem-cell-derived cardiomyocytes in culture: fatal flaw or soluble problem?

Cardiomyocytes from human pluripotent stem cells (hPSC-CMs) are increasingly used to model cardiac disease, test drug efficacy and for safety pharmacology. Nevertheless, a major hurdle to more extensive use is their immaturity and similarity to fetal rather than adult cardiomyocytes. Here, we provide an overview of the strategies currently being used to increase maturation in culture, which include prolongation of time in culture, exposure to electrical stimulation, application of mechanical strain, growth in three-dimensional tissue configuration, addition of non-cardiomyocytes, use of hormones and small molecules, and alteration of the extracellular environment. By comparing the outcomes of these studies, we identify the approaches most likely to improve functional maturation of hPSC-CMs in terms of their electrophysiology and excitation-contraction coupling.

Contractile Defect Caused by Mutation in MYBPC3 Revealed under Conditions Optimized for Human PSC-Cardiomyocyte Function

Summary Maximizing baseline function of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is essential for their effective application in models of cardiac toxicity and disease. Here, we aimed to identify factors that would promote an adequate level of function to permit robust single-cell contractility measurements in a human induced pluripotent stem cell (hiPSC) model of hypertrophic cardiomyopathy (HCM). A simple screen revealed the collaborative effects of thyroid hormone, IGF-1 and the glucocorticoid analog dexamethasone on the electrophysiology, bioenergetics, and contractile force generation of hPSC-CMs. In this optimized condition, hiPSC-CMs with mutations in MYBPC3, a gene encoding myosin-binding protein C, which, when mutated, causes HCM, showed significantly lower contractile force generation than controls. This was recapitulated by direct knockdown of MYBPC3 in control hPSC-CMs, supporting a mechanism of haploinsufficiency. Modeling this disease in vitro using human cells is an important step toward identifying therapeutic interventions for HCM.

PGC-1α and Reactive Oxygen Species Regulate Human Embryonic Stem Cell-Derived Cardiomyocyte Function

Summary Diminished mitochondrial function is causally related to some heart diseases. Here, we developed a human disease model based on cardiomyocytes from human embryonic stem cells (hESCs), in which an important pathway of mitochondrial gene expression was inactivated. Repression of PGC-1α, which is normally induced during development of cardiomyocytes, decreased mitochondrial content and activity and decreased the capacity for coping with energetic stress. Yet, concurrently, reactive oxygen species (ROS) levels were lowered, and the amplitude of the action potential and the maximum amplitude of the calcium transient were in fact increased. Importantly, in control cardiomyocytes, lowering ROS levels emulated this beneficial effect of PGC-1α knockdown and similarly increased the calcium transient amplitude. Our results suggest that controlling ROS levels may be of key physiological importance for recapitulating mature cardiomyocyte phenotypes, and the combination of bioassays used in this study may have broad application in the analysis of cardiac physiology pertaining to disease.

Generation of induced pluripotent stem cells from human foetal fibroblasts using the Sleeping Beauty transposon gene delivery system

Transposon gene delivery systems offer an alternative, non-viral-based approach to generate induced pluripotent stem cells (iPSCs). Here we used the Sleeping Beauty (SB) transposon to generate four human iPSC lines from foetal fibroblasts. In contrast to other gene delivery systems, the SB transposon does not exhibit an integration bias towards particular genetic elements, thereby reducing the risk of insertional mutagenesis. Furthermore, unlike the alternative transposon piggyBac, SB has no SB-like elements within the human genome, minimising the possibility of mobilising endogenous transposon elements. All iPSC lines exhibited the expected characteristics of pluripotent human cells, including the ability to differentiate to derivatives of all three germ layers in vitro. Re-expression of the SB transposase in the iPSCs after reprogramming resulted in the mobilisation of some of the transposons. These results indicate that the SB transposon system is a useful addition to methods for generating human iPSCs, both for basic and applied biomedical research, and in the context of future therapeutic application.

TECRL, a new life‐threatening inherited arrhythmia gene associated with overlapping clinical features of both LQTS and CPVT

Genetic causes of many familial arrhythmia syndromes remain elusive. In this study, whole‐exome sequencing (WES) was carried out on patients from three different families that presented with life‐threatening arrhythmias and high risk of sudden cardiac death (SCD). Two French Canadian probands carried identical homozygous rare variant in TECRL gene (p.Arg196Gln), which encodes the trans‐2,3‐enoyl‐CoA reductase‐like protein. Both patients had cardiac arrest, stress‐induced atrial and ventricular tachycardia, and QT prolongation on adrenergic stimulation. A third patient from a consanguineous Sudanese family diagnosed with catecholaminergic polymorphic ventricular tachycardia (CPVT) had a homozygous splice site mutation (c.331+1G>A) in TECRL. Analysis of intracellular calcium ([Ca2+]i) dynamics in human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) generated from this individual (TECRLHom‐hiPSCs), his heterozygous but clinically asymptomatic father (TECRLHet‐hiPSCs), and a healthy individual (CTRL‐hiPSCs) from the same Sudanese family, revealed smaller [Ca2+]i transient amplitudes as well as elevated diastolic [Ca2+]i in TECRLHom‐hiPSC‐CMs compared with CTRL‐hiPSC‐CMs. The [Ca2+]i transient also rose markedly slower and contained lower sarcoplasmic reticulum (SR) calcium stores, evidenced by the decreased magnitude of caffeine‐induced [Ca2+]i transients. In addition, the decay phase of the [Ca2+]i transient was slower in TECRLHom‐hiPSC‐CMs due to decreased SERCA and NCX activities. Furthermore, TECRLHom‐hiPSC‐CMs showed prolonged action potentials (APs) compared with CTRL‐hiPSC‐CMs. TECRL knockdown in control human embryonic stem cell‐derived CMs (hESC‐CMs) also resulted in significantly longer APs. Moreover, stimulation by noradrenaline (NA) significantly increased the propensity for triggered activity based on delayed afterdepolarizations (DADs) in TECRLHom‐hiPSC‐CMs and treatment with flecainide, a class Ic antiarrhythmic drug, significantly reduced the triggered activity in these cells. In summary, we report that mutations in TECRL are associated with inherited arrhythmias characterized by clinical features of both LQTS and CPVT. Patient‐specific hiPSC‐CMs recapitulated salient features of the clinical phenotype and provide a platform for drug screening evidenced by initial identification of flecainide as a potential therapeutic. These findings have implications for diagnosis and treatment of inherited cardiac arrhythmias.

The sinus venosus myocardium contributes to the atrioventricular canal: potential role during atrioventricular node development?

The presence of distinct electrophysiological pathways within the atrioventricular node (AVN) is a prerequisite for atrioventricular nodal reentrant tachycardia to occur. In this study, the different cell contributions that may account for the anatomical and functional heterogeneity of the AVN were investigated. To study the temporal development of the AVN, the expression pattern of ISL1, expressed in cardiac progenitor cells, was studied in sequential stages performing co‐staining with myocardial markers (TNNI2 and NKX2‐5) and HCN4 (cardiac conduction system marker). An ISL1+/TNNI2+/HCN4+ continuity between the myocardium of the sinus venosus and atrioventricular canal was identified in the region of the putative AVN, which showed a pacemaker‐like phenotype based on single cell patch‐clamp experiments. Furthermore, qPCR analysis showed that even during early development, different cell populations can be identified in the region of the putative AVN. Fate mapping was performed by in ovo vital dye microinjection. Embryos were harvested and analysed 24 and 48 hrs post‐injection. These experiments showed incorporation of sinus venosus myocardium in the posterior region of the atrioventricular canal. The myocardium of the sinus venosus contributes to the atrioventricular canal. It is postulated that the myocardium of the sinus venosus contributes to nodal extensions or transitional cells of the AVN since these cells are located in the posterior region of the AVN. This finding may help to understand the origin of atrioventricular nodal reentrant tachycardia.

Strategies for rapidly mapping proviral integration sites and assessing cardiogenic potential of nascent human induced pluripotent stem cell clones.

Recent methodological advances have improved the ease and efficiency of generating human induced pluripotent stem cells (hiPSCs), but this now typically results in a greater number of hiPSC clones being derived than can be wholly characterized. It is therefore imperative that methods are developed which facilitate rapid selection of hiPSC clones most suited for the downstream research aims. Here we describe a combination of procedures enabling the simultaneous screening of multiple clones to determine their genomic integrity as well as their cardiac differentiation potential within two weeks of the putative reprogrammed colonies initially appearing. By coupling splinkerette-PCR with Ion Torrent sequencing, we could ascertain the number and map the proviral integration sites in lentiviral-reprogrammed hiPSCs. In parallel, we developed an effective cardiac differentiation protocol that generated functional cardiomyocytes within 10 days without requiring line-specific optimization for any of the six independent human pluripotent stem cell lines tested. Finally, to demonstrate the scalable potential of these procedures, we picked 20 nascent iPSC clones and performed these independent assays concurrently. Before the clones required passaging, we were able to identify clones with a single integrated copy of the reprogramming vector and robust cardiac differentiation potential for further analysis.

Altered calcium handling and increased contraction force in human embryonic stem cell derived cardiomyocytes following short term dexamethasone exposure

One limitation in using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) for disease modeling and cardiac safety pharmacology is their immature functional phenotype compared with adult cardiomyocytes. Here, we report that treatment of human embryonic stem cell derived cardiomyocytes (hESC-CMs) with dexamethasone, a synthetic glucocorticoid, activated glucocorticoid signaling which in turn improved their calcium handling properties and contractility. L-type calcium current and action potential properties were not affected by dexamethasone but significantly faster calcium decay, increased forces of contraction and sarcomeric lengths, were observed in hESC-CMs after dexamethasone exposure. Activating the glucocorticoid pathway can thus contribute to mediating hPSC-CMs maturation.

Readthrough-Promoting Drugs Gentamicin and PTC124 Fail to Rescue Nav1.5 Function of Human-Induced Pluripotent Stem Cell–Derived Cardiomyocytes Carrying Nonsense Mutations in the Sodium Channel Gene SCN5A

Background—Several compounds have been reported to induce translational readthrough of premature stop codons resulting in the production of full-length protein by interfering with ribosomal proofreading. Here we examined the effect of 2 of these compounds, gentamicin and PTC124, in human-induced pluripotent stem cell (hiPSC)–derived cardiomyocytes bearing nonsense mutations in the sodium channel gene SCN5A, which are associated with conduction disease and potential lethal arrhythmias. Methods and Results—We generated hiPSC from 2 patients carrying the mutations R1638X and W156X. hiPSC-derived cardiomyocytes from both patients recapitulated the expected electrophysiological phenotype, as evidenced by reduced Na+ currents and action potential upstroke velocities compared with hiPSC-derived cardiomyocytes from 2 unrelated control individuals. While we were able to confirm the readthrough efficacy of the 2 drugs in Human Embryonic Kidney 293 cells, we did not observe rescue of the electrophysiological phenotype in hiPSC-derived cardiomyocytes from the patients. Conclusions—We conclude that these drugs are unlikely to present an effective treatment for patients carrying the loss-of-function SCN5A gene mutations examined in this study.