| 生长方案: |
Protocols for maintenance of hPSCs and generation of lung organoids were slightly modified from previous studies. The hESC line RUES2 was cultured on irradiated mouse embryonic fibroblasts (Global Stem, cat. no. GSC-6001G) at a density of 20,000- 25,000 cells/cm2 in a medium of DMEM/F12, 20% knockout serum replacement (Life Technologies), 0.1 mMmercaptoethanol (Sigma Aldrich) and 20 ng/ml bFGF (R&D Systems), and medium was changed daily. hESC cultures were maintained in an undifferentiated state at 37 in a 5% CO2/air environment until stem cells reached about 90% confluence. hESC differentiation into endoderm was performed in serum-free differentiation (SFD) medium of DMEM/F12 (3:1) (Life Technologies) supplemented with N2 (Life Technologies), B27, 50 /ml ascorbic acid, 2 mM Glutamax, 0.4 monothioglycerol, 0.05% BSA at 37 n a 5% CO2/5% O2/95% N2 environment. hESCs were treated with Accutase and plated onto low attachment 6-well plates (Corning Incorporated, Tewksbury MA), resuspended in endoderm induction medium containing 10 Y-27632, 0.5 ng/ml human BMP4, 2.5 ng/ml human bFGF, 100 ng/ml human Activin A, 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 hrs, and then switched to 36 hrs 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, 10 ng/ml human FGF10, 10 ng/ml human KGF, 10 ng/ml human BMP-4 and 50-60 nM all-trans retinoic acid (ATRA), in SFD medium for 80 d. The day 105 cultures were 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 either a combination of five factors (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 FGF-10; 10 ng/ml human KGF, and DCI (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). |
