α mtg

CCE, a mouse embryonic stem cell line derived from 129/Sv mouse strain, was obtained from ATCC. mESCs were maintained on gelatin-coated plates in feeder-free Iscove's modified Dulbecco's medium (IMDM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (ES qualified, Invitrogen), 1% nonessential amino acids, 1% penicillin-streptomycin, 4.5 × 10⁻⁴ M α-monothioglycerol (α-MTG; Sigma), 1,000 U/mL leukemia inhibitory factor (LIF). Cardiac differentiation of mESCs was performed as previously described (Yang et al., 2014 ). In brief, mESCs were developed into EBs (1,250 cells/drop) in a 3D hanging-drop culture for 4 days with LIF-free differentiation medium (IMDM supplemented with 10% FBS, 1% nonessential amino acids, 1% penicillin-streptomycin, and 4.5 × 10⁻⁴ M α-MTG). The early EBs were either plated onto gelatin-coated 12-well culture plates for adherent differentiation or put into a rotary system for 3D microgravity culture with the same differentiation medium for 8 days. EBs were treated with nicotine at a serial concentration (0.01–10 μM) in the presence or absence of 10 μM global nAChR antagonist Hexa during 12 days of differentiation.

NKX2‐5‐eGFP human ES cells 42 (stable reporter line generated from wild‐type HES3 cells 71) were cultured in Essential 8™ medium (Gibco) on Matrigel (BD, Corning) without penicillin/streptomycin. Cells were differentiated in a monolayer toward cardiomyocytes as previously described 45. In short, human ES cells were cultured in E8 until 60% confluent. Cells were then supplemented with 1% DMSO enriched E8 medium for 24 h. On D0, cells were put on BPEL medium supplemented with Activin A (20 ng/ml, R&D Systems), BMP4 (20 ng/ml, R&D Systems), and CHIR99021 (1.5 μM, Axon Medchem). At D3, medium was changed to BPEL with XAV939 (5 μM, Tocris), and on D6, BPEL without any supplements was used. BPEL medium: IMDM, no phenol red (Gibco) and F12 Ham's F12 nutrient Mix (Gibco) in a 1:1 ratio supplemented with 5% (v/v) PFHM‐II (Gibco), 0.25% (w/v) BSA, 1% (v/v) Chemically Defined Lipid Concentrate (Gibco), 0.1% ITS‐X (Gibco), 450 μM α‐MTG (Sigma), 2 mM GlutaMax, 50 μg/ml L‐ascorbic acid 2‐phosphate (Sigma), and 0.25% penicillin/streptomycin (10,000 U/ml, Gibco).