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MCL1 is an anti-apoptotic protein that is frequently amplified in cancer and confers resistance to current standard of care. Therefore, MCL1 is an attractive anti-cancer target. Here we describe ANJ810 as a novel, potent, and selective MCL1 inhibitor and its key design principle of a rapid systemic clearance to potentially minimize AUC-driven toxicities associated with MCL1 inhibition. ANJ810 induced rapid cell killing within 4 hours in vitro but in the same 4-hour window had no impact on cell viability or troponin I release in hiPSC-derived cardiomyocytes, even at supra-pharmacologic concentrations. In vivo ANJ810 induced efficacy in xenograft hematological and solid tumor models despite the short residence time of ANJ810 in plasma. In totality our data support the hypothesis that short-term inhibition of MCL1 with ANJ810 can induce cell apoptosis in tumor cells while maintaining an acceptable safety profile. We therefore intend to advance ANJ810 to clinical trials.

Aims Human-induced pluripotent stem cell-cardiomyocytes (hiPSC-CMs) are widely used to study arrhythmia-associated mutations in ion channels. Among these, the cardiac sodium channel SCN5A undergoes foetal-to-adult isoform switching around birth. Conventional hiPSC-CM cultures, which are phenotypically foetal, have thus far been unable to capture mutations in adult gene isoforms. Here, we investigated whether tri-cellular cross-talk in a three-dimensional (3D) cardiac microtissue (MT) promoted post-natal SCN5A maturation in hiPSC-CMs. Methods and results We derived patient hiPSC-CMs carrying compound mutations in the adult SCN5A exon 6B and exon 4. Electrophysiological properties of patient hiPSC-CMs in monolayer were not altered by the exon 6B mutation compared with isogenic controls since it is not expressed; further, CRISPR/Cas9-mediated excision of the foetal exon 6A did not promote adult SCN5A expression. However, when hiPSC-CMs were matured in 3D cardiac MTs, SCN5A underwent isoform switch and the functional consequences of the mutation located in exon 6B were revealed. Up-regulation of the splicing factor muscleblind-like protein 1 (MBNL1) drove SCN5A post-natal maturation in microtissues since its overexpression in hiPSC-CMs was sufficient to promote exon 6B inclusion, whilst knocking-out MBNL1 failed to foster isoform switch. Conclusions Our study shows that (i) the tri-cellular cardiac microtissues promote post-natal SCN5A isoform switch in hiPSC-CMs, (ii) adult splicing of SCN5A is driven by MBNL1 in these tissues, and (iii) this model can be used for examining post-natal cardiac arrhythmias due to mutations in the exon 6B. Translational perspective The cardiac sodium channel is essential for conducting the electrical impulse in the heart. Postnatal alternative splicing regulation causes mutual exclusive inclusion of fetal or adult exons of the corresponding gene, SCN5A. Typically, immature hiPSCCMs fall short in studying the effect of mutations located in the adult exon. We describe here that an innovative tri-cellular three-dimensional cardiac microtissue culture promotes hiPSC-CMs maturation through upregulation of MBNL1, thus revealing the effect of a pathogenic genetic variant located in the SCN5A adult exon. These results help advancing the use of hiPSC-CMs in studying adult heart disease and for developing personalized medicine applications.

MCL1 is an anti-apoptotic protein inhibiting cancer cell death. It is frequently amplified or overexpressed in cancer and confers resistance to relevant standard of care. Therefore, MCL1 is an attractive target to potentially re-sensitize tumors to conventional chemotherapy and targeted agents. Here we describe ANJ810 as a novel, potent, and selective MCL1 inhibitor which specifically induces apoptosis in cancer cells. ANJ810 potently and reversibly binds to the BH3-binding groove of human MCL1 (KD = 0.3 nM), thereby inhibiting the interaction with the BH3-only protein NOXA (IC50 = 0.4 nM). ANJ810 shows a 4-log-fold greater selectivity to MCL1 over other anti-apoptotic proteins like Bcl-xL or Bcl-2. In cells, ANJ810 treatment rapidly (< 15 min) disrupts the MCL1-BAK interaction with an IC50 < 10 nM, resulting in caspase-3 activation and cancer cell death. Knockout of BAK and BAX completely rescues cells from ANJ810 induced killing, indicating that initiation of cell death occurs through the intrinsic apoptotic pathway. Screening over 750 cell lines (PRISM), we found that ANJ810 induced anti-cancer activity in 222 cell lines with an IC50 < 1 μM across multiple solid and hematological cancers, including breast, lung, melanoma, sarcoma, lymphoma, and leukemia. The top genomic feature that correlates with sensitivity to ANJ810 treatment is the ratio of Bcl-xL/BAK expression. A key design principle of ANJ810 is its rapid systemic clearance to potentially minimize exposure-driven toxicities associated with MCL1 inhibition. ANJ810 induces efficient cancer cell killing within 4 hours in vitro but has no impact on cell viability or troponin I release in hiPSC-derived cardiomyocytes at supra-pharmacologic concentrations. In vivo, i.v. bolus injections of ANJ810 lead to short plasma residence time, yet are efficacious in xenograft models of multiple myeloma, DLBCL, NSCLC and HCC. ANJ810 will test the hypothesis in human clinical trials that short-term inhibition of MCL1 can overcome tumor resistance with an acceptable safety profile to improve current standard of care. Citation Format: Ulrike Rauh, Guo Wei, Michael Serrano-Wu, Georgios Kosmidis, Stefan Kaulfuss, Franziska Siegel, Kai Thede, James McFarland, Christopher Lemke, Nicolas Werbeck, Katrin Nowak-Reppel, Sabine Pillari, Stephan Menz, Matthias Ocker, Brian Hubbard, Virendar Kaushik, Karl Ziegelbauer, Todd Golub. ANJ810 is a highly selective novel MCL1 inhibitor with optimized in vivo clearance showing robust efficacy in preclinical solid and hematological tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 506.

Εισαγωγή: Αν και έχουν παρουσιαστεί διάφορες μελέτες μέχρι και σήμερα που εξετάζουν την ενδοοικογενειακή βία μέσα στον πληθυσμό της Ελλάδας με σκοπό να συσχετιστεί η κοινωνικοδημογραφική βάση των πληροφοριών που παρατίθεται με τον θύτη και το θύμα, οι προσεγγίσεις τους είναι κατά βάση κοινωνιολογικές. Η παρούσα μελέτη επιχειρεί να εξετάσει το φαινόμενο της ενδοοικογενειακής βίας μέσα από την κλινική ιατροδικαστική πρακτική συνδυάζοντας και συσχετίζοντας τα ιατροδικαστικά περιστατικά με διάφορα δημογραφικά χαρακτηριστικά.Μεθοδολογία: Εφαρμόστηκε μια αναδρομική στατιστική ανάλυση 184 περιστατικών ενδοοικογενειακής βίας που εξετάστηκαν κατά την περίοδο 1996-2010 από το Εργαστήριο Ιατροδικαστικής του Πανεπιστημίου Αθηνών και στη συνέχεια επιχειρήθηκε μια συσχέτιση των δημογραφικών δεδομένων του θύτη και του θύματος με διάφορες ιατροδικαστικές παραμέτρους.Αποτελέσματα: Εκτός από μια ευρεία γκάμα στατιστικών αποτελεσμάτων, το πιο ενδιαφέρον εύρημα της παρούσας εργασίας είναι ο στατιστικός συσχετισμός ανάμεσα στο περιστατικό ενδοοικογενειακής βίας και η κοινωνικοοικονομική διαστρωμάτωση των θυμάτων, όπως αυτή αντανακλάται από το επάγγελμά τους. Σε ότι αφορά τους συσχετισμούς των σωματικών βλαβών με κοινωνικοοικονομικές παραμέτρους, αποδείχθηκε ότι το επάγγελμα του θύματος και το φύλο του θύτη σχετίζονται σε στατιστικά σημαντικό βαθμό με τον χαρακτηρισμό της βλάβης (p=0,026 και p=0,023 αντίστοιχα), αφού στα περιστατικά με τις σοβαρότερες βλάβες τα θύματα ήταν κατά πλειοψηφία χαμηλότερου κοινωνικοοικονομικού επιπέδου, όπως αυτό αντικατοπτρίζεται από το επάγγελμα ενώ σε ποσοστό (96,4%) τα θύματα-γυναίκες είχαν κακοποιηθεί από άνδρες. Συμπεράσματα: Η βία μεταξύ ενηλίκων όπως και η βία στα παιδιά, αποτελούν σοβαρά θέματα δημόσιας υγείας που επηρεάζουν πολλές γυναίκες, άνδρες και παιδιά στην Ελλάδα. Τα ευρήματα ενδοοικογενειακής βίας αυξάνονται όταν το επίπεδο διαβίωσης του θύματος μειώνεται. Αυτό έχει ιδιαίτερη σημασία στις μέρες μας λόγω της δεινής οικονομικής κατάστασης του Ελληνικού πληθυσμού.

Safety pharmacology is an essential part of drug development aiming to identify, evaluate and investigate undesirable pharmacodynamic properties of a drug primarily prior to clinical trials. In particular, cardiovascular adverse drug reactions (ADR) have halted many drug development programs. Safety pharmacology has successfully implemented a screening strategy to detect cardiovascular liabilities, but there is room for further refinement. In this setting, we present the INSPIRE project, a European Training Network in safety pharmacology for Early Stage Researchers (ESRs), funded by the European Commission's H2020-MSCA-ITN programme. INSPIRE has recruited 15 ESR fellows that will conduct an individual PhD-research project for a period of 36 months. INSPIRE aims to be complementary to ongoing research initiatives. With this as a goal, an inventory of collaborative research initiatives in safety pharmacology was created and the ESR projects have been designed to be complementary to this roadmap. Overall, INSPIRE aims to improve cardiovascular safety evaluation, either by investigating technological innovations or by adding mechanistic insight in emerging safety concerns, as observed in the field of cardio-oncology. Finally, in addition to its hands-on research pillar, INSPIRE will organize a number of summer schools and workshops that will be open to the wider community as well. In summary, INSPIRE aims to foster both research and training in safety pharmacology and hopes to inspire the future generation of safety scientists.

Aim The advanced glycation end products (AGEs) are among the mechanisms responsible for the pathogenesis and development of chronic kidney disease. The aim of this study was to estimate the dietary AGE intake and to assess its correlation with hematological and biochemical markers of patients with end-stage renal disease (ESRD) undergoing hemodialysis. Materials and methods For this study, a structured questionnaire of the exogenous AGEs was developed, whose reliability and validity were evaluated in the pilot phase of the study including 50 participants. The questionnaire was issued to 605 participants (305 ESRD patients undergoing hemodialysis and 300 controls), and a blood sample was obtained through which hematological and biochemical markers were analyzed. Results It was noted that patients with ESRD consume large quantities of dietary AGEs not only in absolute values but also in comparison with control subjects (p = 0.001), attributed mainly to the methods of product processing as well as cooking. It was also ascertained that dietary AGEs were correlated (p < 0.005) with fasting glucose and glycated hemoglobin (HbA1c) and with lipidemic profile markers, such as triglyceride, as well as inflammation markers, such as erythrocyte sedimentation rate, ferritin, and C-reactive protein. All the aforementioned markers show abnormally increased levels in patients with ERSD and diabetes compared with healthy subjects. Conclusion Patients with ESRD consuming foods favoring AGE formations combined with increased endogenous AGE burden the body with their harmful action. If the specific group of patients adopt dietary habits contributing to the containment or the inhibition of AGE formation, then this would lead to the improvement of their hematological and biochemical markers and in terms of the effects of AGEs on their health is deemed imperative through the creation of consulting and prevention programs.

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.

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.

Background Human induced pluripotent stem cell–derived cardiomyocytes (hi PSC ‐ CM s) can recapitulate features of ion channel mutations causing inherited rhythm disease. However, the lack of maturity of these cells is considered a significant limitation of the model. Prolonged culture of hi PSC ‐ CM s promotes maturation of these cells. We studied the electrophysiological effects of the I230T mutation in the sodium channel gene SCN 5A in hi PSC ‐ CM s generated from a homozygous (I230T homo ) and a heterozygous (I230T het ) individual from a family with recessive cardiac conduction disease. Since the I230T mutation occurs in the developmentally regulated “adult” isoform of SCN 5A, we investigated the relationship between the expression fraction of the adult SCN 5A isoform and the electrophysiological phenotype at different time points in culture. Methods and Results After a culture period of 20 days, sodium current ( I N a ) was mildly reduced in I230T homo hi PSC ‐ CM s compared with control hi PSC ‐ CM s, while I230T het hi PSC ‐ CM s displayed no reduction in I N a . This coincided with a relatively high expression fraction of the “fetal” SCN 5A isoform compared with the adult isoform as measured by quantitative polymerase chain reaction. Following prolonged culture to 66 days, the fraction of adult SCN 5A isoform increased; this was paralleled by a marked decrease in I N a in I230T homo hi PSC ‐ CM s, in line with the severe clinical phenotype in homozygous patients. At this time in culture, I230T het hi PSC ‐ CM s displayed an intermediate loss of I N a , compatible with a gene dosage effect. Conclusions Prolonged culture of hi PSC ‐ CM s leads to an increased expression fraction of the adult sodium channel isoform. This new aspect of electrophysiological immaturity should be taken into account in studies that focus on the effects of SCN 5A mutations in hi PSC ‐ CM s.

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.

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., Graphical Abstract, Highlights • PGC-1α regulates a mitochondrial biogenesis program in hESC-derived cardiomyocytes • The PGC-1α program can result in potentially detrimental increased ROS levels • Controlling ROS is essential for maximizing the calcium transient • PGC-1α deficiency is exposed by hypertrophic or beta-adrenergic stress/stimulation, Mitochondrial function is critically important for the heart. In this study, Mummery and colleagues identify the gene PGC-1α as a key regulator of mitochondrial function in hESC-derived cardiomyocytes, controlling respiration and ROS production. Investigation of this phenotype exposed a trade-off between energetic capacity and oxidative stress. Controlling this balance may help improve PSC-derived cardiomyocyte function.

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.

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.

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.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 NaWe 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.

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.

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.

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., Graphical Abstract, Highlights • T3+IGF-1+ dexamethasone improves the electrophysiology of hPSC cardiomyocytes • These factors synergistically enhance bioenergetics and contractile force generation • Cardiomyocytes with HCM-causing mutations have a contractile defect, Birket et al. identify a combination of factors that cooperatively improve the function of human pluripotent stem cell (hiPSC)-derived cardiomyocytes. Optimizing the system facilitated the identification of a contraction force defect in a model of hypertrophic cardiomyopathy (HCM), a disease affecting ∼1:500 of the population.

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.