**3. PSC-derived cardiomyocytes for toxicity testing**

Human Stringent cosmetics legislation amending directives especially within the European Union (EU) related to complete replacement of animal models in cosmetic industry safety testing by alternative methods has emphasized an urgent need for the development of reduction, refinement and replacement (3R) of the existing animal studies [25]. In order to fill gaps in non-animal alternative methods and to focus on complex RDT a research initiative called "Safety Evaluation Ultimately Replacing Animal Testing 1 (SEURAT-1)" composed of six complimentary research projects was launched in 2011 and jointly funded by the European Commission's FP7 HEALTH Programme and Cosmetics Europe (http://www.seurat-1.eu/). Embryonic Stem cell-based Novel Alternative Testing Strategies (ESNATS) is a European Union's Seventh Framework Programme (FP7), which focuses on developing a human ESC based novel toxicity test platforms to accelerate drug development. Human ESC based in vitro reproductive toxicity, neurotoxicity, toxicogenomics, proteomics and kinetics were tested for their predictive value in the identification of toxicity endpoints (http://www.esnats.eu). The RDT delivers the No Observable Adverse Effect (NOAEL), which is used in calculation of the substance safety parameters [25]. PSC-derived models hold a great potential for refinement of current models of cardiotoxicity. For many toxicology applications, a homogeneous defined population of specific cell types is required which stem cells can provide.

Validity of human PSC-derived cardiomyocytes (CM) for toxicity testing and safety pharma‐ cology has been investigated in several studies [26-28]. The susceptibility of disease-specific human iPSC-CMs to toxicity compared to healthy human PSC-CMs was evaluated recently by Joseph Wu laboratory [29]. This group showed that disease-specific human iPSC-CM are 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‐ sponses in the endpoints that are taken into consideration.

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

Human Stringent cosmetics legislation amending directives especially within the European Union (EU) related to complete replacement of animal models in cosmetic industry safety testing by alternative methods has emphasized an urgent need for the development of reduction, refinement and replacement (3R) of the existing animal studies [25]. In order to fill gaps in non-animal alternative methods and to focus on complex RDT a research initiative called "Safety Evaluation Ultimately Replacing Animal Testing 1 (SEURAT-1)" composed of six complimentary research projects was launched in 2011 and jointly funded by the European Commission's FP7 HEALTH Programme and Cosmetics Europe (http://www.seurat-1.eu/). Embryonic Stem cell-based Novel Alternative Testing Strategies (ESNATS) is a European Union's Seventh Framework Programme (FP7), which focuses on developing a human ESC based novel toxicity test platforms to accelerate drug development. Human ESC based in vitro reproductive toxicity, neurotoxicity, toxicogenomics, proteomics and kinetics were tested for their predictive value in the identification of toxicity endpoints (http://www.esnats.eu). The RDT delivers the No Observable Adverse Effect (NOAEL), which is used in calculation of the substance safety parameters [25]. PSC-derived models hold a great potential for refinement of current models of cardiotoxicity. For many toxicology applications, a homogeneous defined

RDT in human toxicological endpoints is still challenging.

184 Pluripotent Stem Cell Biology - Advances in Mechanisms, Methods and Models

**3. PSC-derived cardiomyocytes for toxicity testing**

population of specific cell types is required which stem cells can provide.

Validity of human PSC-derived cardiomyocytes (CM) for toxicity testing and safety pharma‐ cology has been investigated in several studies [26-28]. The susceptibility of disease-specific human iPSC-CMs to toxicity compared to healthy human PSC-CMs was evaluated recently by Joseph Wu laboratory [29]. This group showed that disease-specific human iPSC-CM are 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 mature repolarization phenotype in CMs [12].

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 maturation of Ca2+handling [38]. Further improvements of differentiation methods will enable generation of more homogeneous and mature CM populations thus increasing their validity for RDT testing. The overall predictability of drug efficacy and toxicity using iPSC-CMs and disease specific iPSC-CMs has been recently reported by several groups [29, 39-41]. An absolute requirement for CMs to be used for RDT is to be able to stably maintain the beating cardiac phenotype for a prolonged period of time under defined conditions. Both hiPSC-CMs and hESC-CMs display beat rate variability similar to that of a human heart sinoatrial node [42]. However, recently, variability in action potentials and sodium currents in response to lidocaine and tetrodotoxin was shown in late stage in vitro differentiated human iPSC-CMs [32], thus warranting some caution and further analyses.

assessment. The suitability of hepatocyte-like cells derived from human PSCs for toxicity

Human Pluripotent Stem Cell Applications in Drug Discovery and Toxicology – An overview

http://dx.doi.org/10.5772/58485

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Proteomics, genomics and metabonomics, either alone or in combination have the potential for developing biomarkers in applied toxicology. Toxicogenomics refers to the areas men‐ tioned above and is a thorough-mean for hi-throughput discovery of biomarkers using latest technologies [50]. Transcriptomics measures the levels of both coding and non-coding RNAs using hi-throughput technology such as microarrays. This whole genome gene expression analysis can measure the levels of expression of a gene at any stage, in any tissue and in any vitro model. Examples of toxicogenomics applications include prediction of genotoxicity or carcinogenicity, target organ toxicity and endocrine disruption. Expression profiling of any selected cellular systems exposed to new test substances is compared against controls to identify, classify and validate toxic compound and its effects. Bioinformatic analyses of the data sets obtained from above can be used to predict the patterns and signatures of a toxin (e.g. biological processes or signaling pathways affected by a toxin). Furthermore, the data sets can be matched up against existing databases for predicting and carving out a mode-of-action for the toxin. The main disadvantage of this approach is limited reproducibility and also it is semi quantitative and detects only changes in gene expression. Therefore, mRNA expression profiling cannot be used as a standalone method in identifying potential biomarkers of RDT. EU FP7 project Predict-IV is evaluating the integration of 'omics' technologies, biomarkers and high content imaging for the early prediction of toxicity of pharmaceuticals in vitro. The aim is to identify general molecular response pathways that result from toxic drug effects that are independent of the cell/tissue type [51]. Detection of endpoints and biomarkers of RDT using in vitro systems (DETECTIVE) is a unique large scale SEURAT-1 cluster project aimed at establishing screening pipeline of high content, high throughput as well as classical functional and "-omics" technologies to detect human biomarkers for RDT in in vitro test system (http:// www.detect-iv-e.eu/). Other-omics technologies such as microRNA analysis and epigenetics

Drug induced toxicity can also exhibit various effects at the proteome level. Classification of such endpoints is difficult using traditional RDT methods. Proteomics improves the classifi‐ cation by identifying individual proteins or such protein panels that reflect the specific toxic pathway mechanisms. Proteomics-based in vitro toxicity assays measure drug-induced changes by comparing in vitro to in vivo effects thus validating the suitability of in vitro models. There is an absolute need for integration of standard RDT tests with the 'omics' applications. Current proteomic technologies include gel-based (1-DE or 2-DE) and gel-free (LC-MS/MS) techniques [17]. Recently thalidomide-specific proteomics signatures during

testing and drug discovery were systematically studied by several groups [47-49].

**5. Omics strategies to develop biomarkers for RDT**

**5.1. Toxicogenomics**

also play a vital role.

**5.2. Proteomics**

Bioanalytics is a very promising tool in the application of in vitro cardiotoxicty assays. Novel bioanalytical tools for discovery of biomarkers of cardiotoxicity include the field potential QT scanning, using cellular oxygen uptake for monitoring the metabolic state of CMs, using surface plasmon resonance (SPR) biosensing for key CM biomarkers, and also exploiting realtime multi-wavelength fluorimetry [12]. Novel imaging technologies and physiological analyses such as impedance measurements [43] and microelectrode arrays (MEAs) [44] give an insight into major in vitro cellular events such as migration, proliferation, cell morphology, cell–cell interactions and colony formation, relevant to biomarker discovery.

#### **4. Stem cell-derived hepatocytes for toxicity testing**

So far, hepatotoxicity is evaluated on day 28 or 90 in in vivo RDT tests by analysis of clinical parameters, hematology, and histopathology. RDT tests evaluate chronic effects on organ toxicity to establish a NOAEL which is used in calculation of the substance safety parameters [25]. The extrapolation of the quantitative risk assessment for cosmetic ingredients using data derived from animal studies to in vitro systems could be done by considering a margin of safety (MoS) value of at least 100 for intra-species and inter-species variation [25]. Human PSCs represent a promising human cellular model which could help in increasing the safety and predictability of RDT testing. Combined with this cell model, toxicogenomic technologies would help predict biomarkers in an evidence-based approach. During these RDT tests, the animals are observed for indications of toxicity. Afterwards, necropsy, blood analysis and histopathology of the organs of the animals are performed [17]. However, these parameters can turn out to be insensitive and potentially generate false negative results [15, 16]. Unex‐ pected hepatotoxicity may be seen in the clinical trials or even when the product is already on the market because careful examinations of idiosyncratic (person specific) or non-idiosyncratic inter-drug interactions are either ignored or overseen [45]. This is also probably because of dose-dependent reactions and other unknown peculiar drug interactions. There is need for novel screening methods that can address these hepato-toxicological hazards early in the development [46]. Most studies relied on the use of liver slices as an in vitro model for toxicity testing due to limited availability of tissue samples. However, the human PSC-derived hepatocytes have the potential to replace these in vitro models and be applied for toxicity assessment. The suitability of hepatocyte-like cells derived from human PSCs for toxicity testing and drug discovery were systematically studied by several groups [47-49].
