Type 4

Trachea and airway draining lymph node (ADLN) were prepared as single cell suspensions as described (von Garnier et al. 2005). Briefly, pooled (5 mice/group) trachea or ADLN were collagenase digested (Type IV; 1.5 mg/mL; Worthington Biochemical, Lakewood, NJ) and DNAse (0.1 mg/mL; Sigma Aldrich, Sydney, NSW, Australia), then washed in glucose potassium sodium buffer (GKN) as previously described (von Garnier et al. 2005).

Human ESCs were detached with 2 mg/mL collagenase Type IV (Worthington Biochemical Corporation, Lakewood, NJ, USA), for 30 min at 37 °C. EBs were formed from the detached ESCs and were cultured in suspension for 4 days in DMEM/F12 (Thermo Fisher Scientific) containing 10 μg/mL human insulin, 9 μg/mL transferrin, 14 ng/mL selenite, 5 μM PKCβ inhibitor, and 1 μM DMH1 (all from Sigma-Aldrich). The culture medium was changed daily. On Day 4 of culture, EBs were transferred to Matrigel-coated dishes with NPC specification medium containing 1% N2 supplement (Thermo Fisher Scientific), 20 ng/mL bFGF (CHAbiotech, Pangyo, Korea), and 25 μg/mL human insulin (Sigma-Aldrich). The medium was changed every day for 5 days to generate neural rosettes.