**1. Introduction**

The first generation of induced pluripotent stem cells (iPSCs) by Yamanka and co-workers in 2006 was a milestone for stem cell research as it allows the in vitro production of human cells without ethical concerns. Like embryonic stem cells, iPSCs have the capability to differentiate into any cell type, including cardiomyocytes, therefore providing an easy accessible cellular source for the generation of cardiac organoids and tissue structures [1–3].

One possible application for iPSC-derived cardiomyocytes (CMs) is their use in cell therapy replacing damaged tissue by in vitro generated CMs. As cardiovascular diseases are the major cause of death worldwide such regenerative approaches are needed for the development of novel treatment options. The potential and feasibility of iPSC-CM transplantation has been investigated in small and large animal models [4–7].

Thereby, an important future option of iPSC-CMs will be their generation from patient specific tissue enabling the implementation of autologous cell transplantation strategies. In this respect, iPSC-CMs can be used for the development of personalized drug screening approaches and clinically relevant diseases models. Therefore, iPSCs enable cost-effective methods to identify potential drug targets, even more accurately than animal models or other in vitro cell systems. Successful pre-clinical application of iPSC-derived cardiomyocytes for drug screening assays has been lately demonstrated by the CiPA initiative, which was initiated to assess the proarrhythmic risk of novel cardio therapeutics. A myriad of studies investigated in vitro drug effects on different ion channels of iPSC-CMs [8–11], reflecting the importance of electrophysiological measurements using stem cell derived cardiac cells.

However, the maturation of iPSC derived CMs is still a critical point for their application in cardiovascular research as well as for clinical applications. Besides metabolic and structural maturation, proper ion channel composition is crucial for the development of a mature cardiac phenotype. During the last decade, extensive analyses have been performed on the electrophysiological properties of iPSC-CMs [12–15]. Several ion channels and ion currents have been found to be present in iPSC-CMs, including sodium (INa), potassium (IK1 and IKr), L-type and T-type calcium channels, etc. Although multiple differentiation protocols have been developed, researchers failed to generate fully mature cardiomyocytes in vitro, possessing identical electrophysiological properties as their native adult counterparts [16–18]. Moreover, it has largely been shown that iPSC-CMs represent a heterogeneous population of electrophysiological phenotypes, i.e., atrial, ventricular and nodal-like cells [19], each characterized by a specific electrical profile. Therefore, it is important to obtain electrophysiological data for detailed characterization of iPSC-CMs, in particular when differentiation into a certain cardiac subtype is desired [20,21].

Typical approaches to investigate the electrophysiological properties of stem cell derived CMs will be discussed in the following paragraph.

#### **2. Methods for Electrophysiological Characterization of iPSC-CMs**

Several different techniques exist to study the electrophysiological properties of cardiac cells, including patch clamp analysis, MEA measurement and fluorescence dye-based assessment of the membrane potential. Each of these techniques has its own advantages and limitations, which are described in detail in the following.

#### *2.1. Patch Clamping*

Patch clamping is the gold standard technique for the acquisition of ion current data and detailed measurement of action potential (AP) properties in individual cells. The basic principle of patch clamp relies on a blunt ended glass pipette that is sealed onto the cellular membrane to obtain a so-called gigaseal [22].

In the "current patch clamp" mode the membrane potential is recorded while the current applied by the patch pipette is controlled by the operator [23]. The current patch clamp technique allows detection of APs that occur spontaneously or after stimulation with a current change induced by the recording pipette. Considering the fact that iPSC-CMs also contain non-beating populations, current patch clamp methodology allows the detection of AP patterns in these quiescent cells [23,24]. Moreover, detailed AP features, such as AP duration, amplitude, beating rate and mean diastolic potential can reliably be acquired with current patch clamping [25].

When precise characterization of ion channel subtypes is desired, "voltage patch clamp" is performed to measure individual ion currents. Unlike in the current patch clamp mode, the operator keeps the membrane potential at a certain value that enables detection of the net membrane current. In hiPSC-CMs, voltage patch clamp has been successfully applied to obtain data about ion channel density, voltage dependency and activation/deactivation characteristics [23].

However, these manual patch clamp methods are complex, technically challenging procedures that require high operator skills as well as a biophysical background for data interpretation. Another limitation is the low throughput since measurements are usually performed on the single cell level. Therefore, automated patch clamp devices have been developed to overcome the aforementioned drawbacks of manual patch clamp approaches [26]. Automatic platforms profoundly increase the efficiency of electrophysiological data recording by assessment of 10–700 cells at the same time [27]. However, while automated systems are capable to analyze hundreds of cells under variable experimental conditions, the accuracy of obtained data is reduced if compared to manual patch clamping [28,29]. High-throughput analysis is realized by analysis of single cell suspensions, in contrast to manual patch clamping where cells are usually processed in an adherent state. Recently developed systems are equipped with temperature control, optical stimulation and internal perfusion systems to ensure

high data quality and reproducibility [27,30]. Data consistency and robustness is further determined by the homogeneity and density of the applied single cell suspension—a point that is particularly important for iPSC-CMs that are sensitive to dissociation as it can affect membrane proteins and electrical physiology of the cell, including ion channel expression [31,32]. Automatic techniques also do not provide the possibility of selective cell capturing. Hence, the system demands highly purified cell populations, which could be challenging when working with CMs differentiated from iPSCs that commonly represent a mixture of different cardiac subtypes [29].

#### *2.2. Optical Recordings of the Membrane Potential*

An indirect technique to assess electrophysiological data of iPSC-CMs is the application of voltage-sensitive dyes that change fluorescence intensity or emission spectra upon alteration of the membrane potential. Considering fluorescence microscopy as one of the most commonly used methods in cell research, utilizing voltage-sensitive dyes is operationally simple and does not require special instrumentation. Moreover, it is less invasive and it enables monitoring of voltage dynamics over thousands of cells with very high temporal resolution [33]. Several studies have proven feasibility of voltage sensor probes for drug screening experiments in iPSC-CMs [34–36]. Recently, Takaki and colleagues [37] applied voltage sensitive dyes for the identification of distinct cardiac subtypes in an iPSC-CM population. Further, the authors were able to detect differences in the AP pattern in iPSC-CMs obtained from patients suffering from the long QT syndrome, compared to control cells.

Alternatively, voltage sensitive probes can be engineered as fluorescent proteins that are stably expressed in target cells. Compared to voltage-sensitive dyes, these proteins possess lower phototoxicity, thus, facilitating long-term measurements. These genetically encoded probes are designed by conjugating a voltage-sensing domain to a single fluorescent protein, a fluorescence resonance energy transfer (FRET) pair or rhodopsin proteins [38,39]. Changes of the membrane potential induce conformational rearrangement of the voltage-sensor, which in turn modulates the emission spectra of the attached fluorescent protein. The latest generation of genetically encoded voltage sensors, such as ArcLight, Archer1 or QuasAr1, show large fluorescence alteration upon depolarizing events (40%–80% for a 100 mV depolarization) associated with faster on/off kinetics (1–10 ms). Shaheen et al. generated ArcLight expressing human iPSC-CMs to establish a 2D cardiac tissue platform for optical mapping and pharmacological studies [40]. Former data confirmed the suitability of genetically encoded voltage sensors for iPSC-CM drug screening applications and disease modeling attributed to altered AP phenotypes [41–44]. However, there are certain limitations of this technology. Like fluorescence dyes, genetically encoded voltage indicators provide only relative, not absolute values for the membrane potential [45]. The lower on/off kinetics increase the probability of losing high frequency AP elements [41,45]. Furthermore, introduction of voltage-sensitive proteins like ArcLight could affect the electrophysiological properties of iPSC-CMs. This needs to be carefully addressed by the operator as well as proper folding and membrane integration of the voltage sensitive probe.
