Search

In this issue of Cell Stem Cell, Mills et al. (2019) use multidimensional functional screening to identify pro-proliferative compounds in cell-cycle-arrested human cardiac organoids. Using this model, the authors identify two hit compounds that restart cardiomyocyte proliferation by synergistically activating the mevalonate pathway and cell-cycle-related pathways.

Background— The ability to derive human induced pluripotent stem (hiPS) cell lines by reprogramming of adult fibroblasts with a set of transcription factors offers unique opportunities for basic and translational cardiovascular research. In the present study, we aimed to characterize the cardiomyocyte differentiation potential of hiPS cells and to study the molecular, structural, and functional properties of the generated hiPS-derived cardiomyocytes. Methods and Results— Cardiomyocyte differentiation of the hiPS cells was induced with the embryoid body differentiation system. Gene expression studies demonstrated that the cardiomyocyte differentiation process of the hiPS cells was characterized by an initial increase in mesoderm and cardiomesoderm markers, followed by expression of cardiac-specific transcription factors and finally by cardiac-specific structural genes. Cells in the contracting embryoid bodies were stained positively for cardiac troponin-I, sarcomeric α-actinin, and connexin-43. Reverse-transcription polymerase chain reaction studies demonstrated the expression of cardiac-specific sarcomeric proteins and ion channels. Multielectrode array recordings established the development of a functional syncytium with stable pacemaker activity and action potential propagation. Positive and negative chronotropic responses were induced by application of isoproterenol and carbamylcholine, respectively. Administration of quinidine, E4031 ( I Kr blocker), and chromanol 293B ( I Ks blocker) significantly affected repolarization, as manifested by prolongation of the local field potential duration. Conclusions— hiPS cells can differentiate into myocytes with cardiac-specific molecular, structural, and functional properties. These results, coupled with the potential of this technology to generate patient-specific hiPS lines, hold great promise for the development of in vitro models of cardiac genetic disorders, for drug discovery and testing, and for the emerging field of cardiovascular regenerative medicine.

Pro-arrhythmia (development of cardiac arrhythmias as a pharmacological side effect) has become the single most common cause of the withdrawal or restrictions of previously marketed drugs. The development of new medications, free from these side effects, is hampered by the lack of an in vitro assay for human cardiac tissue. We hypothesized that human embryonic stem cell-derived cardiomyocytes (hESC-CMs) assessed with a combination of single cell electrophysiology and microelectrode array (MEA) mapping can serve as a novel model for electrophysiological drug screening. Current-clamp studies revealed that E-4031 and Sotalol (IKr blockers) significantly increased hESC-CM's action potential duration and also induced after-depolarizations (the in vitro correlates of increased arrhythmogenic potential). Multicellular aggregates of hESC-CMs were then analyzed with the MEA technique. Application of class I (Quinidine, Procaineamide) and class III (Sotalol) antiarrhythmic agents, E-4031, and Cisapride (a noncardiogenic agent known to lengthen QT) resulted in dose-dependent prolongation of the corrected field potential duration (cFPD). We next utilized the MEA technique to also assess pharmacological effects on conduction. Activation maps demonstrated significant conduction slowing following administration of Na channel blockers (Quinidine and Propafenone) and of the gap junction blocker (1-heptanol). While most attention has been focused on the prospects of using hESC-derived cardiomyocytes for regenerative medicine, this study highlights the possible utilization of these unique cells also for cardiac electrophysiological studies, drug screening, and target validation.

Myocardial cell replacement strategies are emerging as novel therapeutic paradigms for heart failure but are hampered by the paucity of sources for human cardiomyocytes. Human embryonic stem cells (hESC) are pluripotent stem cell lines derived from human blastocysts that can be propagated, in culture, in the undifferentiated state under special conditions and coaxed to differentiate into cell derivatives of all three germ layers, including cardiomyocytes. The current review describes the derivation and properties of the hESC lines and the different cardiomyocyte differentiation system established so far using these cells. Data regarding the structural, molecular, and functional properties of the hESC-derived cardiomyocytes is provided as well as description of the methods used to achieve cardiomyocyte enrichment and purification in this system. The possible applications of this unique differentiation system in several cardiovascular research and applied areas are discussed. Specific emphasis is put on the descriptions of the efforts performed to date to assess the feasibility of this emerging technology in the fields of cardiac cell replacement therapy and tissue engineering. Finally, the obstacles remaining on the road to clinical translation are described as well as the steps required to fully harness the potential of this new technology.

Human embryonic stem cells (hESC) are pluripotent lines that can differentiate in vitro into cell derivatives of all three germ layers, including cardiomyocytes. Successful application of these unique cells in the areas of cardiovascular research and regenerative medicine has been hampered by difficulties in identifying and selecting specific cardiac progenitor cells from the mixed population of differentiating cells. We report the generation of stable transgenic hESC lines, using lentiviral vectors, and single-cell clones that express a reporter gene (eGFP) under the transcriptional control of a cardiac-specific promoter (the human myosin light chain-2V promoter). Our results demonstrate the appearance of eGFP-expressing cells during the differentiation of the hESC as embryoid bodies (EBs) that can be identified and sorted using FACS (purity95%, viability85%). The eGFP-expressing cells were stained positively for cardiac-specific proteins (93%), expressed cardiac-specific genes, displayed cardiac-specific action-potentials, and could form stable myocardial cell grafts following in vivo cell transplantation. The generation of these transgenic hESC lines may be used to identify and study early cardiac precursors for developmental studies, to robustly quantify the extent of cardiomyocyte differentiation, to label the cells for in vivo grafting, and to allow derivation of purified cell populations of cardiomyocytes for future myocardial cell therapy strategies.

Transplantation of a tissue-engineered heart muscle represents a novel experimental therapeutic paradigm for myocardial diseases. However, this strategy has been hampered by the lack of sources for human cardiomyocytes and by the scarce vasculature in the ischemic area limiting the engraftment and survival of the transplanted muscle. Beyond the necessity of endothelial capillaries for the delivery of oxygen and nutrients to the grafted muscle tissue, interactions between endothelial and cardiomyocyte cells may also play a key role in promoting cell survival and proliferation. In the present study, we describe the formation of synchronously contracting engineered human cardiac tissue derived from human embryonic stem cells containing endothelial vessel networks. The 3D muscle consisted of cardiomyocytes, endothelial cells (ECs), and embryonic fibroblasts (EmFs). The formed vessels were further stabilized by the presence of mural cells originating from the EmFs. The presence of EmFs decreased EC death and increased EC proliferation. Moreover, the presence of endothelial capillaries augmented cardiomyocyte proliferation and did not hamper cardiomyocyte orientation and alignment. Immunostaining, ultrastructural analysis (using transmission electron microscopy), RT-PCR, pharmacological, and confocal laser calcium imaging studies demonstrated the presence of cardiac-specific molecular, ultrastructural, and functional properties of the generated tissue constructs with synchronous activity mediated by action potential propagation through gap junctions. In summary, this is the first report of the construction of 3D vascularized human cardiac tissue that may have unique applications for studies of cardiac development, function, and tissue replacement therapy.

The recent establishment of the human embryonic stem cell lines and the demonstration of their ability to differentiate in vitro to cardiomyocytes brings a unique promise to both basic and clinical research. The present report describes the characteristics of the human embryonic stem cell lines and focuses on the structural and functional properties of their cardiomyocyte derivatives. In addition, the possible signals and cues involved in the commitment and early differentiation of cardiomyocytes will be discussed. Finally, the potential applications of this unique differentiating system in several research and clinical areas are discussed, with special emphasis on the steps required to fully harness their potential for myocardial regeneration strategies.

Background— Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a primary heart muscle disorder resulting from desmosomal protein mutations. ARVC is characterized pathologically by fibrofatty infiltration and clinically by arrhythmias and sudden cardiac death. We aimed to establish a patient-/disease-specific human induced pluripotent stem cell (hiPSC) model of ARVC. Methods and Results— Dermal fibroblasts were obtained from 2 patients with ARVC with plakophilin-2 ( PKP2 ) mutations, reprogrammed to generate hiPSCs, coaxed to differentiate into cardiomyocytes (CMs), and then compared with healthy control hiPSC-derived CMs (hiPSC-CMs). Real-time polymerase chain reaction showed a significant decrease in the expression of PKP2 in the ARVC-hiPSC-CMs. Immunostainings revealed reduced densities of PKP2, the associated desmosomal protein plakoglobin, and the gap-junction protein connexin-43. Electrophysiological assessment demonstrated prolonged field potential rise time in the ARVC-hiPSC-CMs. Transmission electron microscopy identified widened and distorted desmosomes in the ARVC-hiPSC-CMs. Clusters of lipid droplets were identified in the ARVC-CMs that displayed the more severe desmosomal pathology. This finding was associated with upregulation of the proadipogenic transcription factor peroxisome proliferator-activated receptor-γ. Exposure of the cells to apidogenic stimuli augmented desmosomal distortion and lipid accumulation. The latter phenomenon was prevented by application of a specific inhibitor of glycogen synthase kinase 3β (6-bromoindirubin-3'-oxime). Conclusions— This study highlights the unique potential of the hiPSC technology for modeling inherited cardiac disorders in general and ARVC specifically. The hiPSC-CMs were demonstrated to recapitulate the ARVC phenotype in the dish, provide mechanistic insights into early disease pathogenesis, and provide a unique platform for drug discovery and testing in this disorder.

Cell replacement strategies are promising interventions aiming to improve myocardial performance. Yet, the electrophysiological impact of these approaches has not been elucidated. We assessed the electrophysiological consequences of grafting of two candidate cell types, that is, skeletal myoblasts and human embryonic stem cell-derived cardiomyocytes (hESC-CMs). The fluorescently labeled (DiO) candidate cells were grafted into the rat's left ventricular myocardium. Two weeks later, optical mapping was performed using the Langendorff-perfused rat heart preparation. Images were obtained with appropriate filters to delineate the heart's anatomy, to identify the DiO-labeled cells, and to associate this information with the voltage-mapping data (using the voltage-sensitive dye PGH-I). Histological examination revealed the lack of gap junctions between grafted skeletal myotubes and host cardiomyocytes. In contrast, positive Cx43 immunostaining was observed between donor and host cardiomyocytes in the hESC-CMs-transplanted hearts. Optical mapping demonstrated either normal conduction (four of six) or minimal conduction slowing (two of six) at the hESC-CMs engraftment sites. In contrast, marked slowing of conduction or conduction block was seen (seven of eight) at the myoblast transplantation sites. Ventricular arrhythmias could not be induced in the hESC-CM hearts following programmed electrical stimulation but were inducible in 50% of the myoblast-engrafted hearts. In summary, a unique method for assessment of the electrophysiological impact of myocardial cell therapy is presented. Our results demonstrate the ability of hESC-CMs to functionally integrate with host tissue. In contrast, transplantation of cells that do not form gap junctions (skeletal myoblats) led to localized conduction disturbances and to the generation of a proarrhythmogenic substrate.

A decade has passed since the initial derivation of human embryonic stem cells (hESC). The ensuing years have witnessed a significant progress in the development of methodologies allowing cell cultivation, differentiation, genetic manipulation, and in vivo transplantation. Specifically, the potential to derive human cardiomyocytes from the hESC lines, which can be used for several basic and applied cardiovascular research areas including in the emerging field of cardiac regenerative medicine, attracted significant attention from the scientific community. This resulted in the development of protocols for the cultivation of hESC and their successful differentiation toward the cardiomyocyte lineage fate. In this chapter, we will describe in detail methods related to the cultivation, genetic manipulation, selection, and in vivo transplantation of hESC-derived cardiomyocytes.

The derivation of the hESC lines and the resulting cardiomyocyte differentiation system may bring a unique value to several basic and applied research fields. Research based on the cells may help to elucidate the mechanisms involved in early human cardiac lineage commitment, differentiation, and maturation. Moreover, this research may promote the discovery of novel growth and transcriptional factors using gene trapping techniques, functional genomics, and proteomics as well as providing a novel in vitro model for drug development and testing. Finally, the ability to generate, in vitro for the first time, human cardiac tissue provides an exciting and promising cell source for the emerging discipline of regenerative medicine and myocardial repair.

Cell therapy is emerging as a promising strategy for myocardial repair. This approach is hampered, however, by the lack of sources for human cardiac tissue and by the absence of direct evidence for functional integration of donor cells into host tissues. Here we investigate whether cells derived from human embryonic stem (hES) cells can restore myocardial electromechanical properties. Cardiomyocyte cell grafts were generated from hES cells in vitro using the embryoid body differentiating system. This tissue formed structural and electromechanical connections with cultured rat cardiomyocytes. In vivo integration was shown in a large-animal model of slow heart rate. The transplanted hES cell-derived cardiomyocytes paced the hearts of swine with complete atrioventricular block, as assessed by detailed three-dimensional electrophysiological mapping and histopathological examination. These results demonstrate the potential of hES-cell cardiomyocytes to act as a rate-responsive biological pacemaker and for future myocardial regeneration strategies.

Human embryonic stem cells are capable of unlimited proliferation in culture in the undifferentiated state and under the proper conditions can differentiate into different cell types including spontaneously beating cardiac myocytes (Kehat et al. 2001, 2002). Spontaneous beating or automaticity is not normally exhibited by mature atrial or ventricular myocytes. In contrast, embryonic heart cells display spontaneous activity (DeHaan & Gottlieb, 1968). The main requirement for automaticity is the presence of inward current at diastolic potentials. Although two recent reports of human embryonic stem cell-derived cardiac myocytes (hES-CMs) document action potentials (He et al. 2003; Mummery et al. 2003), there are no studies of ionic currents in hES-CMs. Moreover, the more thoroughly studied mouse ES-derived CMs exhibit AP morphologies that broadly reflect either atrial-like, ventricular-like, or nodal-like parameters (Hescheler et al. 1999; Sachinidis et al. 2003) that differ markedly from their human counterparts. The cardiac Na+ channel (termed NaV1.5) is expressed in relatively high density on surface membrane of mature heart cells in atria and ventricle. Despite the high NaV1.5 expression these cell types are not normally automatic because a high density of inward rectifier K+ channels (Kir) clamps the membrane potential to a value near the K+ reversal potential. At such hyperpolarized potentials the NaV1.5 channel's open probability approaches 0. In quiescent, mature heart cells, the initiating depolarization originates from neighbouring cells via gap junctions. Depolarization of membrane potential (Vm) activates NaV1.5 rapidly, driving the rapid AP upstroke, and within a few milliseconds of sustained depolarization NaV1.5 inactivates. Thus, NaV1.5 serves the role of generating a pathway for a rapid influx of depolarizing current. The maximum diastolic potential (MDP) is a key control point for NaV1.5, if MDP is relatively depolarized then NaV1.5 will be largely inactivated and unable to contribute to the AP upstroke. For this reason, there is a correlation between Na+ channel current density (not simply channel density), and the maximum upstroke velocity of the cellular AP (dV/dtmax). Moreover, in the developing heart there is a an increase in dV/dtmax from < 20 to 100–150 V s−1 that is concomitant with the onset of TTX sensitivity (McDonald et al. 1973), an increase in Na+ current density (Fujii et al. 1988), and a negative shift in the MDP (McDonald et al. 1973; DeHaan, 1980; Sperelakis, 1984). Given the exciting potential use of hES-CMs as replacement tissue in diseased heart (Gepstein, 2002; Kehat & Gepstein, 2003), and as an in vitro model for the study of early human cardiac development it is important to characterize their functional properties. We assessed the electrical properties of these cells in spontaneously beating embryoid bodies (EBs) using a multielectrode array mapping technique and detailed patch-clamp recordings and pharmacologically dissected the critical pathways in these structures. Our results provide the first description of the ionic currents in hES-CMs and show that the basis for spontaneous electrical activity in these cells is the absence of Kir conductance, a phenomenon that provides the substrate for a relatively large voltage-gated Na+ current to drive activity.