Human-induced pluripotent stem cell (hiPSC) technologies have provided access to in vitro models of inaccessible human cardiomyocytes (CMs), providing new insights into human disease mechanisms, therapy strategies, and cardiac toxicology. However, the robustness of reproducible outcomes and integration of data among research groups are hampered due to the variation between cell lines, clones, and batches-to-batch differences. These variable outcomes in hiPSC models are caused by differences in human donors, genetic stability, and experimental variability, which affect morphology, cellular heterogeneity, transcript and protein abundance, and differentiation potency. This review summarizes the usage of hiPSC-CMs obtained from multiple lines and evaluates the corresponding experimental variation between studies to perform in-depth in vitro power calculations. Our meta-analyses show that although 4 or more hiPSC lines are used in 21 published case-control studies, these reports still contain high heterogeneity between functional parameters. In specific CM readouts, the SD is >40%, meaning that the variation between different cell lines is larger than the effect of the studied mutation, drug response, or toxicity. Results indicate a need for careful selection of hiPSC lines, controls, and readout stability and these insights will further guide the power of hiPSC lines in biomedical applications.

AIMS:Titin truncating variants (TTNtv) are a major genetic cause of dilated cardiomyopathy (DCM), accounting for approximately 25% of familial cases. Atrial fibrillation (AF) frequently occurs in DCM patients carrying TTNtv and may precede overt ventricular dysfunction, suggesting an atrial-specific disease mechanism. How TTNtv increase susceptibility to AF, particularly in the absence of established DCM, remains incompletely understood. This study aimed to define the cellular and molecular mechanisms by which a clinically relevant TTNtv predisposes to atrial arrhythmogenesis. METHODS AND RESULTS:We introduced a patient-associated TTNtv (TTN c.59926+1G>A) into human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-CMs). TTNtv hiPSC-CMs exhibited proarrhythmic electrophysiological alterations, including increased spontaneous beating frequency, abnormal sodium channel kinetics, and heightened sensitivity to cholinergic agonists. In silico simulations demonstrated that heightened cholinergic sensitivity was sufficient to trigger AF under conditions of sinus tachycardia. RNA sequencing revealed dysregulation of sarcomere assembly and extracellular matrix pathways, and TTNtv hiPSC-CMs showed structurally shortened sarcomeres. Engineered heart tissues composed of TTNtv hiPSC-CMs co-cultured with cardiac fibroblasts demonstrated reduced contractile force and increased secretion of collagen, fibronectin-1 and TGF-β1, consistent with activation of profibrotic signalling. Together, these findings indicate that a TTNtv can cause intrinsic atrial electrical instability and promote pro-fibrotic signalling. CONCLUSION:Our results identify atrial electrophysiological abnormalities and profibrotic remodelling as key mechanisms by which TTNtv increase AF risk, even in the absence of overt DCM. These findings support a primary atrial contribution to TTNtv-associated arrhythmogenesis and provide mechanistic insight into AF as an early clinical manifestation in carriers.

Human induced pluripotent stem cell (hiPSC) technologies offer human-relevant cardiac models for biomedical applications. However, workflows for differentiation of cardiac stromal cells and fabrication of engineered heart tissue (EHT) commonly rely on animal serum, contrary to growing policy demands to reduce use of these products. Applying marker analysis via COL-I, DDR2 and GATA4 for cardiac fibroblasts or CD31, CD34 and CD144 for endothelial cells, we tailored Panexin, a defined serum substitute, to support high efficiency differentiation of cardiac stromal lineages to 85% purity without additional purification steps. We evaluated fabrication of EHTs using hiPSC-cardiomyocytes only (monoculture) or further combined with cardiac fibroblasts and endothelial cells (triculture; 70%:15%:15%, respectively). Panexin poorly supported fabrication and contractility of EHTs, a finding unaltered by modulating spontaneous cardiac myofibroblast activation via TGFβ inhibition. In contrast, human serum enabled fabrication of mono- and tri-culture EHTs, wherein constructs made without TGFβ signalling inhibition delivered the strongest contractile forces and exceeded comparator tissues engineered using animal serum. Our data show that iterative evaluation of serum substitutes, human serum, cell combinations and signalling pathway modulators can mitigate use of animal serum for functional EHT generation, aligning with the UK government's roadmap for alternative methods.

In vitro gene editing using isogenic pairs of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has demonstrated the feasibility of introducing or correcting specific pathogenic variants. These successes represent a key first step towards therapeutic genome editing for cardiomyopathies, showing that precise, variant-specific interventions are achievable. To translate in vitro findings to the clinic, it is essential to develop robust disease models that yield meaningful, translatable data. The next challenge is systematically identifying disease-causing variants amenable to gene editing with strong pre-clinical support. Therefore, we conducted a systematic search of published studies on isogenic hiPSC-CM pairs in cardiomyopathy research with specific criteria, including (likely) pathogenic variants causing cardiomyopathy, correction and/or introduction of variants, differentiation into CMs, and functional follow-up. We systematically assessed 785 papers and highlighted 101 studies meeting our inclusion criteria reporting 69 patients carrying 56 unique variants across 31 genes, most commonly MYH7, MYBPC3, and DMD. This expanded to 91 variants across 38 genes upon inclusion of the introduced variants in a donor line. However, reported clinical data were often incomplete, underscoring the need for standardized phenotypic documentation. We reveal a lack of patient details, which creates an incomplete picture of underlying disease variables that hinder the design of targeted personalized treatments. Omitted key clinical data can lead to misinterpretations or overlooked variables that impact treatment outcomes. This systematic review integrates current evidence from successful in vitro studies using isogenic hiPSC-CM models and proposes a reporting framework for variant prioritization and the rigorous application of isogenic controls in cardiomyopathy research.

AIMS:Electrical integration of human-induced pluripotent stem-cell-derived cardiomyocyte (hiPSC-CM)-based tissue with the host myocardium is a requirement of successful regeneration therapy. This study was designed to identify electrical coupling in the acute phase (1-2 h) post-grafting using an ex vivo model. METHODS AND RESULTS:Small, engineered heart tissues (mini-EHTs), consisting of ∼50 000 hiPSC-CMs on a hydrogel (spontaneous rate 0.34 ± 0.05 Hz), were loaded with Cal520-AM. EHTs were implanted sub-epicardially into a Langendorff-perfused rabbit heart after blebbistatin treatment. For up to 100 min, a continuous pseudo-electrocardiogram was recorded during sinus rhythm (rate 2.0-3.5 Hz). At 25 min intervals, EHT calcium transients (CaTs) were recorded for 10-20 s (no contraction group). To study the influence of mechanical activity, blebbistatin was washed off after implantation (contraction recovery group). Periodic entrainment of EHTs with the myocardium was detected less often (P = 0.011) in the no contraction group (1/9 hearts) than in the contraction recovery group (5/6 hearts). The average coupling delay (QRS-CaT) and the difference in consecutive delays (Δdelay) were 89 ± 50 and 10 ± 3 ms, respectively (n = 12 traces; N = 6 hearts). Coupling ratios (QRS:CaT) varied from 2:1 to 4:1. These coupling parameters were not significantly different in the two experimental groups. Modelling of hiPSC-CM tissue separated by a 25 μm saline gap from the myocardium demonstrated field-effect coupling with similarly variable activation delays. Importantly, coupling failed with a gap of 100 μm. CONCLUSION:EHT entrainment is possible immediately after grafting and has features compatible with field-effect coupling. Sensitivity to the gap dimensions may explain why entrainment is more common in actively contracting myocardium.

AIMS:To investigate the regulatory role of NANOG in genes associated with stemness, symmetric division, and therapeutic resistance in colorectal cancer stem-like cells (CRC-SCs), with a focus on ERK/GSK-3β/β-catenin signalling and epithelial-mesenchymal transition (EMT), in order to evaluate the translational potential of targeting NANOG-associated signalling pathways. METHODS:Stemness, signalling activity, and cell division modes were analysed using 3D colonospheres enriched for CRC-SCs. Drug responses to the MEK inhibitor U0126 and the GSK-3β inhibitor TDZD-8 were assessed in CRC patient-derived organoids (PDOs), alongside molecular assays, immunohistochemistry with H-score quantification in xenograft models, and molecular dynamics simulations. RESULTS:NANOG overexpression enhanced the expression of stemness-associated genes, promoted symmetric cell division, and activated ERK/GSK-3β signalling, contributing to increased sphere formation. Inhibition of MEK and GSK-3β reduced EMT, cell proliferation, and symmetric division in CRC-SCs. NANOG-mediated dysregulation of ERK/GSK-3β altered β-catenin signalling and disrupted E-cadherin-dependent cell-cell adhesion. Molecular simulations and drug assays demonstrated that TDZD-8 and U0126 interfere with NANOG-DNA binding and β-catenin/E-cadherin interactions. CONCLUSIONS:NANOG drives CRC-SC maintenance via ERK/GSK-3β/β-catenin signalling and EMT modulation. This study offers significant insights into the translational impact of targeting NANOG and its downstream pathways with small-molecule inhibitors U0126 and TDZD-8 and presents a promising strategy to reduce CRC-SCs stemness, functionality, and tumourigenicity.

Background:Missense variants of Z-disk protein, alpha-actinin-2 (ACTN2), have been linked to hypertrophic cardiomyopathy (HCM). A novel ACTN2 missense variant, M228T, was identified in family members presenting with HCM and/or atrial arrhythmias. Embryonic lethality was previously shown in mice expressing this variant homozygously, whereas heterozygous (Het) expression did not manifest an overt HCM phenotype. Importantly, the atrial arrhythmias observed in the identified family have not been explored in the context of M228T, despite many patients exhibiting electrical abnormalities prior to the clinical onset of HCM. Methods:Six-month-old Het M228T and wild-type (WT) mice were used to evaluate electrophysiological properties using electrocardiography (ECG) and atrial optical mapping. Echocardiography and strain analysis were employed to assess cardiac structure and function. Results:Het mice exhibited a prolongation in action potential duration and depolarisation time at 30, 50, and 70 % repolarisation in both the left and right atria. No significant alterations in atrial conduction velocity were observed. No changes in atrial ECG parameters were detected. Het mice displayed no evidence of structural remodelling, nor were there any changes in systolic parameters or overt diastolic dysfunction, as assessed by conventional echocardiography and strain analysis. Signs of contractile dyssynchrony were present, specifically at the apex relative to WT controls. Conclusion:The Het M228T mouse model demonstrated atrial electrical alterations that occurred independently of any overt cardiac structural or functional remodelling. These findings may support the causative role for atrial electric phenotypes identified in a subset of patients carrying the variant.

The emphasis in human pluripotent stem cell (hPSC) technologies has shifted from cell therapy to in vitro disease modelling and drug screening. This review examines why this shift has occurred, and how current technological limitations might be overcome to fully realise the potential of hPSCs. Details are provided for all disease-specific human induced pluripotent stem cell lines spanning a dozen dysfunctional organ systems. Phenotype and pharmacology have been examined in only 17 of 63 lines, primarily those that model neurological and cardiac conditions. Drug screening is most advanced in hPSC-cardiomyocytes. Responses for almost 60 agents include examples of how careful tests in hPSC-cardiomyocytes have improved on existing in vitro assays, and how these cells have been integrated into high throughput imaging and electrophysiology industrial platforms. Such successes will provide an incentive to overcome bottlenecks in hPSC technology such as improving cell maturity and industrial scalability whilst reducing cost.