Human-Engineered Atrial Tissue for Studying Atrial Fibrillation.

This chapter details the generation of atrial fibrin-based engineered heart tissue (EHT) in standard 24-well format as a 3D model for the human atrium. Compared to 2D cultivation, human-induced pluripotent stem cells (hiPSCs)-derived atrial cardiomyocytes demonstrated a higher degree of maturation in 3D format. Furthermore, we have demonstrated in previous work that the model displayed atrial characteristics in terms of contraction and gene expression patterns, electrophysiology, and pharmacological response.

Human Engineered Heart Tissue Patches Remuscularize the Injured Heart in a Dose-Dependent Manner.

Background: Human engineered heart tissue (EHT) transplantation represents a potential regenerative strategy for patients with heart failure and has been successful in preclinical models. Clinical application requires upscaling, adaptation to good manufacturing practices, and determination of the effective dose.

Methods: Cardiomyocytes were differentiated from 3 different human induced pluripotent stem cell lines including one reprogrammed under good manufacturing practice conditions.

Comparison of 10 Control hPSC Lines for Drug Screening in an Engineered Heart Tissue Format.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are commercially available, and cardiac differentiation established routine. Systematic evaluation of several control hiPSC-CM is lacking. We investigated 10 different control hiPSC-CM lines and analyzed function and suitability for drug screening.

Cell Banking of hiPSCs: A Practical Guide to Cryopreservation and Quality Control in Basic Research.

The reproducibility of stem cell research relies on the constant availability of quality-controlled cells. As the quality of human induced pluripotent stem cells (hiPSCs) can deteriorate in the course of a few passages, cell banking is key to achieve consistent results and low batch-to-batch variation. Here, we provide a cost-efficient route to generate master and working cell banks for basic research projects.

Isogenic models of hypertrophic cardiomyopathy unveil differential phenotypes and mechanism-driven therapeutics.

Background: Hypertrophic cardiomyopathy (HCM) is a prevalent and complex cardiovascular condition. Despite being strongly associated with genetic alterations, wide variation of disease penetrance, expressivity and hallmarks of progression complicate treatment. We aimed to characterize different human isogenic cellular models of HCM bearing patient-relevant mutations to clarify genetic causation and disease mechanisms, hence facilitating the development of effective therapeutics.

Blinded, Multicenter Evaluation of Drug-induced Changes in Contractility Using Human-induced Pluripotent Stem Cell-derived Cardiomyocytes.

Animal models are 78% accurate in determining whether drugs will alter contractility of the human heart. To evaluate the suitability of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for predictive safety pharmacology, we quantified changes in contractility, voltage, and/or Ca2+ handling in 2D monolayers or 3D engineered heart tissues (EHTs). Protocols were unified via a drug training set, allowing subsequent blinded multicenter evaluation of drugs with known positive, negative, or neutral inotropic effects.

Regulation of I and force by PDEs in human-induced pluripotent stem cell-derived cardiomyocytes.

Background And Purpose: Phosphodiesterases (PDEs) are important regulators of β-adrenoceptor signalling in the heart. While PDE4 is the most important isoform that regulates I and force in rodent cardiomyocytes, the dominant isoform in adult human cardiomyocytes is PDE3.

Experimental Approach: Given the potential of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for biomedical research, this study characterized the contribution of PDE3 and PDE4 isoforms to the regulation of I and force in hiPSC-CMs in an engineered heart tissue (EHT) model.

Case Report on: Very Early Afterdepolarizations in HiPSC-Cardiomyocytes-An Artifact by Big Conductance Calcium Activated Potassium Current (I).

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent an unlimited source of human CMs that could be a standard tool in drug research. However, there is concern whether hiPSC-CMs express all cardiac ion channels at physiological level and whether they might express non-cardiac ion channels. In a control hiPSC line, we found large, "noisy" outward K currents, when we measured outward potassium currents in isolated hiPSC-CMs.

Force and Calcium Transients Analysis in Human Engineered Heart Tissues Reveals Positive Force-Frequency Relation at Physiological Frequency.

Force measurements in ex vivo and engineered heart tissues are well established. Analysis of calcium transients (CaT) is complementary to force, and the combined analysis is meaningful to the study of cardiomyocyte biology and disease. This article describes a model of human induced pluripotent stem cell cardiomyocyte-derived engineered heart tissues (hiPSC-CM EHTs) transduced with the calcium sensor GCaMP6f followed by sequential analysis of force and CaT.

Phosphomimetic cardiac myosin-binding protein C partially rescues a cardiomyopathy phenotype in murine engineered heart tissue.

Phosphorylation of cardiac myosin-binding protein C (cMyBP-C), encoded by MYBPC3, increases the availability of myosin heads for interaction with actin thus enhancing contraction. cMyBP-C phosphorylation level is lower in septal myectomies of patients with hypertrophic cardiomyopathy (HCM) than in non-failing hearts. Here we compared the effect of phosphomimetic (D282) and wild-type (S282) cMyBP-C gene transfer on the HCM phenotype of engineered heart tissues (EHTs) generated from a mouse model carrying a Mybpc3 mutation (KI).

Author Correction: Differentiation of cardiomyocytes and generation of human engineered heart tissue.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Correction: Analysis of Tyrosine Kinase Inhibitor-Mediated Decline in Contractile Force in Rat Engineered Heart Tissue.

[This corrects the article DOI: 10.1371/journal.pone.

Isogenic Pairs of hiPSC-CMs with Hypertrophic Cardiomyopathy/LVNC-Associated ACTC1 E99K Mutation Unveil Differential Functional Deficits.

Hypertrophic cardiomyopathy (HCM) is a primary disorder of contractility in heart muscle. To gain mechanistic insight and guide pharmacological rescue, this study models HCM using isogenic pairs of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the E99K-ACTC1 cardiac actin mutation. In both 3D engineered heart tissues and 2D monolayers, arrhythmogenesis was evident in all E99K-ACTC1 hiPSC-CMs.

Piezo-bending actuators for isometric or auxotonic contraction analysis of engineered heart tissue.

Engineered heart tissue (EHT) has proven as valuable tool for disease modelling, drug safety screening, and cardiac repair. Especially in combination with the stem cell technology, these in vitro models of the human heart have generated interest not only of basic cardiovascular researchers but also of regulatory authorities responsible for drug safety. A main limitation of 3D-based assays for evaluating cardiotoxicity is their limited throughput.

Satellite cells delivered in their niche efficiently generate functional myotubes in three-dimensional cell culture.

Biophysical/biochemical cues from the environment contribute to regulation of the regenerative capacity of resident skeletal muscle stem cells called satellites cells. This can be observed in vitro, where muscle cell behaviour is influenced by the particular culture substrates and whether culture is performed in a 2D or 3D environment, with changes including morphology, nuclear shape and cytoskeletal organization. To create a 3D skeletal muscle model we compared collagen I, Fibrin or PEG-Fibrinogen with different sources of murine and human myogenic cells.

CRISPR/Cas9 editing in human pluripotent stem cell-cardiomyocytes highlights arrhythmias, hypocontractility, and energy depletion as potential therapeutic targets for hypertrophic cardiomyopathy.

Aims: Sarcomeric gene mutations frequently underlie hypertrophic cardiomyopathy (HCM), a prevalent and complex condition leading to left ventricle thickening and heart dysfunction. We evaluated isogenic genome-edited human pluripotent stem cell-cardiomyocytes (hPSC-CM) for their validity to model, and add clarity to, HCM.

Methods And Results: CRISPR/Cas9 editing produced 11 variants of the HCM-causing mutation c.

Three-Dimensional Human iPSC-Derived Artificial Skeletal Muscles Model Muscular Dystrophies and Enable Multilineage Tissue Engineering.

Generating human skeletal muscle models is instrumental for investigating muscle pathology and therapy. Here, we report the generation of three-dimensional (3D) artificial skeletal muscle tissue from human pluripotent stem cells, including induced pluripotent stem cells (iPSCs) from patients with Duchenne, limb-girdle, and congenital muscular dystrophies. 3D skeletal myogenic differentiation of pluripotent cells was induced within hydrogels under tension to provide myofiber alignment.

Contractile Work Contributes to Maturation of Energy Metabolism in hiPSC-Derived Cardiomyocytes.

Energy metabolism is a key aspect of cardiomyocyte biology. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising tool for biomedical application, but they are immature and have not undergone metabolic maturation related to early postnatal development. To assess whether cultivation of hiPSC-CMs in 3D engineered heart tissue format leads to maturation of energy metabolism, we analyzed the mitochondrial and metabolic state of 3D hiPSC-CMs and compared it with 2D culture.

Low Resting Membrane Potential and Low Inward Rectifier Potassium Currents Are Not Inherent Features of hiPSC-Derived Cardiomyocytes.

Human induced pluripotent stem cell (hiPSC) cardiomyocytes (CMs) show less negative resting membrane potential (RMP), which is attributed to small inward rectifier currents (I). Here, I was measured in hiPSC-CMs (proprietary and commercial cell line) cultured as monolayer (ML) or 3D engineered heart tissue (EHT) and, for direct comparison, in CMs from human right atrial (RA) and left ventricular (LV) tissue. RMP was measured in isolated cells and intact tissues.

Prominent differences in left ventricular performance and myocardial properties between right ventricular and left ventricular-based pacing modes in rats.

Biventricular pacing is an important modality to improve left ventricular (LV) synchronization and long-term function. However, the biological effects of this treatment are far from being elucidated and existing animal models are limited and demanding. Recently, we introduced an implanted device for double-site epicardial pacing in rats and echocardiographically demonstrated favorable effects of LV and biventricular (LV-based) pacing modes typically observed in humans.

Human iPSC-derived cardiomyocytes cultured in 3D engineered heart tissue show physiological upstroke velocity and sodium current density.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a promising tool for drug testing and modelling genetic disorders. Abnormally low upstroke velocity is a current limitation. Here we investigated the use of 3D engineered heart tissue (EHT) as a culture method with greater resemblance to human heart tissue in comparison to standard technique of 2D monolayer (ML) format.

Differentiation of cardiomyocytes and generation of human engineered heart tissue.

Since the advent of the generation of human induced pluripotent stem cells (hiPSCs), numerous protocols have been developed to differentiate hiPSCs into cardiomyocytes and then subsequently assess their ability to recapitulate the properties of adult human cardiomyocytes. However, hiPSC-derived cardiomyocytes (hiPSC-CMs) are often assessed in single-cell assays. A shortcoming of these assays is the limited ability to characterize the physiological parameters of cardiomyocytes, such as contractile force, due to random orientations.

Blinded Contractility Analysis in hiPSC-Cardiomyocytes in Engineered Heart Tissue Format: Comparison With Human Atrial Trabeculae.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) may serve as a new assay for drug testing in a human context, but their validity particularly for the evaluation of inotropic drug effects remains unclear. In this blinded analysis, we compared the effects of 10 indicator compounds with known inotropic effects in electrically stimulated (1.5 Hz) hiPSC-CM-derived 3-dimensional engineered heart tissue (EHT) and human atrial trabeculae (hAT).

Engineering Cardiac Muscle Tissue: A Maturating Field of Research.

Twenty years after the initial description of a tissue engineered construct, 3-dimensional human cardiac tissues of different kinds are now generated routinely in many laboratories. Advances in stem cell biology and engineering allow for the generation of constructs that come close to recapitulating the complex structure of heart muscle and might, therefore, be amenable to industrial (eg, drug screening) and clinical (eg, cardiac repair) applications. Whether the more physiological structure of 3-dimensional constructs provides a relevant advantage over standard 2-dimensional cell culture has yet to be shown in head-to-head-comparisons.