Lior Gepstein

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.

A Novel Method for Nonfluoroscopic Catheter-Based Electroanatomical Mapping of the Heart: In Vitro and In Vivo Accuracy Results

BACKGROUND Cardiac mapping is essential for understanding the mechanisms of arrhythmias and for directing curative procedures. A major limitation of the current methods is the inability to accurately relate local electrograms to their spatial orientation. The objective of this study was to present and test the accuracy of a new method for nonfluoroscopic, catheter-based, endocardial mapping. METHODS AND RESULTS The method is based on using a new locatable catheter connected to an endocardial mapping and navigating system. The system uses magnetic technology to accurately determine the location and orientation of the catheter and simultaneously records the local electrogram from its tip. By sampling a plurality of endocardial sites, the system reconstructs the three-dimensional geometry of the chamber, with the electrophysiological information color-coded and superimposed on the anatomy. The accuracy of the system was tested in both in vitro and in vivo studies and was found to be highly reproducible (SD, 0.16 +/- 0.02 [mean +/- SEM] and 0.74 +/- 0.13 mm) and accurate (mean errors, 0.42 +/- 0.05 and 0.73 +/- 0.03 mm). In further studies, electroanatomical mapping of the cardiac chambers was performed in 34 pigs. Both the geometry and activation sequence were repeatable in all pigs. CONCLUSIONS The new mapping method is highly accurate and reproducible. The ability to combine electrophysiological and spatial information provides a unique tool for both research and clinical electrophysiology. Consequently, the main shortcomings of conventional mapping-namely, prolonged x-ray exposure, low spatial resolution, and the inability to accurately navigate to a predefined site-can all be overcome with this new method.

Modelling the long QT syndrome with induced pluripotent stem cells

The ability to generate patient-specific human induced pluripotent stem cells (iPSCs) offers a new paradigm for modelling human disease and for individualizing drug testing. Congenital long QT syndrome (LQTS) is a familial arrhythmogenic syndrome characterized by abnormal ion channel function and sudden cardiac death. Here we report the development of a patient/disease-specific human iPSC line from a patient with type-2 LQTS (which is due to the A614V missense mutation in the KCNH2 gene). The generated iPSCs were coaxed to differentiate into the cardiac lineage. Detailed whole-cell patch-clamp and extracellular multielectrode recordings revealed significant prolongation of the action-potential duration in LQTS human iPSC-derived cardiomyocytes (the characteristic LQTS phenotype) when compared to healthy control cells. Voltage-clamp studies confirmed that this action-potential-duration prolongation stems from a significant reduction of the cardiac potassium current IKr. Importantly, LQTS-derived cells also showed marked arrhythmogenicity, characterized by early-after depolarizations and triggered arrhythmias. We then used the LQTS human iPSC-derived cardiac-tissue model to evaluate the potency of existing and novel pharmacological agents that may either aggravate (potassium-channel blockers) or ameliorate (calcium-channel blockers, KATP-channel openers and late sodium-channel blockers) the disease phenotype. Our study illustrates the ability of human iPSC technology to model the abnormal functional phenotype of an inherited cardiac disorder and to identify potential new therapeutic agents. As such, it represents a promising paradigm to study disease mechanisms, optimize patient care (personalized medicine), and aid in the development of new therapies.

Tissue Engineering of Vascularized Cardiac Muscle From Human Embryonic Stem Cells

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.

Cardiomyocyte Differentiation of Human Induced Pluripotent Stem Cells

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 (IKr blocker), and chromanol 293B (IKs 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.

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)

Derivation and Potential Applications of Human Embryonic Stem Cells

Embryonic stem cells are pluripotent cell lines that are derived from the blastocyst-stage early mammalian embryo. These unique cells are characterized by their capacity for prolonged undifferentiated proliferation in culture while maintaining the potential to differentiate into derivatives of all three germ layers. During in vitro differentiation, embryonic stem cells can develop into specialized somatic cells, including cardiomyocytes, and have been shown to recapitulate many processes of early embryonic development. The present review describes the derivation and unique properties of the recently described human embryonic stem cells as well as the properties of cardiomyocytes derived using this unique differentiating system. The possible applications of this system in several cardiac research areas, including developmental biology, functional genomics, pharmacological testing, cell therapy, and tissue engineering, are discussed. Because of their combined ability to proliferate indefinitely and to differentiate to mature tissue types, human embryonic stem cells can potentially provide an unlimited supply of cardiomyocytes for cell therapy procedures aiming to regenerate functional myocardium. However, many obstacles must still be overcome on the way to successful clinical utilization of these cells.