Using induced pluripotent stem cells for drug discovery in arrhythmias.

INTRODUCTION: Arrhythmias are disturbances in the normal rhythm of the heart and account for significant cardiovascular morbidity and mortality worldwide. Historically, preclinical research has been anchored in animal models, though physiological differences between these models and humans have limited their clinical translation. The discovery of human induced pluripotent stem cells (iPSC) and subsequent differentiation into cardiomyocyte has led to the development of new in vitro models of arrhythmias with the hope of a new pathway for both exploration of pathogenic variants and novel therapeutic discovery. AREAS COVERED: The authors describe the latest two-dimensional in vitro models of arrhythmias, several examples of the use of these models in drug development, and the role of gene editing when modeling diseases. They conclude by discussing the use of three-dimensional models in the study of arrythmias and the integration of computational technologies and machine learning with experimental technologies. EXPERT OPINION: Human iPSC-derived cardiomyocytes models have significant potential to augment disease modeling, drug discovery, and toxicity studies in preclinical development. While there is initial success with modeling arrhythmias, the field is still in its nascency and requires advances in maturation, cellular diversity, and readouts to emulate arrhythmias more accurately.

A multi-organ chip with matured tissue niches linked by vascular flow.

Engineered tissues can be used to model human pathophysiology and test the efficacy and safety of drugs. Yet, to model whole-body physiology and systemic diseases, engineered tissues with preserved phenotypes need to physiologically communicate. Here we report the development and applicability of a tissue-chip system in which matured human heart, liver, bone and skin tissue niches are linked by recirculating vascular flow to allow for the recapitulation of interdependent organ functions. Each tissue is cultured in its own optimized environment and is separated from the common vascular flow by a selectively permeable endothelial barrier. The interlinked tissues maintained their molecular, structural and functional phenotypes over 4 weeks of culture, recapitulated the pharmacokinetic and pharmacodynamic profiles of doxorubicin in humans, allowed for the identification of early miRNA biomarkers of cardiotoxicity, and increased the predictive values of clinically observed miRNA responses relative to tissues cultured in isolation and to fluidically interlinked tissues in the absence of endothelial barriers. Vascularly linked and phenotypically stable matured human tissues may facilitate the clinical applicability of tissue chips.

Machine Learning Techniques to Classify Healthy and Diseased Cardiomyocytes by Contractility Profile.

Cardiomyocytes derived from human induced pluripotent stem (iPS) cells enable the study of cardiac physiology and the developmental testing of new therapeutic drugs in a human setting. In parallel, machine learning methods are being applied to biomedical science in unprecedented ways. Machine learning has been used to distinguish healthy from diseased cardiomyocytes using calcium (Ca2+) transient signals. Most Ca2+ transient signals are obtained via terminal assays that do not permit longitudinal studies, although some recently developed options can circumvent these concerns. Here, we describe the use of machine learning to identify healthy and diseased cardiomyocytes according to their contractility profiles, which are derived from brightfield videos. This noncontact, label-free approach allows for the continued cultivation of cells after they have been evaluated for use in other assays and can be readily extended to organs-on-chip. To demonstrate utility, we assessed contractility profiles of cardiomyocytes obtained from patients with Timothy Syndrome (TS), a long QT disease which can lead to fatal arrhythmias, and from healthy individuals. The videos were processed and classified using machine learning methods and their performance was evaluated according to several parameters. The trained algorithms were able to distinguish the TS cardiomyocytes from healthy controls and classify two different healthy controls. The proposed computational machine learning evaluation of human iPS cell-derived cardiomyocytes' contractility profiles has the potential to identify other genetic proarrhythmic events, screen therapeutic agents for inducing or suppressing long QT events, and predict drug-target interactions. The same approach could be readily extended to the evaluation of engineered cardiac tissues within single-tissue and multi-tissue organs-on-chip.

Integrated human organ-on-a-chip model for predictive studies of anti-tumor drug efficacy and cardiac safety.

Traditional drug screening models are often unable to faithfully recapitulate human physiology in health and disease, motivating the development of microfluidic organs-on-a-chip (OOC) platforms that can mimic many aspects of human physiology and in the process alleviate many of the discrepancies between preclinical studies and clinical trials outcomes. Linsitinib, a novel anti-cancer drug, showed promising results in pre-clinical models of Ewing Sarcoma (ES), where it suppressed tumor growth. However, a Phase II clinical trial in several European centers with patients showed relapsed and/or refractory ES. We report an integrated, open setting, imaging and sampling accessible, polysulfone-based platform, featuring minimal hydrophobic compound binding. Two bioengineered human tissues - bone ES tumor and heart muscle - were cultured either in isolation or in the integrated platform and subjected to a clinically used linsitinib dosage. The measured anti-tumor efficacy and cardiotoxicity were compared with the results observed in the clinical trial. Only the engineered tumor tissues, and not monolayers, recapitulated the bone microenvironment pathways targeted by linsitinib, and the clinically-relevant differences in drug responses between non-metastatic and metastatic ES tumors. The responses of non-metastatic ES tumor tissues and heart muscle to linsitinib were much closer to those observed in the clinical trial for tissues cultured in an integrated setting than for tissues cultured in isolation. Drug treatment of isolated tissues resulted in significant decreases in tumor viability and cardiac function. Meanwhile, drug treatment in an integrated setting showed poor tumor response and less cardiotoxicity, which matched the results of the clinical trial. Overall, the integration of engineered human tumor and cardiac tissues in the integrated platform improved the predictive accuracy for both the direct and off-target effects of linsitinib. The proposed approach could be readily extended to other drugs and tissue systems.

Engineering of human cardiac muscle electromechanically matured to an adult-like phenotype.

The application of tissue-engineering approaches to human induced pluripotent stem (hiPS) cells enables the development of physiologically relevant human tissue models for in vitro studies of development, regeneration, and disease. However, the immature phenotype of hiPS-derived cardiomyocytes (hiPS-CMs) limits their utility. We have developed a protocol to generate engineered cardiac tissues from hiPS cells and electromechanically mature them toward an adult-like phenotype. This protocol also provides optimized methods for analyzing these tissues' functionality, ultrastructure, and cellular properties. The approach relies on biological adaptation of cultured tissues subjected to biomimetic cues, applied at an increasing intensity, to drive accelerated maturation. hiPS cells are differentiated into cardiomyocytes and used immediately after the first contractions are observed, when they still have developmental plasticity. This starting cell population is combined with human dermal fibroblasts, encapsulated in a fibrin hydrogel and allowed to compact under passive tension in a custom-designed bioreactor. After 7 d of tissue formation, the engineered tissues are matured for an additional 21 d by increasingly intense electromechanical stimulation. Tissue properties can be evaluated by measuring contractile function, responsiveness to electrical stimuli, ultrastructure properties (sarcomere length, mitochondrial density, networks of transverse tubules), force-frequency and force-length relationships, calcium handling, and responses to ß-adrenergic agonists. Cell properties can be evaluated by monitoring gene/protein expression, oxidative metabolism, and electrophysiology. The protocol takes 4 weeks and requires experience in advanced cell culture and machining methods for bioreactor fabrication. We anticipate that this protocol will improve modeling of cardiac diseases and testing of drugs.

Author Correction: Advanced maturation of human cardiac tissue grown from pluripotent stem cells.

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

Advanced maturation of human cardiac tissue grown from pluripotent stem cells.

Cardiac tissues generated from human induced pluripotent stem cells (iPSCs) can serve as platforms for patient-specific studies of physiology and disease1-6. However, the predictive power of these models is presently limited by the immature state of the cells1, 2, 5, 6. Here we show that this fundamental limitation can be overcome if cardiac tissues are formed from early-stage iPSC-derived cardiomyocytes soon after the initiation of spontaneous contractions and are subjected to physical conditioning with increasing intensity over time. After only four weeks of culture, for all iPSC lines studied, such tissues displayed adult-like gene expression profiles, remarkably organized ultrastructure, physiological sarcomere length (2.2 µm) and density of mitochondria (30%), the presence of transverse tubules, oxidative metabolism, a positive force-frequency relationship and functional calcium handling. Electromechanical properties developed more slowly and did not achieve the stage of maturity seen in adult human myocardium. Tissue maturity was necessary for achieving physiological responses to isoproterenol and recapitulating pathological hypertrophy, supporting the utility of this tissue model for studies of cardiac development and disease.