One day before initiating NCSC differentiation, hPSCs maintained in E8 medium were singularized using Accutase and seeded at 9.1 × 104 cells/cm2 onto Matrigel-coated plates with E8 + 10 μM Y27632. NCSC differentiation was initiated the next day by switching the medium to E6, which is DMEM/F12 basal medium supplemented with l-ascorbic acid-2-phosphate magnesium (64 mg/liter), sodium selenium (14 μg/liter), insulin (19.4 mg/liter), NaHCO3 (543 mg/liter), and transferrin (10.7 mg/liter). E6 was supplemented with heparin sodium salt from porcine mucosa (22.5 mg/liter) to stabilize FGF2, 1 μM CHIR99021, 10 μM SB431542 (Tocris), FGF2 (10 μg/liter), and 1 μM dorsomorphin, hereafter labeled E6-CSFD. Cells were expanded by replacing E6-CSFD daily and passaging cells every time cells reached 100% confluence to fresh Matrigel-coated plates. During passaging, cells were singularized using Accutase and replated at a splitting density of one 6-well to six new 6-wells in E6-CSFD medium. Cells were generally passaged without 10 μM Y27632. However, to increase IMR90C4 cell line survival during first passaging following NCSC differentiation initiation, IMR90C4 cells were replated in E6-CSFD + 10 μM Y27632. Subsequent IMR90C4 NCSC expansion passages were replated without Y27632. Cells were typically passaged 2 to 3 days following NCSC differentiation initiation and subsequently passaged every 3 to 6 days depending on cell growth kinetics.

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