Selection of Human Myocardial Precursors Figure 6. hMPs give rise to multiple CM subtypes. aMHC-GFP or wild type hESCs were differentiated for 21 days, and beating GFP+ or wild type hEBs were manually dissected for further analysis. (A) GFP+ hEBs were analyzed for expression of Islet-1 (ISL-1), sarcolipin (SLN), Na+/K+ hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4), myosin light chain-2 atrial (MLC2a), MLC2v, aMHC and cTnT, relative to undifferentiated hESCs. Gene expression patterns consistent with embryonic atrium, sinoatrial (SA) node, left (LV) and right (RV) ventricle were seen. Data shown represent mean6s.e.m. for at least three hEBs with similar expression profiles for these seven genes. (B) A typical GFP+ hEB cultured on a gelatin-coated microelectrode array for 2 days is shown. (C) Sample field potential (FP) tracings recorded at arrayed microelectrodes are shown for HL-1 cells and typical aMHC-GFP+ or wild type hEBs. (D) Field potentials of averaged HL-1 cultures or hEBs were analyzed, and the number of tracings with a given FP duration (FPdur) or decay of extracellular potential (FPrise) were plotted as a function FPdur (left) or FPrise (right). Compared to the homogeneous FP recordings of HL-1 cultures, both aMHC-GFP+ (n = 7) and wild type (n = 5) hEBs showed similarly heterogeneous, averaged FP recordings, consistent with a heterogeneous distribution of action potential durations extracellular potential decay. doi:10.1371/journal.pone.0016004.g006 upregulated by day 8 of hMP differentiation (Table 2). In addition, secreted Frizzled-related proteins (SFRPs) that are known to inhibit Wnt signaling and regulate CM proliferation, were downregulated at day 8, but then upregulated in the case of SFRP3 at day 14 (Table 2). CM migration during heart development has been shown to rely on transforming growth factor b (TGFb) signaling [23]. Consistent with its role in cardiogenesis, several effectors and inhibitors of TGFb pathways were upregulated and downregulated, respectively, over the course of hMP differentiation (Table 3). Discussion Although b-myosin heavy chain (bMHC) is the predominant isoform expressed during murine embryonic and fetal develop- ment, the transition from bMHC to aMHC begins as early as day 7.5 p.c. in mouse embryos with the appearance of somites and the onset of cardiac tube formation [11,12]. Expression of both isoforms approaches equivalence as the cardiac tube begins to contract by day 9.5 p.c., and the b/a ratio begins to reverse with aMHC becoming the predominant isoform during postnatal life [11,12]. These observations during mouse development in vivo parallel mouse ESC (mESC) differentiation in vitro. aMHC is expressed by day 8 of mESC differentiation and is expressed exclusively in beating mouse EBs [12]. While many studies have used the murine aMHC promoter to track mature CMs, our data suggest that because of its expression across early and mature stages of CM development even between species, the murine aMHC promoter can be used to track CM differentiation from a PLoS ONE | www.plosone.org 6 January 2011 | Volume 6 | Issue 1 | e16004