Cytotune

In this study, we used nine iPSC-derived lines outlined in Supplementary Table 1. Newly derived lines were reprogrammed using the Nakanishi SeVdp vector Sendai system described previously (Nishimura et al., 2011) or CytoTune® (Life Technologies). Six lines used were derived from skin biopsies: three healthy controls (AH017, SBAD02, NHDF1) and three lines from migraine patients (837, 838, 839), three sisters all belonging to the same family with the F139WfsX24 mutation in KCNK18 gene encoding TRESK. Two additional lines were reprogrammed from blood erythroblasts; BPC345 (healthy control) and BP8512 (containing the C110R TRESK variant). A further iPSC line, RCi002-A, which is from a patient with inherited erythromelalgia and having the F1449V mutation in SCN9A, was obtained from the EBiSC consortium and has been published (Cao et al., 2016 ).

Preparation of iPSC from human primary lymphocytes using Sendai viral vectors (CytoTune™, Life Technologies) has been described24 . All biomaterials were de-identified repository specimens and therefore are exempt from human subjects regulations. Midbrain-like DA neurons were generated using a dual SMAD inhibition protocol26 (link). Excitatory neurons were induced through a modified protocol27 (link) where during day 5 of the protocol iNs were gently dissociated with accutase (StemCell Technologies), and (7.0 × 10⁴) were co-cultured with mouse glia (3.5 × 10⁴ cells)54 (link). Electrophysiological analyses were performed following at least 30 days of differentiation and maturation. Additional details are found in the Supplemental Methods.

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.

The iPSC lines were produced from skin fibroblasts with Sendai virus vectors (OCT4, SOX2, KLF4, C-MYC; CytoTune; Life Technologies, Carlsbad, CA, USA) and maintained as published before [14 (link)]. Two patient-derived iPSC lines (UTA.10211.EURCAs and UTA.11304.EURCCs) were used in this study, and the lines were characterized as previously described [15 (link)]. Written and informed consent was obtained from all study subjects. The Ethics Committee of Tampere University Hospital approved the study and patient recruitment (approval number: R12123) and all experiments were performed in accordance with relevant guidelines and regulations.