Izhak Kehat

Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes

The study of human cardiac tissue development is hampered by the lack of a suitable in vitro model. We describe the phenotypic properties of cardiomyocytes derived from human embryonic stem (ES) cells. Human ES cells were cultivated in suspension and plated to form aggregates termed embryoid bodies (EBs). Spontaneously contracting areas appeared in 8.1% of the EBs. Cells from the spontaneously contracting areas within EBs were stained positively with anti-cardiac myosin heavy chain, anti--alpha-actinin, anti-desmin, anti--cardiac troponin I (anti-cTnI), and anti-ANP antibodies. Electron microscopy revealed varying degrees of myofibrillar organization, consistent with early-stage cardiomyocytes. RT-PCR studies demonstrated the expression of several cardiac-specific genes and transcription factors. Extracellular electrograms were characterized by a sharp component lasting 30 +/- 25 milliseconds, followed by a slow component of 347 +/- 120 milliseconds. Intracellular Ca(2+) transients displayed a sharp rise lasting 130 +/- 27 milliseconds and a relaxation component lasting 200--300 milliseconds. Positive and negative chronotropic effects were induced by application of isoproterenol and carbamylcholine, respectively. In conclusion, the human ES cell--derived cardiomyocytes displayed structural and functional properties of early-stage cardiomyocytes. Establishment of this unique differentiation system may have significant impact on the study of early human cardiac differentiation, functional genomics, pharmacological testing, cell therapy, and tissue engineering.

Electromechanical integration of cardiomyocytes derived from human embryonic stem cells

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.

High-Resolution Electrophysiological Assessment of Human Embryonic Stem Cell-Derived Cardiomyocytes A Novel In Vitro Model for the Study of Conduction

The goal of the present report was to establish a new in vitro model for the study of impulse propagation in human cardiac tissue. By using the human embryonic stem cell differentiating system, spontaneously contracting areas were generated in three-dimensional differentiating cell aggregates (embryoid bodies). Morphological analysis revealed an isotropic tissue of early-stage cardiac phenotype. Gap junctions, assessed by immunostaining of connexin43 and connexin45, were distributed along the cell borders. High-resolution activation maps demonstrated the presence of a functional syncytium with stable focal activation and conduction properties. Conduction was significantly slower in narrow bands of contracting tissue compared with broad cardiomyocyte regions. Establishment of this unique in vitro human model may be used for the assessment of long-term structure-function relationships, for pharmacological studies, for tissue engineering, and may permit the study of genetically modified cardiomyocytes.

Mechanism of spontaneous excitability in human embryonic stem cell derived cardiomyocytes

Human embryonic stem cell‐derived cardiomyocytes (hES‐CMs) are thought to recapitulate the embryonic development of heart cells. Given the exciting potential of hES‐CMs as replacement tissue in diseased hearts, we investigated the pharmacological sensitivity and ionic current of mid‐stage hES‐CMs (20–35 days post plating). A high‐resolution microelectrode array was used to assess conduction in multicellular preparations of hES‐CMs in spontaneously contracting embryoid bodies (EBs). TTX (10 μm) dramatically slowed conduction velocity from 5.1 to 3.2 cm s−1 while 100 μm TTX caused complete cessation of spontaneous electrical activity in all EBs studied. In contrast, the Ca2+ channel blockers nifedipine or diltiazem (1 μm) had a negligible effect on conduction. These results suggested a prominent Na+ channel current, and therefore we patch‐clamped isolated cells to record Na+ current and action potentials (APs). We found for isolated hES‐CMs a prominent Na+ current (244 ± 42 pA pF−1 at 0 mV; n= 19), and a hyperpolarization‐activated current (HCN), but no inward rectifier K+ current. In cell clusters, 3 μm TTX induced longer AP interpulse intervals and 10 μm TTX caused cessation of spontaneous APs. In contrast nifedipine (Ca2+ channel block) and 2 mm Cs+ (HCN complete block) induced shorter AP interpulse intervals. In single cells, APs stimulated by current pulses had a maximum upstroke velocity (dV/dtmax) of 118 ± 14 V s−1 in control conditions; in contrast, partial block of Na+ current significantly reduced stimulated dV/dtmax (38 ± 15 V s−1). RT‐PCR revealed NaV1.5, CaV1.2, and HCN‐2 expression but we could not detect Kir2.1. We conclude that hES‐CMs at mid‐range development express prominent Na+ current. The absence of background K+ current creates conditions for spontaneous activity that is sensitive to TTX in the same range of partial block of NaV1.5; thus, the NaV1.5 Na+ channel is important for initiating spontaneous excitability in hES‐derived heart cells.

Identification and selection of cardiomyocytes during human embryonic stem cell differentiation

Human embryonic stem cells (hESC) are pluripotent lines that can differentiate in vitro into cell derivatives of all three germ layers, including cardiomy‐ocytes. 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 (purity>95%, viability>85%). 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.—Huber, I., Itzhaki, I., Caspi, O., Arbel, G., Tzukerman, M., Gepstein, A., Habib, M., Yankelson, L., Kehat, I., Gepstein, L. Identification and selection of cardiomy‐ocytes during human embryonic stem cell differentiation. FASEB J. 21, 2551–2563 (2007)

In vitro electrophysiological drug testing using human embryonic stem cell derived cardiomyocytes.

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-CMs 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.

Extracellular Signal-Regulated Kinases 1 and 2 Regulate the Balance Between Eccentric and Concentric Cardiac Growth

Rationale: An increase in cardiac afterload typically produces concentric hypertrophy characterized by an increase in cardiomyocyte width, whereas volume overload or exercise results in eccentric growth characterized by cellular elongation and addition of sarcomeres in series. The signaling pathways that control eccentric versus concentric heart growth are not well understood. Objective: To determine the role of extracellular signal-regulated kinase 1 and 2 (ERK1/2) in regulating the cardiac hypertrophic response. Methods and Results: Here, we used mice lacking all ERK1/2 protein in the heart (Erk1−/− Erk2fl/fl-Cre) and mice expressing activated mitogen-activated protein kinase kinase (Mek)1 in the heart to induce ERK1/2 signaling, as well as mechanistic experiments in cultured myocytes to assess cellular growth characteristics associated with this signaling pathway. Although genetic deletion of all ERK1/2 from the mouse heart did not block the cardiac hypertrophic response per se, meaning that the heart still increased in weight with both aging and pathological stress stimulation, it did dramatically alter how the heart grew. For example, adult myocytes from hearts of Erk1−/− Erk2fl/fl-Cre mice showed preferential eccentric growth (lengthening), whereas myocytes from Mek1 transgenic hearts showed concentric growth (width increase). Isolated adult myocytes acutely inhibited for ERK1/2 signaling by adenoviral gene transfer showed spontaneous lengthening, whereas infection with an activated Mek1 adenovirus promoted constitutive ERK1/2 signaling and increased myocyte thickness. A similar effect was observed in engineered heart tissue under cyclic stretching, where ERK1/2 inhibition led to preferential lengthening. Conclusions: Taken together, these data demonstrate that the ERK1/2 signaling pathway uniquely regulates the balance between eccentric and concentric growth of the heart.Rationale An increase in cardiac afterload typically produces concentric hypertrophy characterized by an increase in cardiomyocyte width, while volume overload or exercise results in eccentric growth characterized by cellular elongation and addition of sarcomeres in series. The signaling pathways that control eccentric versus concentric heart growth are not well understood.

ERK1/2 regulate the balance between eccentric and concentric cardiac growth

Rationale: An increase in cardiac afterload typically produces concentric hypertrophy characterized by an increase in cardiomyocyte width, whereas volume overload or exercise results in eccentric growth characterized by cellular elongation and addition of sarcomeres in series. The signaling pathways that control eccentric versus concentric heart growth are not well understood. Objective: To determine the role of extracellular signal-regulated kinase 1 and 2 (ERK1/2) in regulating the cardiac hypertrophic response. Methods and Results: Here, we used mice lacking all ERK1/2 protein in the heart (Erk1−/− Erk2fl/fl-Cre) and mice expressing activated mitogen-activated protein kinase kinase (Mek)1 in the heart to induce ERK1/2 signaling, as well as mechanistic experiments in cultured myocytes to assess cellular growth characteristics associated with this signaling pathway. Although genetic deletion of all ERK1/2 from the mouse heart did not block the cardiac hypertrophic response per se, meaning that the heart still increased in weight with both aging and pathological stress stimulation, it did dramatically alter how the heart grew. For example, adult myocytes from hearts of Erk1−/− Erk2fl/fl-Cre mice showed preferential eccentric growth (lengthening), whereas myocytes from Mek1 transgenic hearts showed concentric growth (width increase). Isolated adult myocytes acutely inhibited for ERK1/2 signaling by adenoviral gene transfer showed spontaneous lengthening, whereas infection with an activated Mek1 adenovirus promoted constitutive ERK1/2 signaling and increased myocyte thickness. A similar effect was observed in engineered heart tissue under cyclic stretching, where ERK1/2 inhibition led to preferential lengthening. Conclusions: Taken together, these data demonstrate that the ERK1/2 signaling pathway uniquely regulates the balance between eccentric and concentric growth of the heart.Rationale An increase in cardiac afterload typically produces concentric hypertrophy characterized by an increase in cardiomyocyte width, while volume overload or exercise results in eccentric growth characterized by cellular elongation and addition of sarcomeres in series. The signaling pathways that control eccentric versus concentric heart growth are not well understood.

Differentiation Pathways in Human Embryonic Stem Cell‐Derived Cardiomyocytes

Abstract: Human embryonic stem (hES) cells are pluripotent cell lines derived from the inner cell mass of the blastocyst‐stage embryo. These unique cell lines can be propagated in the undifferentiated state in culture, while retaining the capacity to differentiate into derivatives of all three germ layers, including cardiomyocytes. The derivation of the hES cell lines presents a powerful tool to explore the early events of cardiac progenitor cell specification and differentiation, and it also provides a novel cell source for the emerging field of cardiovascular regenerative medicine. A spontaneous differentiation system of these stem cells to cardiomyocytes was established and the generated myocytes displayed molecular, structural, and functional properties of early‐stage heart cells. In order to follow the in vitro differentiation process, the temporal expression of signaling molecules and transcription factors governing early cardiac differentiation was examined throughout the process. A characteristic pattern was noted recapitulating the normal in vivo cardiac differentiation scheme observed in other model systems. This review discusses the known pathways involved in cardiac specification and the possible factors that may be used to enhance cardiac differentiation of hES cells, as well as the steps required to fully harness the enormous potential of these unique cells.

Defined engineered human myocardium with advanced maturation for applications in heart failure modelling and repair

Background: Advancing structural and functional maturation of stem cell–derived cardiomyocytes remains a key challenge for applications in disease modeling, drug screening, and heart repair. Here, we sought to advance cardiomyocyte maturation in engineered human myocardium (EHM) toward an adult phenotype under defined conditions. Methods: We systematically investigated cell composition, matrix, and media conditions to generate EHM from embryonic and induced pluripotent stem cell–derived cardiomyocytes and fibroblasts with organotypic functionality under serum-free conditions. We used morphological, functional, and transcriptome analyses to benchmark maturation of EHM. Results: EHM demonstrated important structural and functional properties of postnatal myocardium, including: (1) rod-shaped cardiomyocytes with M bands assembled as a functional syncytium; (2) systolic twitch forces at a similar level as observed in bona fide postnatal myocardium; (3) a positive force-frequency response; (4) inotropic responses to &bgr;-adrenergic stimulation mediated via canonical &bgr;1- and &bgr;2-adrenoceptor signaling pathways; and (5) evidence for advanced molecular maturation by transcriptome profiling. EHM responded to chronic catecholamine toxicity with contractile dysfunction, cardiomyocyte hypertrophy, cardiomyocyte death, and N-terminal pro B-type natriuretic peptide release; all are classical hallmarks of heart failure. In addition, we demonstrate the scalability of EHM according to anticipated clinical demands for cardiac repair. Conclusions: We provide proof-of-concept for a universally applicable technology for the engineering of macroscale human myocardium for disease modeling and heart repair from embryonic and induced pluripotent stem cell–derived cardiomyocytes under defined, serum-free conditions.