A 8301

Human fetal lung within the canalicular stage of lung development was utilized for lung ALI explant cultures (specifically 15–18.5 weeks post conception). Small pieces (<0.5 cm diameter) were dissected from distal regions of the lung and placed on Nucleopore Track-Etched Membrane disks (13 mm, 8 µm pore, poly-carbonate) (Sigma, Cat#WHA110414) floating on top of 500 µl of human lung ALI explant media (Advanced DMEM/F-12 (Thermo Fisher, Cat#12634010), 2 mM Glutamax (Thermo Fisher, Cat#35050061), 15 mM HEPES (Corning, Cat#25060CI), 1× B27 Supplement (Thermo Fisher, Cat#17504044) 1× N-2 Supplement (Thermo Fisher, Cat#17502048), 100 U/mL penicillin-streptomycin (Thermo Fisher, Cat#15140122)) in a 24-well tissue culture plate (Thermo Fisher, Cat#12565163). Where indicated, 1 µM A-8301 (APExBIO Cat#A3133), 100 ng/mL rhTGFβ1 (R&D Systems Cat#240-B-002), 100 ng/mL rhNOGGIN (produced in-house) or 100 ng/mL BMP4 (R&D Systems Cat#314-BP-050) was added to human lung ALI explant media.

AML12 cells were cultured in basic medium containing DMEM/F12, 10% FBS, 1% insulin-transferrin-selenium (ITS) (Sigma, UAS), 40 ng/mL dexamethasone, 1% penicillin, and 1% streptomycin according to ATCC recommendations. For short-term culture, AML12 cells were cultured in basic medium containing LPS (100 ng/mL) for the indicated amounts of time and then collected for RNA and protein analysis. For 3D culture, cells were plated on collagen-coated 12-well plates in reprogramming medium containing DMEM/F12, 1% FBS (Gibco), 10 mM nicotinamide (Sigma, UAS), 0.1 μM dexamethasone, 1% ITS, 20 ng/mL EGF (Peprotech, USA), 20 ng/mL HGF (Peprotech, USA), 10 μM Y27632 (APExBIO, USA), 1 μM A83-01 (APExBIO, USA), and 3 μM CHIR99021 (APExBIO, USA) supplemented with or without LPS (100 ng/mL) [23 (link)]. The medium was replaced every 2 days. After 2 weeks, the cells were harvested for the following assays. Mouse hepatic progenitor cells (mHPCs) were isolated and cultured as previously described [21 (link)].