after EtOH treatment in both H1 and H9 hESCs. This is to demonstrate an example of EtOH-induced alterations in gene signatures independent of cell lines. Volcano plots (Fig. 1D) demonstrated similarities in EtOH's effects on expression of genes in the two hESC lines, however, the changes were consistently greater in the H9 compared to the H1 line. Based on these data, we subsequently focused on molecular analysis of differentiation and EtOH application in the H9 hESC line. We also chose to focus on the lower concentration of EtOH (20 mM) because a similar blood level of EtOH, which is just above the driving limit (Jones and Sternebring, 1992), is easily achieved after excessive drinking by women (Bedford et al., 2006). The 24 hr duration of EtOH exposure was chosen because in addition to the low EtOH elimination capacity of the fetus, particularly in the early stages of pregnancy (Pikkarainen, 1971), EtOH remains trapped in the amniotic fluid leading to reabsorption by the fetus and prolonging exposure time (Brien et al., 1983).
