Directed Differentiation of Stem Cells

ETX embryos were generated as described previously^{10 (link)}. Approximately 6000–7000 ES cells, 15,000–19,000 TS cells and 5000–6000 XEN cells were added dropwise into AggreWell plates having 1200 microwells in one well (34411; STEMCELL Technologies). The microwells were treated with rinsing solution (07010; STEMCELL Technologies). Cells were centrifuged at 100g for 3 min. 1.5 ml c-IDG medium containing 7.5 nM ROCK inhibitor (72304; STEMCELL Technologies) was added dropwise to each well. On the following day (day 1), 1 ml medium was removed gently from each well and replaced with 1 ml fresh c-IDG medium without ROCK inhibitor. This step was repeated once to fully remove the ROCK inhibitor. On day 2, 1 ml c-IDG medium was replaced with 1 ml fresh medium. On day 3, the media was replaced with IVC1 medium^{10 (link),24 (link),52 (link)}. IVC1 medium comprises advanced DMEM/F12 (21331-020; Gibco) supplemented with 20% (vol/vol) FBS, 2 mM GlutaMax, 1% vol/vol penicillin–streptomycin, 1× ITS-X (51500-056; Thermo Fisher Scientific), 8 nM β-estradiol, 200 ng ml⁻¹ progesterone and 25 mM N-acetyl-l-cysteine.