Aggrewell 400 system

We generated embryoid bodies (EB) from iPSCs with Aggrewell 400 system (Stemcell Technologies) according to manufacturer's protocol. After formation, EBs were transferred to Ultra-low-adhesion plates (Corning, USA) and cultured in DMEM/F12 (Paneco, Russia), 15% Knock-Out Serum Replacement (Invitrogen, USA), 5% FBS (Hyclone, USA), 0.1 mM β-mercaptoethanol (Sigma, USA), 1% NEAA (Hyclone, USA) and 50 U/ml penicillin-streptomycin (Paneco, Russia). The medium was changed every 2 days. Embryoid bodies were grown for 10 days, transferred to gelatin-coated Petri dishes and cultured for 15–20 days in the same medium.

hESC colony cultures were incubated with the Rho Kinase inhibitor Y-27632 (Rock Inhibitor, 10 μM: Sigma-Aldrich, St Louis, MO, USA) for 1 h and dissociated to single cells using TrypLE™ Express. Cell aggregates (1000–1500 cells) were formed using the AggreWell™400 system (StemCell Technologies) and cultured in Basal Differentiation Media (BDM) with Rock Inhibitor (Table S1). For differentiation, aggregates were cultured in BDM supplemented with combinations of SB43218 (SB: 10 μM), CKI-7 (CKI: 5 μM) and nicotinamide (NIC: 10 mM) (all Sigma-Aldrich). After 6 days, embryoid bodies (EBs) were plated on GFR-MG-coated plates in BDM.
At day 8, expanding EBs were dissociated with collagenase IV (1 mg/mL) and re-plated as clumps of 200 to 500 cells on GFR-MG-coated 24-well dishes. Cells were then cultured in BDM without factors and refed every 2 to 3 days until day 38.