endodermal differentiation of EBs (Mayshar et al., 2011). Another recent study that focused on differentiation of human hepatocytes from EBs demonstrated that a 48 hr EtOH (20 mM) treatment perturbed the differentiation of progenitor cells into hepatocytes (Pal et al., 2012). Given our focus on the early onset of hESC differentiation (2-3 days) versus their longer than 21 days of differentiation, direct comparisons are difficult, nevertheless commonalities include EtOH-induced downregulation of the stem cell transcription marker, OCT4, and downregulation of several differentiation markers associated with the formation of definitive endoderm and functional hepatocytes (GATA6 and SOX17, see Fig. S3). In addition, we used WCGNA to demonstrate many similarities between selective OCT4 knockdown (Wang et al., 2012) and EtOH effects on undifferentiated hESCs (Fig. S4). Overall, published literature support our findings of a selective impact of EtOH on differentiating cells compared to undifferentiated stem cells.
