Homo sapiens
Protocols: Culture media, growth factors and small molecules Serum-free basal media: All human fetal lung bud, and hPSC-derived human lung organoids were grown in serum-free basal media (basal media) with added growth factors. Basal media consisted of Dulbecco's Modified Eagle Medium: Nutrient Mixture F12 (DMEM/F12, ThermoFisher SKU# 12634-010) supplemented with 1X N2 supplement (ThermoFisher Catalog# 17502048) and 1X B27 supplement (ThermoFisher Catalog# 17504044), along with 2mM Glutamax (ThermoFisher Catalog# 35050061), 1x Pennicillin-Streptomycin (ThermoFisher Cat# 15140122) and 0.05% Bovine Serum Albumin (BSA; Sigma product# A9647). BSA was weighed and dissolved in DMEM F/12 media before being passed through a SteriFlip 0.22 uM filter (Millipore Item# EW-29969-24) and being added to basal media. Media was stored at 4 degrees Celsius for up to 1 month. On the day of use, basal media was aliquoted and 50ug/mL Ascorbic acid and 0.4 uM Monothioglycerol was added. Once ascorbic acid and monothioglycerol had been added, media was used within one week. Growth factors and small molecules: Recombinant Human Fibroblast Growth Factor 7 (FGF7) was obtained from R&D systems (R&D #251-KG/CF) and used at a concentration of 10 ng/mL unless otherwise noted. Recombinant Human Fibroblast Growth Factor 10 (FGF10) was obtained either from R&D systems (R&D #345-FG) or generated in house (see below), and used at a concentration of 10 ng/mL (low) or 500 ng/mL (high) unless otherwise noted. Recombinant Human Bone Morphogenic Protein 4 (BMP4) was purchased from R&D systems (R&D Catalog # 314-BP) and used at a concentration of 10 ng/mL. All-trans Retinoic Acid (RA) was obtained from Stemgent (Stemgent Catalog# 04-0021) and used at a concentration of 50 nM. CHIR-99021, a GSK3 inhibitor that stabilizes -CATENIN, was obtained from STEM CELL technologies (STEM CELL Technologies Catalog# 72054) and used at a concentration of 3 uM. Y27632, a ROCK inhibitor (APExBIO Cat# A3008) was used at a concentration of 10uM. Generation and Isolation of human recombinant FGF10: Recombinant human FGF10 was produced in-house, as previously described (Bagai et al., 2002). The recombinant human FGF10 (rhFGF10) expression plasmid pET21d-FGF10 in E. coli strain BL21trxB(DE3) was a gift from James A. Bassuk at the University of Washington School of Medicine. E. coli was grown in standard LB media with peptone derived from meat, carbenicillin and glucose. rhFGF10 expression was induced by addition of isopropyl-1-thio-β-D-galactopyranoside (IPTG). rhFGF10 was purified by using a HiTrap-Heparin HP column (GE Healthcare, 17040601) with step gradients of 0.5M to 2M LiCl. From a 200 ml culture, 3 mg of at least 98% pure rFGF-10 (evaluated by SDS-PAGE stained with Coomassie Blue R-250) was purified. rFGF10 was compared to commercially purchased human FGF10 (R&D Systems) to test/validate activity based on the ability to phosphorylate ERK1/2 in an A549 alveolar epithelial cell line (ATCC Cat#CCL-185) as assessed by western blot analysis. RNA was isolated using the mirVana RNA isolation kit, following the “Total RNA” isolation protocol (Thermo-Fisher Scientific, Waltham MA). RNA sequencing library preparation and sequencing was carried out by the University of Michigan DNA Sequencing Core and Genomic Analysis Services (https://seqcore.brcf.med.umich.edu/).