Background: Functional and molecular integrity of cardiomyocytes (CMs) derived from induced pluripotent stem (iPS) cells is essential for their use in tissue repair, disease modelling and drug screening. In this study we compared global transcriptomes of beating clusters (BCs) microdissected from differentiating human iPS cells and embryonic stem (ES) cells. Results: Hierarchical clustering and principal component analysis revealed that iPS-BCs and ES-BCs cluster together, are similarly enriched for cardiospecific genes and differ in expression of only 1.9% of present transcripts. Similarly, sarcomeric organization, electrophysiological properties and calcium handling of iPS-CMs were indistinguishable from those of ES-CMs. Gene ontology analysis revealed that among 204 genes that were upregulated in iPS-BCs vs ES-BCs the processes related to extracellular matrix, cell adhesion and tissue development were overrepresented. Interestingly, 47 of 106 genes that were upregulated in undifferentiated iPS vs ES cells remained enriched in iPS-BCs vs ES-BCs. Most of these genes were found to be highly expressed in fibroblasts used for reprogramming and 34% overlapped with the recently reported iPS cell-enriched genes. Conclusions: These data suggest that iPS-BCs are transcriptionally highly similar to ES-BCs. However, iPS-BCs appear to share some somatic cell signature with undifferentiated iPS cells. Thus, iPS-BCs may not be perfectly identical to ES-BCs. These minor differences in the expression profiles may occur due to differential cellular composition of iPS-BCs and ES-BCs, due to retention of some genetic profile of somatic cells in differentiated iPS cell-derivatives, or both.

Cardiac dysfunction is one of the main cause of drug candidate failures in the preclinical and/or clinical studies and responsible for the retraction of large number of drugs from the market. The prediction of arrhythmic risk based on preclinical trials during drug development remains limited despite intensive and costly investigation. Moreover, methods for analyzing beating behavior of cardiomyocytes (CMs) in culture to diagnose arrhythmias are not well developed. In this study, we combined two emerging technologies, induced pluripotent stem (iPS) cell-derived CMs and impedance-based real-time (xCELLigence RTCA Cardio Instrument) monitoring of CM electrical activity, to assess the effect of drugs known affect cardiac activity such as isoproterenol, carbachol, terfenadine, sotalol and doxorubicin. Cells were exposed to a drug in a single dose or repeated dose scenarios and data were analyzed using RTCA Cardio software, Poincaré plot and detrended fluctuation analysis. The results revealed significant changes in beating parameters of iPS-CMs induced by reference compounds. Heptanol, gap junction blocker, completely disrupted the synchronous beating pattern of iPS-CMs. Decrease of beating rate, amplitude and beat-to-beat signal variations of iPS-CMs monolayer observed in the presence of doxorubicin revealed severe abnormality detected by the system. Additionally, the irregular beating rhythms recorded in the presence of Terfenadine and Sotalol at high concentration, reflect abnormalities in cell contraction and/or relaxation which may lead to arrhythmia. All these results indicated that xCELLigence RTCA Cardio system combined with iPS cells, has the potential to be an attractive high-throughput tool for studying CMs during prolonged culture times and to screen potential drugs for cardiotoxic side effects.

Introduction: Induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) may be suitable for myocardial repair. While their functional and structural properties have been extensively investigated, their response to ischemia-like conditions has not yet been clearly defined. Methods: iPS-CMs were differentiated and enriched from murine induced pluripotent stem cells expressing enhanced green fluorescent protein (eGFP) and puromycin resistance genes under the control of an α-myosin heavy chain (α-MHC) promoter. iPS-CMs maturity and function were characterized by microscopy, real-time PCR, calcium transient recordings, electrophysiology, and mitochondrial function assays, and compared to those from neonatal murine cardiomyocytes. iPS-CMs as well as neonatal murine cardiomyocytes were exposed for 3 hours to hypoxia (1% O₂) and glucose/serum deprivation, and viability, apoptosis markers, reactive oxygen species, mitochondrial membrane potential and intracellular stress signaling cascades were investigated. Then, the iPS-CMs response to mesenchymal stromal cell-conditioned medium was determined. Results: iPS-CMs displayed key morphological and functional properties that were comparable to those of neonatal cardiomyocytes, but several parameters indicated an earlier iPS-CMs maturation stage. During hypoxia and glucose/serum deprivation, iPS-CMs exhibited a significantly higher proportion of poly-caspase-active, 7-aminoactinomycin D-positive and TUNEL-positive cells than neonatal cardiomyocytes. The average mitochondrial membrane potential was reduced in “ischemic” iPS-CMs but remained unchanged in neonatal cardiomyocytes; reactive oxygen species production was only increased in “ischemic” iPS-CMs, and oxidoreductase activity in iPS-CMs dropped more rapidly than in neonatal cardiomyocytes. In iPS-CMs, hypoxia and glucose/serum deprivation led to upregulation of Hsp70 transcripts and decreased STAT3 phosphorylation and total PKCε protein expression. Treatment with mesenchymal stromal cell-conditioned medium preserved oxidoreductase activity and restored pSTAT3 and PKCε levels. Conclusion: iPS-CMs appear to be particularly sensitive to hypoxia and nutrient deprivation. Counteracting the ischemic susceptibility of iPS-CMs with mesenchymal stromal cell-conditioned medium may help enhance their survival and efficacy in cell-based approaches for myocardial repair.

Embryonic stem cell (ESC) derived tissue is a promising tool to be used in different clinical, preclinical and also scientific settings, for example as in vivo biological pacemaker, preclinical drug safety screening tool or ultimately as part of a cell replacement therapy. However, before ESC derived tissue can be used routinely for these purposes in humans, further studies are needed. In this context, the aims of the present study were to examine the effect of antiarrhythmic drugs on human ESC (hESC) und human induced pluripotent stem cell (hiPSC) derived cardiomyocytes by analyzing the beat rate variability (BRV), which can be considered as the in vitro equivalent of the heart rate variability (HRV) in vivo. Short-term recordings of extracellular field potentials of spontaneously beating cardiomyocytes derived from hESCs and hiPSCs were made using Microelectrode Arrays (MEA). The effect of Flecainide, Ivabradine and Metoprolol was tested. The offline analysis of the BRV was mainly focused on time domain methods. Additionally a non-linear analysis method was used. The evaluation of the Poincaré-Plots of the measurements without pharmacological intervention revealed that the vast majority of the scatter plots have a similar, ellipsoid shape. Flecainide and Ivabradine influenced BRV parameters significantly, whereas Metoprolol did not alter the BRV markedly. We detected remarkable similarities between the BRV of hESC and hiPSC derived cardiomyocytes in vitro and the HRV in vivo. The effect of antiarrhythmic drugs on spontaneously beating cardiomyocytes derived from hESC and hiPSC was generally consistent with clinical experiences and also with our previous study based on murine ESC derived cardiomyocytes. In conclusion, our study points out the great potential of hESC and hiPSC derived tissue to be used routinely for many different applications in medicine and science.

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) might provide cells to repopulate injured myocardium. Electrical coupling of these cells to the host myocardium is a prerequisite for improved functionality. The aim of this study was to investigate electrical interaction of hESC-CMs with myocardial tissue and to identify factors challenging functional integration. Beating clusters containing hESC-CMs were cocultured in vitro with viable slices of late-stage embryonic murine ventricles. Field potentials recorded with micro-electrode arrays and video data were analyzed. The effects of heptanol, electrical pacing, beta-adrenergic, and muscarinic stimulation on coupling were studied. Beating clusters integrated morphologically and functionally resulting in a synchronized beating pattern after two to four days of coculture. Heptanol-induced conduction block between transplanted cells and host tissue and immunoreactivity for connexin43 suggested electrical coupling via gap junctions. Beta-adrenergic or muscarinic stimulation induced uncoupling and arrhythmias probably due to genetically determined differences of hormonal modulation of spontaneous beating rates of transplanted cells and host tissue. HESC-CMs can integrate functionally and develop synchronized beating. Interventions unraveling the different electrophysiological properties of transplanted and host tissue induce functional disintegration. Successful cellular replacement has to improve coupling but should also aim to transplant cardiomyocytes with similar electrophysiological properties as the host tissue.