**2. Human pluripotent stem cells for repeated dose toxicity assessment**

Human pluripotent stem cells (PSC) offer with their ability to recapitulate the most essential steps of embryonic development and give rise to different mature cell types in vitro an optimal human cellular model, which could help in increasing the safety and predictability of RDT testing leading to low late stage attrition of compounds. Combined with this cell model, toxicogenomic technologies would help predict biomarkers in an evidence-based approach.

So far, the safety assessment for novel drug candidates includes in vivo RDT tests in rodent and non-rodent models. The drawbacks of RDT studies include false negative results and unexpected humans toxicity of compounds that were judged to be safe in preclinical studies [15, 16]. Such unexpected toxicity is one of the major reasons for the withdrawal of a drug from the market. The heart and liver are often target organs in toxicology. Novel in vitro screening methods are, thus, required to classify toxic compounds earlier in development, which would lead to safer drugs, more efficient drug discovery process, lower costs and reduced laboratory animal use [17]. There is an increasing interest from biopharmaceutical industry to develop such test systems by using derivatives of human ESCs or iPSCs.

The iPSCs have a clear advantage over ESCs as they do not involve ethical issues. The generation of iPSCs involves reorganization of condensed chromatin to open state chromatin, which is aided by histone acetylation. Epigenetic factors are crucial for iPSC generation and maintenance of their pluripotent state. Although the epigenetic state of iPSCs largely resembles that of ESCs, iPSCs also have a unique DNA methylation patterns they retain epigenetic memory of the respective somatic tissue of origin which might influence their differentiation potential and affect the quality and quantity of cells for RDT [18]. On the other side, it is also well known that different agents, so called epimutagens, can cause DNA methylation and histone modification changes leading to disease [19]. These epigenetic modifications directly affect transcription factors and other chromatin binding proteins that regulate cell type-specific gene expression. The detection of biomarkers related to epigenetic modifications in RDT would be of great importance, but until now there are no systematic studies conducted in this direction. In addition, employing of iPSCs and their derivatives for this purpose poses a great challenge because genetic and epigenetic variations in iPSCs associated with reprogramming and in vitro manipulation may compromise their utility for downstream applications [20]. The lesser the variation in epigenetic changes in iPSCs the greater will be the specificity in in vitro toxicological studies. Recently, Planello and coworkers demonstrated that the choice of reprogramming factors greatly influences the DNA methylation abnormalities in iPSCs. Even highly selected iPSC lines have been shown to retain epigenetic signature of donor cell [21]. Gupta et al have shown that global transcriptional profiles of human iPSCs and ESCs are very similar and that this similarity also exists between the corresponding beating clusters derived from them [22]. They have also shown that some fibroblasts-specific mRNA expression partners were retained in the iPSCs derived from them. Significant proportion of these genes were also shown to be expressed at the same level in iPSC-derived but not in ESC-derived beating clusters indicating the retention of epigenetic memory even in the differentiated and highly enriched iPSC derivatives. Likewise, several microRNA expression profiling studies have shown the subtle differences between iPSC derivatives [23]. Hence, the iPSCs may not represent an ideal platform for RDT testing. With the current pace of iPSC research it may be possible to create iPSCs with little or no epigenetic anomalies. Polo and coworkers have shown that this retained epigenetic memory of iPSCs in early passages can be erased using extensive continued passage [24]. By using chromatin-modifying compounds like HDAC inhibitors it may be possible to stabilize the epigenetic state of iPSCs and their derivatives and decrease the frequency of heterogeneity within iPSCs. However, using the PSC-derivatives to predict RDT in human toxicological endpoints is still challenging.

more accurate predictors of drug-induced cardiotoxicity than standard hERG-expressing HEK293 cells. This observation suggests that human iPSC-CM may represent a suitable model for evaluation of drug safety and efficacy. However, there is still a need to examine how well the alternative systems can replace the animal models for RDT testing. The traditional repeated-dose toxicological endpoints that relate to cardiotoxicity include histopathological examinations of the heart and electrocardiographic recordings in the non-rodent species [30]. The current regulatory framework guidelines for cardiotoxicity testing include blood pressure, heart rate and electrocardiogram (ECG) parameters as well as repolarization and conductance abnormalities, cardiac output, ventricular contractility and vascular resistance. The limitations of RDT testing in vivo clearly encouraged the scientific community to identify and develop alternative in vitro methodologies to thoroughly estimate the integrated and complex re‐

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Drugs exerting toxic effects on cardiovascular system have shown to affect the heart function in a way that includes changes in the contractility, cardiac rhythm, blood pressure and ischemia [31]. Such toxic effects have led these drugs to be withdrawn, requiring expansion of rules on cardiotoxicity testing. A new application for CMs derived from human ESCs and iPSCs has surfaced because of the lack of availability of human primary material for cardiotoxicity testing and their ability to overcome species variability. In vitro cardiotoxicity testing applications using human PSC-CMs is very advantageous and complimentary to the existing RDT appli‐ cations. Endpoints such as action potential parameters, metabolic activity, membrane leakage, energy content and intracellular calcium handling can be monitored for assessing cardiotox‐ icity. As mentioned above, the effect of new drugs on cardiac electrophysiology (i.e. changes in ventricular repolarization) is a focus for tight control. The balanced concerted activity of several cardiac ion channels is important for proper ventricular repolarization and alterations may lead to ventricular arrhythmias. Therefore, electrophysiological assessment of the proarrhythmic potential of drugs is very relevant in cardiotoxicity assays and human PSCderived CMs are suitable for such assays because they exhibit calcium handling properties, ion channel activity and regulatory protein expression important for the development of a

Recently, several studies evaluated the potential of human iPSC-CMs for pharmacological screening-assays and drug discovery applications [32-34]. However, the utility of iPSC-CMs to accurately predict toxicity in humans may be limited by their immature character [35]. Current differentiation protocols give rise to heterogeneous phenotypes of spontaneously beating human PSC-CMs with structural proteins, Ca2+release units, ion channels, action potentials, and hormonal response being similar to that of native fetal CMs. However, the electrophysiological and structural properties of PSC-CMs do not fully resemble those of adult CMs. Therefore, the model based on human PSC-CMs must be improved before it can represent an ideal platform for cardiac RDT. The mentioned issues can be solved by following measures: a) by modulating cellular signaling pathways it is possible to get a homogeneous CM population [36] b) with the application of tissue engineering it is possible to create a 3D tissue constructs which provide microenvironment similar to native heart thus helping in structural maturation of CMs [37] and c) prolonged culturing of iPSC-CMs can increase the

sponses in the endpoints that are taken into consideration.

mature repolarization phenotype in CMs [12].
