Impaired Reorganization of Centrosome Structure Underlies Human Infantile Dilated Cardiomyopathy.

BACKGROUND: During cardiomyocyte maturation, the centrosome, which functions as a microtubule organizing center in cardiomyocytes, undergoes dramatic structural reorganization where its components reorganize from being localized at the centriole to the nuclear envelope. This developmentally programmed process, referred to as centrosome reduction, has been previously associated with cell cycle exit. However, understanding of how this process influences cardiomyocyte cell biology, and whether its disruption results in human cardiac disease, remains unknown. We studied this phenomenon in an infant with a rare case of infantile dilated cardiomyopathy (iDCM) who presented with left ventricular ejection fraction of 18% and disrupted sarcomere and mitochondria structure. METHODS: We performed an analysis beginning with an infant who presented with a rare case of iDCM. We derived induced pluripotent stem cells from the patient to model iDCM in vitro. We performed whole exome sequencing on the patient and his parents for causal gene analysis. CRISPR/Cas9-mediated gene knockout and correction in vitro were used to confirm whole exome sequencing results. Zebrafish and Drosophila models were used for in vivo validation of the causal gene. Matrigel mattress technology and single-cell RNA sequencing were used to characterize iDCM cardiomyocytes further. RESULTS: Whole exome sequencing and CRISPR/Cas9 gene knockout/correction identified RTTN, the gene encoding the centrosomal protein RTTN (rotatin), as the causal gene underlying the patient's condition, representing the first time a centrosome defect has been implicated in a nonsyndromic dilated cardiomyopathy. Genetic knockdowns in zebrafish and Drosophila confirmed an evolutionarily conserved requirement of RTTN for cardiac structure and function. Single-cell RNA sequencing of iDCM cardiomyocytes showed impaired maturation of iDCM cardiomyocytes, which underlie the observed cardiomyocyte structural and functional deficits. We also observed persistent localization of the centrosome at the centriole, contrasting with expected programmed perinuclear reorganization, which led to subsequent global microtubule network defects. In addition, we identified a small molecule that restored centrosome reorganization and improved the structure and contractility of iDCM cardiomyocytes. CONCLUSIONS: This study is the first to demonstrate a case of human disease caused by a defect in centrosome reduction. We also uncovered a novel role for RTTN in perinatal cardiac development and identified a potential therapeutic strategy for centrosome-related iDCM. Future study aimed at identifying variants in centrosome components may uncover additional contributors to human cardiac disease.

Nonclinical evaluation of chronic cardiac contractility modulation on 3D human engineered cardiac tissues.

INTRODUCTION: Cardiac contractility modulation (CCM) is a medical device-based therapy delivering non-excitatory electrical stimulations to the heart to enhance cardiac function in heart failure (HF) patients. The lack of human in vitro tools to assess CCM hinders our understanding of CCM mechanisms of action. Here, we introduce a novel chronic (i.e., 2-day) in vitro CCM assay to evaluate the effects of CCM in a human 3D microphysiological system consisting of engineered cardiac tissues (ECTs). METHODS: Cryopreserved human induced pluripotent stem cell-derived cardiomyocytes were used to generate 3D ECTs. The ECTs were cultured, incorporating human primary ventricular cardiac fibroblasts and a fibrin-based gel. Electrical stimulation was applied using two separate pulse generators for the CCM group and control group. Contractile properties and intracellular calcium were measured, and a cardiac gene quantitative PCR screen was conducted. RESULTS: Chronic CCM increased contraction amplitude and duration, enhanced intracellular calcium transient amplitude, and altered gene expression related to HF (i.e., natriuretic peptide B, NPPB) and excitation-contraction coupling (i.e., sodium-calcium exchanger, SLC8). CONCLUSION: These data represent the first study of chronic CCM in a 3D ECT model, providing a nonclinical tool to assess the effects of cardiac electrophysiology medical device signals complementing in vivo animal studies. The methodology established a standardized 3D ECT-based in vitro testbed for chronic CCM, allowing evaluation of physiological and molecular effects on human cardiac tissues.

Real-time visualization of titin dynamics reveals extensive reversible photobleaching in human induced pluripotent stem cell-derived cardiomyocytes.

Fluorescence recovery after photobleaching (FRAP) has been useful in delineating cardiac myofilament biology, and innovations in fluorophore chemistry have expanded the array of microscopic assays used. However, one assumption in FRAP is the irreversible photobleaching of fluorescent proteins after laser excitation. Here we demonstrate reversible photobleaching regarding the photoconvertible fluorescent protein mEos3.2. We used CRISPR/Cas9 genome editing in human induced pluripotent stem cells (hiPSCs) to knock-in mEos3.2 into the COOH terminus of titin to visualize sarcomeric titin incorporation and turnover. Upon cardiac induction, the titin-mEos3.2 fusion protein is expressed and integrated in the sarcomeres of hiPSC-derived cardiomyocytes (CMs). STORM imaging shows M-band clustered regions of bound titin-mEos3.2 with few soluble titin-mEos3.2 molecules. FRAP revealed a baseline titin-mEos3.2 fluorescence recovery of 68% and half-life of ~1.2 h, suggesting a rapid exchange of sarcomeric titin with soluble titin. However, paraformaldehyde-fixed and permeabilized titin-mEos3.2 hiPSC-CMs surprisingly revealed a 55% fluorescence recovery. Whole cell FRAP analysis in paraformaldehyde-fixed, cycloheximide-treated, and untreated titin-mEos3.2 hiPSC-CMs displayed no significant differences in fluorescence recovery. FRAP in fixed HEK 293T expressing cytosolic mEos3.2 demonstrates a 58% fluorescence recovery. These data suggest that titin-mEos3.2 is subject to reversible photobleaching following FRAP. Using a mouse titin-eGFP model, we demonstrate that no reversible photobleaching occurs. Our results reveal that reversible photobleaching accounts for the majority of titin recovery in the titin-mEos3.2 hiPSC-CM model and should warrant as a caution in the extrapolation of reliable FRAP data from specific fluorescent proteins in long-term cell imaging.

Matrigel Mattress: A Method for the Generation of Single Contracting Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

RATIONALE: The lack of measurable single-cell contractility of human-induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) currently limits the utility of hiPSC-CMs for evaluating contractile performance for both basic research and drug discovery. OBJECTIVE: To develop a culture method that rapidly generates contracting single hiPSC-CMs and allows quantification of cell shortening with standard equipment used for studying adult CMs. METHODS AND RESULTS: Single hiPSC-CMs were cultured for 5 to 7 days on a 0.4- to 0.8-mm thick mattress of undiluted Matrigel (mattress hiPSC-CMs) and compared with hiPSC-CMs maintained on a control substrate (<0.1-mm thick 1:60 diluted Matrigel, control hiPSC-CMs). Compared with control hiPSC-CMs, mattress hiPSC-CMs had more rod-shape morphology and significantly increased sarcomere length. Contractile parameters of mattress hiPSC-CMs measured with video-based edge detection were comparable with those of freshly isolated adult rabbit ventricular CMs. Morphological and contractile properties of mattress hiPSC-CMs were consistent across cryopreserved hiPSC-CMs generated independently at another institution. Unlike control hiPSC-CMs, mattress hiPSC-CMs display robust contractile responses to positive inotropic agents, such as myofilament calcium sensitizers. Mattress hiPSC-CMs exhibit molecular changes that include increased expression of the maturation marker cardiac troponin I and significantly increased action potential upstroke velocity because of a 2-fold increase in sodium current (INa). CONCLUSIONS: The Matrigel mattress method enables the rapid generation of robustly contracting hiPSC-CMs and enhances maturation. This new method allows quantification of contractile performance at the single-cell level, which should be valuable to disease modeling, drug discovery, and preclinical cardiotoxicity testing.

Pulsed electric field performance calculator tool based on an in vitro human cardiac model.

Introduction: Pulsed Field Ablation (PFA) is a novel non-thermal method for cardiac ablation, relying on irreversible electroporation induced by high-energy pulsed electric fields (PEFs) to create localized lesions in the heart atria. A significant challenge in optimizing PFA treatments is determining the lethal electric field threshold (EFT), which governs ablation volume and varies with PEF waveform parameters. However, the proprietary nature of device developer's waveform characteristics and the lack of standardized nonclinical testing methods have left optimal EFTs for cardiac ablation uncertain. Methods: To address this gap, we introduced a laboratory protocol employing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in monolayer format to evaluate the impact of a range of clinically relevant biphasic pulse parameters on lethal EFT and adiabatic heating (AH). Cell death areas were assessed using fluorescent dyes and confocal microscopy, while lethal EFTs were quantified through comparison with electric field numerical simulations. Results and conclusion: Our study confirmed a strong correlation between cell death in hiPSC-CMs and the number and duration of pulses in each train, with pulse repetition frequency exerting a comparatively weaker influence. Fitting of these results through machine learning algorithms were used to develop an open-source online calculator. By estimating lethal EFT and associated temperature increases for diverse pulse parameter combinations, this tool, once validated, has the potential to significantly reduce reliance on animal models during early-stage device de-risking and performance assessment. This tool also offers a promising avenue for advancing PFA technology for cardiac ablation medical devices to enhance patient outcomes.

Human in vitro neurocardiac coculture (ivNCC) assay development for evaluating cardiac contractility modulation.

Two of the most prominent organ systems, the nervous and the cardiovascular systems, are intricately connected to maintain homeostasis in mammals. Recent years have shown tremendous efforts toward therapeutic modulation of cardiac contractility and electrophysiology by electrical stimulation. Neuronal innervation and cardiac ganglia regulation are often overlooked when developing in vitro models for cardiac devices, but it is likely that peripheral nervous system plays a role in the clinical effects. We developed an in vitro neurocardiac coculture (ivNCC) model system to study cardiac and neuronal interplay using human induced pluripotent stem cell (hiPSC) technology. We demonstrated significant expression and colocalization of cardiac markers including troponin, α-actinin, and neuronal marker peripherin in neurocardiac coculture. To assess functional coupling between the cardiomyocytes and neurons, we evaluated nicotine-induced ß-adrenergic norepinephrine effect and found beat rate was significantly increased in ivNCC as compared to monoculture alone. The developed platform was used as a nonclinical model for the assessment of cardiac medical devices that deliver nonexcitatory electrical pulses to the heart during the absolute refractory period of the cardiac cycle, that is, cardiac contractility modulation (CCM) therapy. Robust coculture response was observed at 14 V/cm (5 V, 64 mA), monophasic, 2 ms pulse duration for pacing and 20 V/cm (7 V, 90 mA) phase amplitude, biphasic, 5.14 ms pulse duration for CCM. We observed that the CCM effect and kinetics were more pronounced in coculture as compared to cardiac monoculture, supporting a hypothesis that some part of CCM mechanism of action can be attributed to peripheral nervous system stimulation. This study provides novel characterization of CCM effects on hiPSC-derived neurocardiac cocultures. This innervated human heart model can be further extended to investigate arrhythmic mechanisms, neurocardiac safety, and toxicity post-chronic exposure to materials, drugs, and medical devices. We present data on acute CCM electrical stimulation effects on a functional and optimized coculture using commercially available hiPSC-derived cardiomyocytes and neurons. Moreover, this study provides an in vitro human heart model to evaluate neuronal innervation and cardiac ganglia regulation of contractility by applying CCM pulse parameters that closely resemble clinical setting. This ivNCC platform provides a potential tool for investigating aspects of cardiac and neurological device safety and performance.

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format. The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Human cardiomyocytes are more susceptible to irreversible electroporation by pulsed electric field than human esophageal cells.

Pulse electric field-based (PEF) ablation is a technique whereby short high-intensity electric fields inducing irreversible electroporation (IRE) are applied to various tissues. Here, we implemented a standardized in vitro model to compare the effects of biphasic symmetrical pulses (100 pulses, 1-10 µs phase duration (d), 10-1000 Hz pulse repetition rate (f)) using two different human cellular models: human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and human esophageal smooth muscle cells (hESMCs) cultured in monolayer format. We report the PEF-induced irreversibly electroporated cell monolayer areas and the corresponding electric field thresholds (EFTs) for both cardiac and esophageal cultures. Our results suggest marked cell type specificity with EFT estimated to be 2-2.5 times lower in hiPSC-CMs than in hESMCs when subjected to identical PEF treatments (e.g., 0.90 vs 1.85 kV/cm for the treatment of 100 pulses with d = 5 µs, f = 10 Hz, and 0.65 vs 1.67 kV/cm for the treatment of 100 pulses with d = 10 µs, f = 10 Hz). PEF treatment can result in increased temperature around the stimulating electrodes and lead to unanticipated thermal tissue damage that is proportional to the peak temperature rise and to the duration of the PEF-induced elevated temperatures. In our study, temperature increases ranged from less than 1°C to as high as 30°C, however, all temperature changes were transient and quickly returned to baseline and the highest observed ∆T returned to 50% of its maximum recorded temperature in tens of seconds.

Human in vitro assay for irreversible electroporation cardiac ablation.

Introduction: Pulsed electric field (PEF) cardiac ablation has been recently proposed as a technique to treat drug resistant atrial fibrillation by inducing cell death through irreversible electroporation (IRE). Improper PEF dosing can result in thermal damage or reversible electroporation. The lack of comprehensive and systematic studies to select PEF parameters for safe and effective IRE cardiac treatments hinders device development and regulatory decision-making. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been proposed as an alternative to animal models in the evaluation of cardiac electrophysiology safety. Methods: We developed a novel high-throughput in vitro assay to quantify the electric field threshold (EFT) for electroporation (acute effect) and cell death (long-term effect) in hiPSC-CMs. Monolayers of hiPSC-CMs were cultured in high-throughput format and exposed to clinically relevant biphasic PEF treatments. Electroporation and cell death areas were identified using fluorescent probes and confocal microscopy; electroporation and cell death EFTs were quantified by comparison of fluorescent images with electric field numerical simulations. Results: Study results confirmed that PEF induces electroporation and cell death in hiPSC-CMs, dependent on the number of pulses and the amplitude, duration, and repetition frequency. In addition, PEF-induced temperature increase, absorbed dose, and total treatment time for each PEF parameter combination are reported. Discussion: Upon verification of the translatability of the in vitro results presented here to in vivo models, this novel hiPSC-CM-based assay could be used as an alternative to animal or human studies and can assist in early nonclinical device development, as well as inform regulatory decision-making for cardiac ablation medical devices.

Acute effects of cardiac contractility modulation stimulation in conventional 2D and 3D human induced pluripotent stem cell-derived cardiomyocyte models.

Cardiac contractility modulation (CCM) is a medical device therapy whereby non-excitatory electrical stimulations are delivered to the myocardium during the absolute refractory period to enhance cardiac function. We previously evaluated the effects of the standard CCM pulse parameters in isolated rabbit ventricular cardiomyocytes and 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, on flexible substrate. In the present study, we sought to extend these results to human 3D microphysiological systems to develop a robust model to evaluate various clinical CCM pulse parameters in vitro. HiPSC-CMs were studied in conventional 2D monolayer format, on stiff substrate (i.e., glass), and as 3D human engineered cardiac tissues (ECTs). Cardiac contractile properties were evaluated by video (i.e., pixel) and force-based analysis. CCM pulses were assessed at varying electrical 'doses' using a commercial pulse generator. A robust CCM contractile response was observed for 3D ECTs. Under comparable conditions, conventional 2D monolayer hiPSC-CMs, on stiff substrate, displayed no contractile response. 3D ECTs displayed enhanced contractile properties including increased contraction amplitude (i.e., force), and accelerated contraction and relaxation slopes under standard acute CCM stimulation. Moreover, 3D ECTs displayed enhanced contractility in a CCM pulse parameter-dependent manner by adjustment of CCM pulse delay, duration, amplitude, and number relative to baseline. The observed acute effects subsided when the CCM stimulation was stopped and gradually returned to baseline. These data represent the first study of CCM in 3D hiPSC-CM models and provide a nonclinical tool to assess various CCM device signals in 3D human cardiac tissues prior to in vivo animal studies. Moreover, this work provides a foundation to evaluate the effects of additional cardiac medical devices in 3D ECTs.

Acute effects of cardiac contractility modulation on human induced pluripotent stem cell-derived cardiomyocytes.

Cardiac contractility modulation (CCM) is an intracardiac therapy whereby nonexcitatory electrical simulations are delivered during the absolute refractory period of the cardiac cycle. We previously evaluated the effects of CCM in isolated adult rabbit ventricular cardiomyocytes and found a transient increase in calcium and contractility. In the present study, we sought to extend these results to human cardiomyocytes using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to develop a robust model to evaluate CCM in vitro. HiPSC-CMs (iCell Cardiomyocytes2 , Fujifilm Cellular Dynamic, Inc.) were studied in monolayer format plated on flexible substrate. Contractility, calcium handling, and electrophysiology were evaluated by fluorescence- and video-based analysis (CellOPTIQ, Clyde Biosciences). CCM pulses were applied using an A-M Systems 4100 pulse generator. Robust hiPSC-CMs response was observed at 14 V/cm (64 mA) for pacing and 28 V/cm (128 mA, phase amplitude) for CCM. Under these conditions, hiPSC-CMs displayed enhanced contractile properties including increased contraction amplitude and faster contraction kinetics. Likewise, calcium transient amplitude increased, and calcium kinetics were faster. Furthermore, electrophysiological properties were altered resulting in shortened action potential duration (APD). The observed effects subsided when the CCM stimulation was stopped. CCM-induced increase in hiPSC-CMs contractility was significantly more pronounced when extracellular calcium concentration was lowered from 2 mM to 0.5 mM. This study provides a comprehensive characterization of CCM effects on hiPSC-CMs. These data represent the first study of CCM in hiPSC-CMs and provide an in vitro model to assess physiologically relevant mechanisms and evaluate safety and effectiveness of future cardiac electrophysiology medical devices.

An initial map of chromosomal segmental copy number variations in the chicken.

BACKGROUND: Chromosomal segmental copy number variation (CNV) has been recently recognized as a very important source of genetic variability. Some CNV loci involve genes or conserved regulatory elements. Compelling evidence indicates that CNVs impact genome functions. The chicken is a very important farm animal species which has also served as a model for biological and biomedical research for hundreds of years. A map of CNVs in chickens could facilitate the identification of chromosomal regions that segregate for important agricultural and disease phenotypes. RESULTS: Ninety six CNVs were identified in three lines of chickens (Cornish Rock broiler, Leghorn and Rhode Island Red) using whole genome tiling array. These CNVs encompass 16 Mb (1.3%) of the chicken genome. Twenty six CNVs were found in two or more animals. Whereas most small sized CNVs reside in none coding sequences, larger CNV regions involve genes (for example prolactin receptor, aldose reductase and zinc finger proteins). These results suggest that chicken CNVs potentially affect agricultural or disease related traits. CONCLUSION: An initial map of CNVs for the chicken has been described. Although chicken genome is approximately one third the size of a typical mammalian genome, the pattern of chicken CNVs is similar to that of mammals. The number of CNVs detected per individual was also similar to that found in dogs, mice, rats and macaques. A map of chicken CNVs provides new information on genetic variations for the understanding of important agricultural traits and disease.

International Multisite Study of Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Drug Proarrhythmic Potential Assessment.

To assess the utility of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as an in vitro proarrhythmia model, we evaluated the concentration dependence and sources of variability of electrophysiologic responses to 28 drugs linked to low, intermediate, and high torsades de pointes (TdP) risk categories using two commercial cell lines and standardized protocols in a blinded multisite study using multielectrode array or voltage-sensing optical approaches. Logistical and ordinal linear regression models were constructed using drug responses as predictors and TdP risk categories as outcomes. Three of seven predictors (drug-induced arrhythmia-like events and prolongation of repolarization at either maximum tested or maximal clinical exposures) categorized drugs with reasonable accuracy (area under the curve values of receiver operator curves ∼0.8). hiPSC-CM line, test site, and platform had minimal influence on drug categorization. These results demonstrate the utility of hiPSC-CMs to detect drug-induced proarrhythmic effects as part of the evolving Comprehensive In Vitro Proarrhythmia Assay paradigm.

Production of Single Contracting Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Matrigel Mattress Technique.

This unit describes the published Matrigel mattress method. Briefly, we describe the preparation of the mattress, replating of the human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) on the Matrigel mattress, and hiPSC-CM mattress maintenance. Adherence to this protocol will yield individual, robustly shortening hiPSC-CMs, which can be used for downstream applications. © 2017 by John Wiley & Sons, Inc.

Combinatorial polymer matrices enhance in vitro maturation of human induced pluripotent stem cell-derived cardiomyocytes.

Cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs) hold great promise for modeling human heart diseases. However, iPSC-CMs studied to date resemble immature embryonic myocytes and therefore do not adequately recapitulate native adult cardiomyocyte phenotypes. Since extracellular matrix plays an essential role in heart development and maturation in vivo, we sought to develop a synthetic culture matrix that could enhance functional maturation of iPSC-CMs in vitro. In this study, we employed a library of combinatorial polymers comprising of three functional subunits - poly-ε-caprolacton (PCL), polyethylene glycol (PEG), and carboxylated PCL (cPCL) - as synthetic substrates for culturing human iPSC-CMs. Of these, iPSC-CMs cultured on 4%PEG-96%PCL (each % indicates the corresponding molar ratio) exhibit the greatest contractility and mitochondrial function. These functional enhancements are associated with increased expression of cardiac myosin light chain-2v, cardiac troponin I and integrin alpha-7. Importantly, iPSC-CMs cultured on 4%PEG-96%PCL demonstrate troponin I (TnI) isoform switch from the fetal slow skeletal TnI (ssTnI) to the postnatal cardiac TnI (cTnI), the first report of such transition in vitro. Finally, culturing iPSC-CMs on 4%PEG-96%PCL also significantly increased expression of genes encoding intermediate filaments known to transduce integrin-mediated mechanical signals to the myofilaments. In summary, our study demonstrates that synthetic culture matrices engineered from combinatorial polymers can be utilized to promote in vitro maturation of human iPSC-CMs through the engagement of critical matrix-integrin interactions.

Combinatorial polymer electrospun matrices promote physiologically-relevant cardiomyogenic stem cell differentiation.

Myocardial infarction results in extensive cardiomyocyte death which can lead to fatal arrhythmias or congestive heart failure. Delivery of stem cells to repopulate damaged cardiac tissue may be an attractive and innovative solution for repairing the damaged heart. Instructive polymer scaffolds with a wide range of properties have been used extensively to direct the differentiation of stem cells. In this study, we have optimized the chemical and mechanical properties of an electrospun polymer mesh for directed differentiation of embryonic stem cells (ESCs) towards a cardiomyogenic lineage. A combinatorial polymer library was prepared by copolymerizing three distinct subunits at varying molar ratios to tune the physicochemical properties of the resulting polymer: hydrophilic polyethylene glycol (PEG), hydrophobic poly(ε-caprolactone) (PCL), and negatively-charged, carboxylated PCL (CPCL). Murine ESCs were cultured on electrospun polymeric scaffolds and their differentiation to cardiomyocytes was assessed through measurements of viability, intracellular reactive oxygen species (ROS), α-myosin heavy chain expression (α-MHC), and intracellular Ca(2+) signaling dynamics. Interestingly, ESCs on the most compliant substrate, 4%PEG-86%PCL-10%CPCL, exhibited the highest α-MHC expression as well as the most mature Ca(2+) signaling dynamics. To investigate the role of scaffold modulus in ESC differentiation, the scaffold fiber density was reduced by altering the electrospinning parameters. The reduced modulus was found to enhance α-MHC gene expression, and promote maturation of myocyte Ca(2+) handling. These data indicate that ESC-derived cardiomyocyte differentiation and maturation can be promoted by tuning the mechanical and chemical properties of polymer scaffold via copolymerization and electrospinning techniques.