3f). We found that cells differentiated in purified nicotine were not significantly different on the basis of all these endpoints compared to control EPZ020411 (hydrochloride) samples (S2 Fig). These initial studies illustrated the cytotoxic effect of tobacco cigarette extract exposure on developing hESCs and revealed its inhibitory effect on cardiomyocyte differentiation.
Cardiac developmental defects observed in zebrafish treated with cigarette smoke. (a) Representative whole mount images of zebrafish at 72 hpe showing normal, mild, intermediate, and severe cardiac developmental defects. v = ventricle, At = atrium. (b-c) Analysis of percent zebrafish with heart defects (b) severity of heart defects and (c). (d) Analysis of heart function in control, e-cigarette and tobacco treated groups at 72 hpe. (e) Quantitative RT-PCR analysis (fold change from control) of a panel of genes with critical roles in early heart development at 24 hpe. n ! 3 (independent experiments with each n containing between 248 animals per treatment). For qRT-PCR, n = 3 with each n consisting of 285 embryos from independent breeding pools. P 0.05, hpe = hours post exposure; N = Nicotine, E = E-cigarette, T = Tobacco. Analysis of e-cigarette and tobacco cigarette on hESC cardiac differentiation. (a) Timeline of differentiation protocol for cardiac directed differentiation of hESCs. (b-e) Analysis of cardiac endpoints including intrinsic beating rate (b) cardiomyocyte yield (c), cardiomyocyte purity (d), and cardiomyocyte immaturity based on percent cTnT+/SMA+ (e) and representative flow cytometry plots (f) on day 14 of differentiation with increasing doses of purified e-cigarette and tobacco cigarette extracts.
Given results from initial studies looking at increasing doses of nicotine from different cigarette sources, a dose of 6.8 M nicotine was chosen to compare the effects of e-cigarette aerosol extract and tobacco cigarette smoke extract to control samples during a time course analysis. To determine the impact of cigarette smoke treatment on different stages of cardiac differentiation, RNA samples were harvested at day 2 (mesoderm), day 5 (cardiac progenitor cell), and day 14 (definitive fetal-stage cardiomyocytes). Quantitative RT-PCR analyses were performed to determine the transcript abundance of a panel of genes known to have critical roles in cell fate decisions or to participate in the functional development of the cardiomyocyte at each of these stages. During the transition through mesoderm on day 2, we assessed expression 
of the pan mesendoderm marker Brachyury T and found no difference between control, e-cigarette aerosol extract and tobacco cigarette smoke extract treated samples (Fig 4a). However, genes involved in patterning anterior primitive streak-derived mesendoderm development including the bicoid homeobox protein Goosecoid (GSC) and NODAL were significantly higher only in cells treated with tobacco cigarette smoke extract (Fig 4b). Among the transcription factors known to specify the early stages of cardiac development, eomesodermin (EOMES) is known to regulate MESP1 in an axis of signaling to directed pre-cardiac mesoderm fate specification [35]. We found that cells treated with tobacco cigarette smoke extract had significantly higher levels of EOMES and lower levels of MESP1 compared to control and e-cigarette aerosol extract treated samples (Fig 4c). We also analyzed a panel of genes involved in transition through the cardiac progenitor cell stage (day 5). GATA4 a
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