Previous studies have shown that even a single exposure to EtOH during the pre-implantation period enhanced post-implantation fetal death and resorption and retarded normal embryo development (Padmanabhan and Hameed, 1988). Since the pluripotent hESCs are naturally most abundant during the pre-implantation blastocyst stage we hypothesized that EtOH exposure might affect their pluripotency, which in turn would adversely affect their subsequent differentiation. To address this hypothesis, we first examined the effect of EtOH on hESC maintenance by immunofluorescence analysis for stem cell marker proteins. H9 hESCs were treated with EtOH (20 mM) for 24 hrs and subjected to IF analysis. The results showed that the expression of stemness marker proteins (OCT4, TRA-1-60 and SSEA-3) was visibly reduced with EtOH treatment (Fig. S3A). Since fetal alcohol exposure is known to induce programmed cell death in certain embryonic cell populations (Dunty et al., 2001; Ikonomidou et al., 2000; Kilburn et al., 2006), we also examined EtOH's effects on the levels of an early apoptosis marker Annexin V and found noticeable increases in Annexin V immunoreactivity in hESCs after 20 mM EtOH treatment (Fig.
