We observed numerous instances where EtOH

We observed numerous instances where EtOH had a differential effect on the transcriptome and methylome of undifferentiated hESCs compared to EBs, which contain differentiating Sulforhodamine 101 encompassing all somatic cell types (Itskovitz-Eldor et al., 2000) including those of neural and hepatic cell lineages. Recent studies have demonstrated inhibitory effects of pharmacologically relevant EtOH concentrations (20–100mM) on neuronal differentiation of ESCs and specified neural stem cells (NSCs). Specifically, EtOH was shown to inhibit NSC proliferation with dose-dependent increases in apoptosis (Anthony et al., 2008; Fujita et al., 2008; Talens-Visconti et al., 2011), decrease NSC differentiation (Tateno et al., 2004; Zhou et al., 2011) and divert ESC differentiation from neuroectodermal to mesodermal lineage (Ogony et al., 2013; Sanchez-Alvarez et al., 2013). Others utilizing very high EtOH concentrations (300–2000mM) also observed increased apoptosis of mouse blastocysts (Huang et al., 2007) and increased endodermal differentiation of EBs (Mayshar et al., 2011). Another recent study that focused on differentiation of human hepatocytes from EBs demonstrated that a 48hour EtOH (20mM) 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 21days 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 supports our findings of a selective impact of EtOH on differentiating cells compared to undifferentiated stem cells. Our methylome profiling revealed hotspots of DNA methylation on chromosomes 2, 16 and 18. Association of these chromosomes with alcohol dependence has been reported (Dick et al., 2010; Foroud et al., 1998; Treutlein et al., 2009; Wang et al., 2005). Thus, using earlier findings that chromosome 2, especially 2p14-2q14.3, is associated with phenotypes of alcohol dependence, suicide attempts, and conduct disorder, a systematic screen of SNPs with the three comorbid phenotypes yielded evidence of association with 23 genes, likely contributing to the preponderance of reported linkages with alcohol dependence and related phenotypes across chromosome 2 (Dick et al., 2010). Another study utilized a genome-wide association study, discovering 15 SNPs significantly associated with the same allele that had shown expression changes in rat brains after long-term alcohol consumption. In the combined analysis, 2 closely linked intergenic SNPs are located on chromosome region 2q35, which has been implicated in linkage studies for alcohol phenotypes, including the CDH13 and ADH1C genes that have been reported to be associated with alcohol dependence (Treutlein et al., 2009). Linkage analysis also detected loci underlying moderate and severe alcoholism implicating chromosome 16 (Foroud et al., 1998). In other studies, patients with carcinoid tumors had frequent history of alcohol consumption (Wang et al., 2005). Allelic loss of chromosome 11q was present in 21% of tumors, chromosome 16q in 13%, and chromosome 18 in 30% (Wang et al., 2005). In addition to identifying EtOH-induced global changes in genetic and epigenetic molecular landscapes, we were able to narrow down these changes further to four major potential signaling pathways with the help of extensive bioinformatic analyses. To our knowledge, this is the first time that energy metabolism, neuroactive ligand–receptor interaction, vascular smooth muscle contraction, and calcium signaling pathways are implicated in EtOH-induced changes in human embryonic stem cells. We provided strong molecular and bioinformatic evidence that even at a low physiologically relevant dose, EtOH may epigenetically alter key genes such as SLC12A4, P2RX3 and others in glycerophospholipid metabolism and regulation of calcium ion transport and/or calcium-mediated signaling, which ultimately may lead to the significant decrease in the self-renewal capacity of hESCs.