Modeling Alcoholism in iPSC-derived Neurons

Three iPS cell lines were obtained from three healthy patients with no history of alcohol addiction. Briefly, cryopreserved primary lymphocytes were processed for CD4⁺ T-cell selection and Sendai viral reprogramming (CytoTune™, Life Tech), as described previously [56 ]. Pluripotency was confirmed by immunohistochemistry (IHC) for Oct4 and TRA-1-60 (data not shown). NPCs were generated from iPS cells by using the Gibco Neural Induction Medium (MAN0008031, Gibco) according to the manufacturer’s guidelines. iPS cells or NPCs after three passages from neural induction were treated with 70 mM ethanol for 24hr or 7d, by daily full replenishment with new culture medium. It has been reported that the alcohol concentration in culture gradually decreases, with an approximate 19-hr half-life in an unsealed culture dish [40 (link)]. The daily replenishment of the ethanol containing media, followed by a gradual loss by evaporation in unsealed culture dishes mimics the pattern of alcohol exposure of heavy drinkers [40 (link)]. After 7 days in vitro, the cells were plated at a density of 2.5×10⁵ on coverslips in 24-well plates for 24hr and processed for immunostaining, Western Blot, differentiation or peroxide treatment. All results are presented as relative to the average of the iPS cell lines respectively.