To ultimately test the functional pacemaker activity of the

To ultimately test the functional GSK343 activity of the iSABs, we took advantage of the ex vivo model based on cultivated ventricular slices of murine hearts (Halbach et al., 2006; Figure 6A). Although such slices typically have spontaneous beating activity immediately after preparation, they can be induced to robustly contract at up to ∼60 bpm via electrodes and are therefore an ideal system for testing the functionality of our iSABs. Correspondingly, when seeded on the slices, the iSABs were able to attach and persist on the slices as shown by labeling with DiI (d.n.s.). To address the effect of iSAB seeding, we first carefully quantified spontaneous slice activity in naive slices. On day 2 after slice preparation, a maximum of 70% of slices containing at least one area of spontaneous beating activity was observed. This value decreased dramatically on days 3 and 4 (Figure 6B). Yet, whereas coculturing slices with aCaBs did not significantly alter the number of beating areas or beating parameters compared with naive slices, seeding slices with iSABs resulted in a ∼1.5-fold increased beating activity as early as days 1 and 2. Moreover, only slices cocultured with iSABs preserved their spontaneous beating activity and multiple beating sites also on days 3 and 4 (Figures 6B and 6C). Furthermore, the beating rates of iSAB-seeded slices continuously and significantly increased over 4-fold from day 1 until day 4 (Figure 6D), which we did not observe for unseeded and aCaB seeded slices. Loading the iSABs with calcein before transferring them to the slice confirmed the formation of syncytia between iSABs and slice cells, as shown by transmission of the dye over time (Figure 6E). The same iSAB is shown in Figure 6E and Movie S7, demonstrating pacing of the host slice within the respective area. Furthermore, functional coupling is evident from synchronized Ca2+ transients between iSAB and slice myocardium. Therefore, peaks of Ca2+ transients within the slice are smaller but highly synchronous to spontaneous iSAB activity and can be detected within a radius of ∼200 μm (Figure 6F; Movie S8). Thus, the signals were derived from the slice myocardium, especially since no significant outgrowths of iSABs into the slice were observed in staining for the pacemaker cell marker HCN4 (Figure 6G).
Discussion The ability to produce de novo, highly enriched, stem cell-derived populations of cardiac pacemaker cells that bear all the functional parameters of mature nodal cells is of great interest for future cell-based therapies. Such cells might be able to reconstitute a proper cardiac rhythm in the sense of a biological pacemaker. In addition, drug testing in vitro would benefit from the availability of such purified nodal cells. PSCs are the focus of these goals because they have been shown to give rise to any cell type of the mammalian organism, including spontaneously beating cardiomyocytes with molecular and functional properties characteristic of SAN/pacemaker cells (Maltsev et al., 1994; He et al., 2003; Yanagi et al., 2007; Barbuti et al., 2009; Ma et al., 2011; David and Franz, 2012). Typically, however, the cell populations within EBs are highly heterogeneous, which inevitably leads to the requirement of reliable selection and isolation strategies. This applies in particular to the very rare cell type of cardiac nodal cells. Cardiac nodal cells are characterized by a low membrane potential, diastolic depolarization, and low upstroke velocities. A number of currents contribute to diastolic depolarization and to the AP in the SAN, including the pacemaker current If. This current is carried by the HCN channel. The cyclic-AMP binding site on the HCN channel permits catecholamines to modulate activation, and this property might regulate the autonomic responsiveness of the pacemaker mechanism (Rosen, 2005). There are four genes representing the four known isoforms, termed HCN1–HCN4. In the SAN, the predominant isoform by far is HCN4 (Ishii et al., 1999; Stieber et al., 2004).