| 处理方案: |
hPSC differentiation into endoderm was performed in serum-free differentiation (SFD) medium of DMEM/F12 (3:1) (Life Technologies) supplemented with 1N2 (Life Technologies), 1B27 (Life Technologies), 50 g/ml ascorbic acid, 2 mM Glutamax (Gibco), 0.4 monothioglycerol, 0.05% BSA at 37 in a 5% CO2/5% O2/95% N2 environment. hPSCs were treated with Accutase and plated onto low attachment 6-well plates (Corning), re-suspended in endoderm induction medium containing 10 Y-27632, 0.5 ng/ml human BMP4 (R&D Systems), 2.5 ng/ml human bFGF, 100 ng/ml human Activin A (R&D Systems), for 72-76 hours dependent on the formation rates of endoderm cells. On day 3 or 3.5, the endoderm bodies were dissociated into single cells using 0.05% Trypsin/0.02% EDTA and plated onto fibronectin-coated, 24-well tissue culture plates (~100,00050,000 cells/well). For induction of anterior foregut endoderm, the endoderm cells were cultured in SFD medium supplemented with 1.5 dorsomorphin dihydrochloride (R&D Systems) and 10 SB431542 (R&D Systems) for 36 hours, and then switched to 36 hours of 10 SB431542 and 1 IWP2 (R&D Systems) treatment. For induction of early stage lung progenitor cells (day 65), the resulting anterior foregut endoderm was treated with 3 CHIR99021 (CHIR, Stem-RD), 10 ng/ml human FGF10, 10 ng/ml human KGF, 10 ng/ml human BMP4 and 50-60 nM all-trans retinoic acid (ATRA), in SFD medium for day 80. The day 105 culture was maintained in a 5% CO2/air environment. On days 15 and 16, the lung field progenitor cells were replated after one minute trypsinization onto fibronectin-coated plates, in the presence of SFD containing 3 CHIR99021, 10 ng/ml human FGF10, 10 ng/ml human FGF7, 10 ng/ml human BMP4, and 50 nM ATRA. Day 165 cultures of late stage lung progenitor cells were maintained in SFD media containing 3 CHIR99021, 10 ng/ml human FGF10, 10 ng/ml human KGF, in a 5% CO2/air environment. For differentiation of mature lung cells (day 25 to 55) in 3D culture, cultures were re-plated and embedded in 90% Matrigel after brief trypsinization in SFD media containing maturation components containing 3 CHIR99021, 10 ng/ml human FGF10; 10 ng/ml human KGF, 50 nM Dexamethasone, 0.1 mM 8-bromo-cAMP (Sigma Aldrich) and 0.1 mM IBMX (3,7-dihydro-1-methyl-3-(2-methylpropyl)-1H-purine-2,6-dione, Sigma Aldrich). |
