Culture of Human Induced Pluripotent Stem Cell (hiPSC)-Derived AT2 Cells (iAT2 Cells)

IPSCs-derived human alveolar type 2 cells (iAT2s) (differentiated from the SPC2-ST-B2 iPSC line) were expanded as alveolospheres in growth factor reduced Matrigel (Corning, 354230) as described elsewhere (Jacob et al., 2019). The iAT2s were maintained in IMDM medium containing 25% Ham’s F12 (Cellgro, 10–080-CV), 1% B27 supplement, 0.5% N2 supplement, 0.05% BSA (Invitrogen, 15260037), 200 ng/ml Primocin (Invivogen, NC9141851), 1X GlutaMAX, 50 μg/ml ascorbic acid (Sigma, A4544), 0.45 mM monothioglycerol (Sigma, M6145), 3 μM CHIR99021, 10 ng/ml rhKGF (R&D, 251-KG-010), 50 nM Dexamethasone (Sigma, D4902), 10 μM Y27632 (for first 3 days of culture), 0.1 mM 8-bromoadenosine 3′,5′-cyclic monophosphate sodium salt (Sigma, B7880), and 0.1 mM 3-isobutyl-1-methylxanthine (Sigma, I5879).