Embryonic Stem Cell-derived Cardiac Myocytes Research Articles

Calreticulin Is Required for TGF-β-Induced Epithelial-to-Mesenchymal Transition during Cardiogenesis in Mouse Embryonic Stem Cells.

SummaryCalreticulin, a multifunctional endoplasmic reticulum resident protein, is required for TGF-β-induced epithelial-to-mesenchymal transition (EMT) and subsequent cardiomyogenesis. Using embryoid bodies (EBs) derived from calreticulin-null and wild-type (WT) embryonic stem cells (ESCs), we show that expression of EMT and cardiac differentiation markers is induced during differentiation of WT EBs. This induction is inhibited in the absence of calreticulin and can be mimicked by inhibiting TGF-β signaling in WT cells. The presence of calreticulin in WT cells permits TGF-β-mediated signaling via AKT/GSK3β and promotes repression of E-cadherin by SNAIL2/SLUG. This is paralleled by induction of N-cadherin in a process known as the cadherin switch. We show that regulated Ca2+ signaling between calreticulin and calcineurin is critical for the unabated TGF-β signaling that is necessary for the exit from pluripotency and the cadherin switch during EMT. Calreticulin is thus a key mediator of TGF-β-induced commencement of cardiomyogenesis in mouse ESCs.

Bridging Functional and Structural Cardiotoxicity Assays Using Human Embryonic Stem Cell-Derived Cardiomyocytes for a More Comprehensive Risk Assessment.

More relevant and reliable preclinical cardiotoxicity tests are required to improve drug safety and reduce the cost of drug development. Current in vitro testing strategies predominantly take the form of functional assays to predict the potential for drug-induced ECG abnormalities in vivo. Cardiotoxicity can also be structural in nature, so a full and efficient assessment of cardiac liabilities for new chemical entities should account for both these phenomena. As well as providing a more appropriate nonclinical model for in vitro cardiotoxicity testing, human stem cell-derived cardiomyocytes offer an integrated system to study drug impact on cardiomyocyte structure as well as function. Employing human embryonic stem cell-derived cardiacmyocytes (hESC-CMs) on 3 assay platforms with complementary insights into cardiac biology (multielectrode array assay, electrophysiology; impedance assay, cell movement/beating; and high content analysis assay, subcellular structure) we profiled a panel of 13 drugs with well characterized cardiac liabilities (Amiodarone, Aspirin, Astemizole, Axitinib, AZT, Bepridil, Doxorubicin, E-4031, Mexiletine, Rosiglitazone, Sunitinib, Sibutramine, and Verapamil). Our data show good correlations with previous studies and reported clinical observations. Using multiparameter phenotypic profiling techniques we demonstrate the dynamic relationship that exists between functional and structural toxicity, and the benefits of this more holistic approach to risk assessment. We conclude by showing for the first time how the advent of transparent MEA plate technology enables functional and structural cardiotoxic responses to be recorded from the same cell population. This approach more directly links changes in morphology of the hESC-CMs with recorded electrophysiology signatures, offering even greater insight into the wide range of potential drug impacts on cardiac physiology, with a throughput that is more amenable to early drug discovery.

Downregulation of beta-microglobulin to diminish T-lymphocyte lysis of non-syngeneic cell sources of engineered heart tissue constructs

The presence of non-autologous major histocompatibility complex class I (MHC-I) molecules on the surface of the grafted cells is one of the main reasons for their rejection in non-syngeneic hosts. We present a straightforward strategy to decrease the presence of MHC-I by shRNA inhibition of beta-2-microglobulin (B2M), a conservative light chain of MHC-I, on the surface of two main cell types that are used to engineer heart tissue constructs.Engineered heart tissue constructs can be generated by combining mouse WT19 fibroblasts and mouse embryonic stem cell-derived cardiac myocytes (mESC-CM). WT19 fibroblasts were stably transduced with an anti-B2M shRNA, which yielded a cell line with dramatically reduced B2M expression levels (16 ± 11% of mock treated control cell line). Interferon gamma treatment increased the levels of B2M expression by >3-fold in both control and transduced fibroblasts; yet, B2M expression levels still remained very low in the transduced cells. When compared with their unmodified counterparts, transduced fibroblasts caused 5.7-fold lesser activation of cognate T-cells. B2M depletion in mESC-CM was achieved by 72 h transduction with anti-B2M shRNA lentiviral particles. Transduced mESC-CM exhibited regular beating and expressed classical cardiac markers. When compared with their unmodified counterparts, transduced mESC-CM caused 2.5-fold lesser activation of cognate T-cells. In vivo assessment of B2M downregulation was performed by analyzing the preferential survival of B2M-downregulated cells in the intraperitoneal cavity of allogeneic mice. Both B2M-downregulated fibroblasts and B2M-downregulated myocytes survived significantly better when compared to their unmodified counterparts (2.01 ± 0.4 and 5.07 ± 1.6 fold increase in survival, respectively). In contrast, when modified WT19 fibroblasts were injected into the intraperitoneal cavity of syngeneic C57Bl/6 mice, no significant survival advantage was observed. Notably, the preferential survival of B2M-downregulated cells persisted in allogeneic hosts with normal levels of natural killer cells, although the effect was lesser in magnitude.Use of shRNA against beta-2-microglobulin offers a simple and effective approach to minimize immunogenicity of the main cellular components of cardiac tissue constructs in non-syngeneic recipients.

Mouse embryonic stem cell-derived cardiac myocytes in a cell culture dish.

Embryonic stem (ES) cells are pluripotent stem cells capable of self-renewal and have broad differentiation potential yielding cell types from all three germ layers. In the absence of differentiation inhibitory factors, when cultured in suspension, ES cells spontaneously differentiate and form three-dimensional cell aggregates termed embryoid bodies (EBs). Although various methods exist for the generation of EBs, the hanging drop method offers reproducibility and homogeneity from a predetermined number of ES cells. Herein, we describe the in vitro differentiation of mouse embryonic stem cells into cardiac myocytes using the hanging drop method and immunocytochemistry to identify cardiomyogenic differentiation. In brief, ES cells, placed in droplets on the lid of culture dishes following a 2-day incubation, yield embryoid bodies, which are resuspended and plated. 1-2 weeks following plating of the EBs, spontaneous beating areas can be observed and staining for specific cardiac markers can be achieved.

Mechanisms Underlying Spontaneous Beating in Human Embryonic Stem Cell-Derived Cardiac Myocytes

Cardiac myocytes derived from human embryonic stem cells (hESC-CMs) are increasingly considered a promising therapy for a variety of pathologies, but many questions persist regarding the physiology of these cells and how it changes during differentiation. We sought to determine the mechanisms underlying spontaneous beating in hESC-CMs derived from HES2 line. Enrichment for developing cardiomyocytes was achieved by exposing differentiating Embryoid Bodies (EBs) to the following growth factors: BMP4, DKK and VEGF, and DKK, VEGF and bFGF.

Gene-trapped mouse embryonic stem cell-derived cardiac myocytes and human genetics implicate AKAP10 in heart rhythm regulation

Sudden cardiac death due to abnormal heart rhythm kills 400,000-460,000 Americans each year. To identify genes that regulate heart rhythm, we are developing a screen that uses mouse embryonic stem cells (mESCs) with gene disruptions that can be differentiated into cardiac cells for phenotyping. Here, we show that the heterozygous disruption of the Akap10 (D-AKAP2) gene that disrupts the final 51 aa increases the contractile response of cultured cardiac cells to cholinergic signals. In both heterozygous and homozygous mutant mice derived from these mESCs, the same Akap10 disruption increases the cardiac response to cholinergic signals, suggesting a dominant interfering effect of the Akap10 mutant allele. The mutant mice have cardiac arrhythmias and die prematurely. We also found that a common variant of AKAP10 in humans (646V, 40% of alleles) was associated with increased basal heart rate and decreased heart rate variability (markers of low cholinergic/vagus nerve sensitivity). These markers predict an increased risk of sudden cardiac death. Although the molecular mechanism remains unknown, our findings in mutant mESCs, mice, and a common human AKAP10 SNP all suggest a role for AKAP10 in heart rhythm control. Our stem cell-based screen may provide a means of identifying other genes that control heart rhythm.

Calcium Handling in Embryonic Stem Cell–Derived Cardiac Myocytes

Excitation-contraction (EC) coupling is fundamental to the function of cardiac myocytes (CMs). In mature myocytes plasma membrane (PM) L-type Ca(2+) channels function in close juxtaposition to ryanodine receptors (RyR) on the sarcoplasmic reticulum (SR) membrane. Action potentials (APs) cause the opening of PM L-type Ca(2+) channels, which in turn provide trigger Ca(2+) for a larger RyR-mediated SR Ca(2+) release. In contrast, developing myocytes have a less well-developed SR. This incomplete development is observed in early stage and mid-maturation stages of murine embryonic stem cell-derived cardiac myocytes (ESC-CMs). Despite the absence of a well-developed t-tubule system, murine ESC-CMs use internal Ca(2+) stores for EC coupling. Direct measures of Ca(2+) handling, including pharmacological studies and investigation of genetically modified mouse ESC-CMs, established an important contribution of RyR-mediated internal Ca(2+) store to cell function. Similarly, early-stage human ESC-CMs use internal Ca(2+) store and partially share Ca(2+) handling characteristics with murine ESC-CMs. For example, elementary Ca(2+) release events are present in both murine and human ESC-CMs, and it is likely that Ca(2+) handling contributes to automatic rhythm generation in these cells. However, in human ESC-CMs, a unique voltage-gated Na(+) channel window current is critical for spontaneous, rhythmic depolarization. The advent of the murine and human ES cardiomyocyte differentiating systems has provided initial insights into the early steps of development of excitability and electromechanical coupling in the mammalian heart, including patterns of gene expression, myofibrillogenesis, ion channel development and function, and Ca(2+) handling. Here we discuss the information gained from these models to describe the nexus of voltage-gated channel currents and Ca(2+) handling on rhythmic activity.

02 Selection of embryonic stem cell-derived cardiac myocytes for transfer to myocardium

02 Selection of embryonic stem cell-derived cardiac myocytes for transfer to myocardium

MEKK1 suppresses oxidative stress-induced apoptosis of embryonic stem cell-derived cardiac myocytes

A combination of in vitro embryonic stem (ES) cell differentiation and targeted gene disruption has defined complex regulatory events underlying oxidative stress-induced cardiac apoptosis, a model of postischemic reperfusion injury of myocardium. ES cell-derived cardiac myocytes (ESCM) having targeted disruption of the MEKK1 gene were extremely sensitive, relative to wild-type ESCM, to hydrogen peroxide-induced apoptosis. In response to oxidative stress, MEKK1-/- ESCM failed to activate c-Jun kinase (JNK) but did activate p38 kinase similar to that observed in wild-type ESCM. The increased apoptosis was mediated through enhanced tumor necrosis factor alpha production, a response that was positively and negatively regulated by p38 and the MEKK1-JNK pathway, respectively. Thus, MEKK1 functions in the survival of cardiac myocytes by inhibiting the production of a proapoptotic cytokine. MEKK1 regulation of the JNK pathway is a critical response for the protection against oxidative stress-induced apoptosis in cardiac myocytes.

Gene transfer into mouse embryonic stem cell-derived cardiac myocytes mediated by recombinant adenovirus.

The main purpose of this study was to examine, for the first time, the ability of recombinant adenovirus to mediate gene transfer into cardiac myocytes derived from mouse embryonic stem (ES) cells differentiating in vitro. In addition, observations were made on the effect of adenovirus infection on cardiac myocyte differentiation and contractility in this in vitro system of cardiogenesis. ES cell cultures were infected at various times of differentiation with a recombinant adenovirus vector (AdCMVlacZ) containing the bacterial lacZ gene under the control of the cytomegalovirus (CMV) promoter. Expression of the lacZ reporter gene was determined by histochemical staining for beta-galactosidase activity. LacZ expression was not detected in undifferentiated ES cells infected with AdCMVlacZ. In contrast, infection of differentiating ES cell cultures showed increasing transgene expression with continued time in culture. Expression in ES-cell-derived cardiac myocytes was demonstrated by codetection of beta-galactosidase activity and troponin T with indirect immunofluorescence. At 24 h postinfection, approximately 27% of the cardiac myocytes were beta-galactosidase positive, and lacZ gene expression appeared to be stable for up to 21 d postinfection. Adenovirus infection had no apparent effect on the onset, extent, or duration of spontaneously contracting ES-cell-derived cardiomyocytes, indicating that cardiac differentiation and contractile function were not significantly altered in the infected cultures. The demonstration of adenovirus-mediated gene transfer into ES-cell-derived cardiac myocytes will aid studies of gene expression with this in vitro model of cardiogenesis and may facilitate future studies involving the use of these myocytes for grafting experiments in vivo.

Ultrastructure and cell-cell coupling of cardiac myocytes differentiating in embryonic stem cell cultures.

Differentiation cultures of embryonic stem (ES) cells can be a useful in vitro system for understanding cardiac myocyte development. However, cell morphometry, sarcomere development, and functional cell-cell junction formation have not been examined in detail to determine whether ES cell-derived cardiac myocytes exhibit structural and functional characteristics similar to cardiac myocytes within the developing heart. Therefore, we examined cellular dimensions, sarcomere formation, and cell-cell contacts in differentiating cardiac myocytes derived from mouse D3-ES cell cultures. Cells exhibited rod-shaped morphology and had single centrally located nuclei, typical of maturing cardiac myocytes. The cellular dimensions of 59 individual cardiac myocytes within contracting foci of ES cell cultures were analyzed (length = 42.2 +/- 2.1 microns, area = 197 +/- 19 microns2, and diameter = 5.5 +/- 0.3 microns) and found to be similar to myocytes in vivo. Transmission electron micrographs of ES cell-derived cardiac myocytes indicated myofibrillar architecture ranged from sparse and disorganized to densely packed, parallel arrays of myofibrils organized into mature sarcomeres. This pattern of myofibrillar assembly in maturing sarcomeres was similar to that observed during in vivo myocyte differentiation. Another hallmark of cardiac development is the formation of intercalated discs, which functionally couple adjacent cardiac myocytes. Electron micrographs indicated nascent intercalated discs were forming in foci of ES cell-derived cardiac myocytes. In addition, indirect immunostaining with anti-connexin 43 antibody (Ab), a monoclonal Ab to the gap junction component of the intercalated disc, indicated that gap junctions were present in contracting ES cell foci. Furthermore, microinjection of single cardiac myocytes with Lucifer yellow (2.5 microM) resulted in the spread of fluorescence to adjacent cells within a contracting focus, an indication of functional cell-cell coupling across these gap junctions. Together, these results indicate ES cell-derived cardiac myocytes exhibit cell morphology, sarcomere formation, and cell-cell junctions similar to those observed in cardiac myocytes developing in vivo.

Troponin I isoform expression is developmentally regulated in differentiating embryonic stem cell-derived cardiac myocytes.

We studied troponin I (TnI) isoform expression in the mouse embryonic stem (ES) cell model of cardiogenesis as an essential first step to understanding the relationship between TnI isoform transitions and myofibrillar function. Cultures of differentiating ES cells were grown on coverslips to permit microscopic inspection of foci of spontaneously contracting cardiac myocytes developing in culture. TnI expression was followed over time to test whether the cardiac myocytes undergo the developmental pattern of expression characteristic of vertebrate cardiogenesis, in which slow skeletal TnI (ssTnI) is expressed initially, followed by induction of cardiac (cTnI) isoform expression. Cardiac TnI expression was examined using the cardiac-specific, monoclonal TI-1 antibody (Ab) while all striated muscle ThI isoforms were detected using the monoclonal TI-4 Ab. Cardiac-specific TnI expression was detected in only 8% (8/96) of foci contracting less than 5 days while TI-4 positive staining was present in 95% (71/73) of foci. These results indicate that other striated muscle TnI isoforms were being expressed in most of the TI-4 positive staining foci. The proportion of contracting foci expressing the cardiac isoform increased steadily over time, such that 100% of foci contracting more than 20 days (13/13) stained positive with the TI-1 Ab. Dual labeling experiments with both TI-1 and TI-4 anti-TnI Abs in the same culture confirmed that within each foci, the area expressing cTnI increased with the days of spontaneous contraction. Western blot analysis of micro-dissected ES cell derived cardiac myocytes confirmed that TI-4 immunostaining at early developmental time points represented ssTnI, and not the fast skeletal TnI isoform. We conclude that ES cell-derived cardiac myocytes display the developmental induction of cardiac TnI expression characteristic of vertebrate cardiac development. Thus, this model should be useful for studying the regulation and functional significance of TnI isoform expression during in vitro cardiogenesis.

Vital staining of cardiac myocytes during embryonic stem cell cardiogenesis in vitro.

Mouse embryonic stem (ES) cells differentiate in vitro into a variety of cell types, including spontaneously contracting cardiac myocytes. The primary aim of this work was to use vital stain techniques for real-time detection of developing cardiac myocytes in ES cell differentiation cultures. The -440 to +6 human cardiac alpha-actin promoter was used to direct expression of the Escherichia coli reporter gene lacZ (pHCActlacZ) into ES cell-derived cardiac myocytes during cardiogenesis in vitro. Undifferentiated ES cells were electroporated with HCActlacZ together with a plasmid containing the neomycin gene under the direction of the phosphoglycerate kinase promoter, and stable transformants were selected in G418. Individual clones were screened for activation of lacZ gene expression in cardiac myocytes developing in vitro. Results showed that expression of the HCActlacZ reporter construct was activated very early during the ES cell differentiation program, at a time point before the appearance of spontaneous contractile activity. The earliest detection was at day 6 of differentiation, when approximately 25% of the differentiation cultures expressed the reporter construct, with expression increasing to approximately 70% at day 9 and continuing throughout the duration of spontaneous contractile activity exhibited by the ES cell-derived cardiac myocytes. Indirect immunofluorescence assays provide evidence that expression was restricted to the cardiac myocytes in culture. In the present study, we show vital staining of transgene expression in living cardiac myocytes using lipophilic fluorogenic beta-galactopyranoside substrates for real-time detection of the reporter gene during continuous contraction of the ES cell myocytes in vitro. The vital stain approach used in the present study will permit the identification of differentiating ES cells that are committed to the cardiac lineage for analysis of gene expression at early time points of ES cell cardiogenesis and, in addition, will aid in selecting genetically modified ES cell cardiac myocytes for use in functional studies.

Transition in cardiac contractile sensitivity to calcium during the in vitro differentiation of mouse embryonic stem cells.

Mouse embryonic stem (ES) cells differentiate in vitro into a variety of cell types including spontaneously contracting cardiac myocytes. We have utilized the ES cell differentiation culture system to study the development of the cardiac contractile apparatus in vitro. Difficulties associated with the cellular and developmental heterogeneity of this system have been overcome by establishing attached cultures of differentiating ES cells, and by the micro-dissection of the contracting cardiac myocytes from culture. The time of onset and duration of continuous contractile activity of the individual contracting myocytes was determined by daily visual inspection of the cultures. A functional assay was used to directly measure force production in ES cell-derived cardiac myocyte preparations. The forces produced during spontaneous contractions in the membrane intact preparation, and during activation by Ca2+ subsequent to chemical permeabilization of the surface membranes were determined in the same preparation. Results showed a transition in contractile sensitivity to Ca2+ in ES cell-derived cardiac myocytes during development in vitro. Cardiac preparations isolated from culture following the initiation of spontaneous contractile activity showed marked sensitivity of the contractile apparatus to activation by Ca2+. However, the Ca2+ sensitivity of tension development was significantly decreased in preparations isolated from culture following prolonged continuous contractile activity in vitro. The alteration in Ca2+ sensitivity obtained in vitro paralleled that observed during murine cardiac myocyte development in vivo. This provides functional evidence that ES cell-derived cardiac myocytes recapitulate cardiogenesis in vitro. Alterations in Ca2+ sensitivity could be important in optimizing the cardiac contractile response to variations in the myoplasmic Ca2+ transient during embryogenesis. The potential to stably transfect ES cells with cardiac regulatory genes, together with the availability of a functional assay using control and genetically modified ES cell-derived cardiac myocytes, will permit determination of the functional significance of altered cardiac gene expression during cardiogenesis in vitro.