4.3. Directed Differentiation of hESCs

Directed differentiation was carried out using H9 cells without or with pre-treatment of 0.0064% DMSO (Sigma-Aldrich, Munich, Germany) or 10 nM TCDD (Cambridge Isotope Laboratories, Tewksbury, MA, USA) in mTESR^™1 medium for 3 days. Cells were washed with PBS and harvested by GCDR (Gentle Cell Dissociation Reagent, STEMCELL Technologies) and incubated at 37 °C for 8 min in 5% CO₂. Single cell suspensions were generated by gentle pipetting and added to 1 mL KnockOut^™ DMEM (Dulbecco’s modified Eagle medium, Thermo Fisher Scientific, Rockford, IL, USA). Remaining cells were gathered by washing the wells with 1 mL fresh KnockOut^™ D-MEM. Cells were then centrifuged at 300× g for 5 min and resuspended in medium with 10 µM ROCK (Rho-associated protein kinase, Bio-Techne, Minneapolis, MN, USA) inhibitor. Cells were counted using Countess II cell counter (Thermo Fisher Scientific) and transferred to Matrigel^™-coated 6-well plates. For neural differentiation, STEMdiff™ neural induction medium (STEMCELL Technologies) was used. Cells were differentiated for 7 days with daily medium changes according to the manufacturer’s protocol. Pre-treated hESCs were differentiated in the presence of DMSO or 10 nM TCDD. For mesodermal and endodermal differentiation, cells were initially plated in mTESR^™1. The next day, medium was changed to STEMdiff™ Mesodermal Induction Medium or STEMdiff™ Definite Endoderm Kit Medium (both STEMCELL Technologies), respectively. Cells were differentiated for 5 days with daily medium changes according to the manufacturer’s protocol. Pre-treated hESCs were differentiated in the presence of DMSO or 10 nM TCDD. Plating densities are shown in Figure S14.