Selection of Human Myocardial Precursors Figure 5. Reporter activation coincides with early cardiogen- esis. aMHC-GFP hESCs were differentiated for 12 days and the number of hMPs (GFP+aMHC+) cells were scored as a percentage of total hESC number on each day. On days 1–4, before the onset of GFP expression, unsorted differentiating hESCs were analyzed by qPCR. On days 5–12, sorted GFP+ hMPs were analyzed by qPCR for expression of cTnT, NKX2- 5, GATA4 and aMHC, relative to undifferentiated hESCs at day 1. Data shown represent mean6s.e.m. (N = 3). Reporter activation was first observed on day 5, with 15% GFP+ (aMHC+) hESCs seen by day 9 (top). Endogenous aMHC, GATA4 and NKX2-5 expression was observed with activation of the reporter on day 5, while strong expression of cTnT did not appear until day 11 (bottom). doi:10.1371/journal.pone.0016004.g005 (FP) over time (Fig. 6C). This allowed us to measure the size of the largest negative peak (FPmin), the last postive peak of the cycle (FPmax), the time interval between FPmin and FPmax (FPdur), and decay of the extracellular potential (time from the onset of the FP to FPmin = FPrise). Others have shown that FPrise directly correlates to action potential rise time, and FPdur directly correlates to action potential duration [19,20]. Compared to HL-1 cells, which represent a homogenous culture of mouse atrial CMs [21] with FPdur#75 ms and FPrise#50 ms, both aMHC-GFP+ and wild type, beating hEBs demonstrated similarly heterogeneous combi- nations of FPdur (25–250 ms versus 10–475 ms; p = 0.86) and FPrise (10–175 ms versus 10–275 ms; p = 0.83) (Fig. 6D). Expression profiling of human CM differentiation Since we had established that this aMHC-GFP hESC line could identify myocardial precusors capable of giving rise to all embryonic CM subtypes, we used it to examine potential pathways involved in CM differentiation. We isolated hMPs at day 8 of differentiation, after the onset of GATA4, NKX2-5 and aMHC expression, but before cTnT expression or spontaneous contrac- tions (Fig. 5), and at day 14 of differentiation, after the onset of cTnT expression and spontaneous beating (Fig. 1), and compared the gene expression profiles of these two differentiation time points to each other and to undifferentiated hESCs using an array of .41,000 unique sequences representing the whole human genome. Analysis of the 830 most highly differentially expressed genes from biological replicates of each time point resulted in the clustering shown in Fig. 7. This indicated that the reproducibility of hMP differentiation was reasonably robust. Overall, there were 3,279 probes targeting human genes that showed differential expression across the three time points with a false discovery rate ,0.05. The complete data set has been deposited in Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/; Acces- sion Number GPL6480). Analysis of cardiac-specific gene expression demonstrated that hMP differentiation proceeded in a manner commensurate with cardiac morphological development (Table 1). Differentiating hMPs expressed markers of cardiac tube formation (NKX2-5, GATA4) and looping (MEF2c, aMHC) by day 8, and markers of spontaneous contractile activity (cTnT, cTnI) by day 14. In addition, expression levels of atrial (ISL-1, ANF) and right ventricular (ISL-1, HAND2) genes were observed by day 8 and continued to rise through day 14, while expression of genes associated with development of the atrioventricular septum (TBX2) and left ventricle (HAND1, TBX5) were observed by day 14. The role of Wnt signaling during myocardial specification and early cardiac morphogenesis has been described [22]. Wnt ligands and Frizzled receptors specifically have been implicated during early heart development. This is supported by our findings that the expression of Wnt1, 2, 5B and 11, as well as Frizzled 4 and 5 was PLoS ONE | www.plosone.org 5 January 2011 | Volume 6 | Issue 1 | e16004