In this regard murine ESC derivatives

In this regard, murine ESC derivatives bearing EGFP transcriptionally controlled via the promoter of the HCN4 gene were reported to coexpress EGFP, HCN4, and other cardiac markers in spontaneously beating foci (Morikawa et al., 2010). Yet, after the EGFP-positive fraction was subjected to cell sorting, spontaneous APs were rarely observed and, interestingly, most of the cells were positive for nestin, a marker for neurons (Morikawa et al., 2010). This corresponds to the observation that HCN4 is a marker not only for spontaneously active cardiomyocytes but also for nerve cells (Garcia-Frigola et al., 2003). Therefore, alternative promising approaches based on endogenous surface markers (Scavone et al., 2013) and pharmacological administration of small molecules (Wiese et al., 2011; Kleger et al., 2010) have been pursued in addition to direct reprogramming strategies (Bakker et al., 2012; Kapoor et al., 2013). Here, we describe a protocol that combines ESC forward programming using TBX3 as a key transcription factor with Myh6-based antibiotic selection (Klug et al., 1996). This approach consistently led to highly effective generation of nodal-like pacemaker cell GSK343 cost characterized by spontaneously contracting cardiomyocytes with highly synchronized beating rates of 300–400 bpm, approximating those of a mouse heart. Most (>80%) of the cardiomyocytes within these clusters clearly represented the nodal cell type, and, to our knowledge, such high purities have not been achieved before (Kleger et al., 2010; Wiese et al., 2011; Scavone et al., 2013). Although it is technically almost impossible to discriminate between murine SAN and AV node cells, especially on the electrophysiological level, Marger et al. (2011) showed that the two cell types differ in a more easily accessible feature, namely, their spontaneous beating rates after isolation. They observed rates of ∼260 bpm in isolated SAN cells and ∼175 bpm in AV node cells in culture. Isoproterenol stimulation led to beating rates of ∼350 bpm in SAN cells and ∼225 bpm in AV node cells. Our iSAB-derived cells showed rates of ∼305 bpm and >550 bpm without and with isoproterenol stimulation, respectively, thus exceeding the values for SAN cells reported by Marger et al. (2011). Moreover, as our cells provide all other properties required for full functionality (i.e., protein expression patterns and electrophysiological and Ca2+ signaling parameters), we propose to call these engineered pacemaker aggregates induced sinoatrial bodies (iSABs). Furthermore, our iSABs are functional in the sense that they can robustly pace working myocardium ex vivo. Our initial experiments with TBX3-based programming alone led to merely a doubling of functional pacemaker cell numbers in cell aggregates with significantly lower beating frequencies. This may correspond to the insufficiency of TBX3 for fully direct reprogramming of ventricular myocardium to pacemaker cells (Bakker et al., 2012). Yet, the effect of TBX3 in ESCs was drastically further enhanced when we introduced the additional Myh6 promoter-based antibiotic selection plus an additional dissociation step (Figure 7). Our rationale for this was based on recent reports describing the enrichment of ventricular and nodal cardiomyocytes via Myh6-based selection (Klug et al., 1996; Kensah et al., 2013; Otsuji et al., 2010), although this method was originally thought to enrich cardiomyocyte subtypes in an undirected manner. Our approach differs from those described in previous studies, which simulated biological pacemakers by manipulating terminal effector molecules underlying the sarcolemmal electrophysiology instead of de novo generating fully functional nodal cells (Johns et al., 1995; Nuss et al., 1999). In contrast, our protocol leads to cells that exhibit not only electrical oscillations but also the subtle electrophysiological and calcium signaling characteristics and distinctive morphological features of endogenous pacemaker cells. Our technology thus may become important for future alternatives to electronic pacing devices.