br Results Relying on the high

Results Relying on the high conservation of vertebrate TBX3 proteins, we used human TBX3 in murine ESCs due to its specific traceability. We inserted human TBX3 cDNA into the pEF-DEST51 vector (Invitrogen) for EF1α-promoter-based stable and constitutive overexpression in ESCs. Out of 20 independent clones, we selected four clones that represented the entire spectrum, ranging from low to high levels of TBX3 mRNA overexpression based on quantitative RT-PCR (qRT-PCR; Figure 1A). These overexpression levels were confirmed on the protein level (Figure 1B). Fluorescence-activated cell sorting (FACS) analyses showed no influence of TBX3 overexpression on the numbers of pluripotency marker Oct-4/Pou5f1 and Sox2-positive top article (Figure 1C). This corresponds to normal undifferentiated colony growth in leukemia inhibitory factor (LIF)-containing medium (Figure 1B) and is in agreement with our previous findings for MESP1 and NKX2-5 overexpression (David et al., 2008, 2009; David and Franz, 2012). We concluded that similar to what was found for those factors, TBX3 alone was not sufficient to induce differentiation of ESCs. During differentiation, highly and moderately TBX3-overexpressing ESC clones started to contract earlier and showed approximately 5- to 10-fold more contracting areas than controls (Figure 2A). This enhanced cardiac differentiation resembles the effect of MESP1 and NKX2-5 overexpression described in our previous studies (David et al., 2008, 2009; David and Franz, 2012). Correspondingly to their beating activity, Tbx3-overexpressing cardiomyocytes showed a normal pattern of the sarcomeric marker MYH6 (Figure 2B). To further verify the functionality and subtype content of the cardiomyocytes, we analyzed their electrophysiological properties via single-cell patch clamping and “funny channel” density measurements at day 6+12 as previously described (David et al., 2008, 2009, 2013). In general, three cell types that have been described for isolated beating cardiomyocytes obtained from embryoid body (EB) development, namely, ventricle-like, atrial-like, and SA/AV (pacemaker-like) cells, plus intermediate cells were present in preparations from TBX3 cell clones (Figure 2C). The action potentials (APs) generated by the various cell types were typical with respect to their distinct parameters, including the maximum diastolic potential (MDP), diastolic depolarization rate (DDR), overshoot or AP/plateau duration, and cycle length (Figure 2C; Figure S1 and Table S1 available online). However, we found an unusually high proportion of the pacemaker-like subtype in TBX3 programmed cells, representing 38.5% of all cardiomyocytes. These cells lacked a plateau phase, had a positive MDP of >−50 mV, and high DDR values typically exceeding 100 mV/s. In addition, they had the smallest positive overshoot, with a maximum of ∼+20 mV. In contrast, within control cells, only 20% of these pacemaker-like cells were found (Table S2). In accordance with the high proportion of pacemaker cells, cell numbers expressing HCN4 (representing the funny channel) were enhanced among the Tbx3 programmed cardiomyocytes (Figure 2D). To further enhance the yield of the SA/AV subtype achieved via TBX3 programming, we chose to combine this approach with MYH6-promoter-based antibiotic selection (Klug et al., 1996). This was recently described as directed pacemaker cell enrichment (Kensah et al., 2013; Otsuji et al., 2010). We therefore additionally introduced the plasmid containing the Myh6-Neomycin cassette (Klug et al., 1996) into our TBX3 clones. As expected, administration of the antibiotic during differentiation led to an enrichment of beating tissue in Myh6-promoter antibiotic-selected controls. Likewise, Myh6-TBX3 double-transgenic clones were enriched for beating foci (Movies S1 and S2). However, in the latter, spontaneous beating rates were approximately doubled (Figure 3A). After introduction of an additional dissociation step, beating rates increased in Myh6-promoter antibiotic-selected controls, leading to antibiotic-selected cardiac bodies (aCaBs), as well as in Myh6-Tbx3 double-transgenic clones. In the latter, we often found cellular aggregates contracting at 300–400 bpm, which is ∼6-fold faster than wild-type cells. These beating rates come close to murine heart rates (∼500 bpm), indicating the formation of induced SA bodies (iSABs; Figure 3A; Movie S3). The cells within iSABs were positive for HCN4 as well as for Connexins CX45 and CX30.2 (Figure 3B) which is characteristic for pacemaker cells (Kreuzberg et al., 2005; Verheijck et al., 2001).