MCDB 131 W/O L-Glutamin

Protocols for maintenance of hPSCs and generation of lung cells were slightly modified from previous studies (Chen et al., 2019; Huang et al., 2014). 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 mM β-mercaptoethanol (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 °C in a 5% CO2/air environment until stem cells reached about 90% confluence.

Pancreatic endocrine cell differentiation was performed using INSGFP/W MEL-1 cells.

Cells were cultured on Matrigel-coated 6-well plates in StemFlex medium (Gibco Thermo Fisher). Cells were maintained at 37℃ with 5% CO2. hESCs were differentiated using a previously reported strategy (Zeng et al., 2016). In brief, on day 0, cells were exposed to basal medium RPMI 1640 supplemented with 1× Glutamax (Thermo Fisher Scientific), 50 μg/mL Normocin, 100 ng/mL Activin A (R&D systems), and 2 μM of CHIR99021 (GSK3β inhibitor 3, Cayman Chemical ) for 24 h. The medium was changed on day 1 to basal RPMI 1640 medium supplemented with 1× Glutamax (Thermo Fisher Scientific), 50 μg/mL normocin, 0.2% fetal bovine serum (FBS, Corning), 100 ng/mLActivin A (R&D systems) for 2 days.

On day 3, the resulting definitive endoderm cells were cultured in MCDB 131 medium (Thermo Fisher Scientific) supplemented with 1.5 g/L sodium bicarbonate, 1× Glutamax, 10 mM glucose (Sigma Aldrich) at final concentration, 2% bovine serum albumin ( BSA, Lampire), 0.25 mM L-ascorbic acid (Sigma Aldrich) and 50 ng/ml of fibroblast growth factor 7 (FGF-7, Peprotech) for 2 days to acquire primitive gut tube. On day 5, the cells were induced to differentiate to posterior foregut in MCDB 131 medium supplemented with 2.5 g/L sodium bicarbonate, 1× Glutamax, 10 mM glucose at final concentration, 2% BSA, 0.25 mM L-ascorbic acid, 50 ng /ml of FGF-7, 1 μM Retinoic acid (RA; Sigma Aldrich), 100 nM LDN193189 (LDN, Axon Medchem), 1:200 ITS-X (Thermo Fisher Scientific), 200 nM TPPB (Tocris Bioscience) and 0.

On day 7, the cells were induced to differentiate to pancreatic endoderm in MCDB 131 medium supplemented with 2.5 g/L sodium bicarbonate, 1× Glutamax, 10 mM glucose at final concentration, 2% BSA, 0.25 mM L-ascorbic acid, 2 ng /ml of FGF-7, 0.1 μM RA, 200 nM LDN193189, 1:200 ITS-X, 100 nM TPPB and 0.25 μM SANT-1 for 3 days.On day 10, the cells were induced to differentiate to pancreatic endocrine precursors in MCDB 131 medium supplemented with 1.5 g/L sodium bicarbonate, 1× Glutamax, 20 mM glucose at final concentration, 2% BSA, 0.05 μM RA, 100 nM LDN, 1:200 ITS-X, 0.25 μM SANT-1, 1 mM T3 hormone (Sigma Aldrich), 10 μM ALK5 inhibitor II (Cayman Chemical), 10 μM zinc sulfate heptahydrate (Sigma Aldrich) and 10 μg/ml of heparin (Sigma Aldrich) for 3 days.

On day 13, cells were exposed to MCDB 131 medium supplemented with 1.5 g/L sodium bicarbonate, 1× Glutamax, 20 mM glucose at final concentration, 2% BSA, 100 nM LDN193189, 1:200 ITS-X, 1 μM T3, 10 μM ALK5 inhibitor II, 10 μM zinc sulfate, 10 μg/ml of heparin, 100 nM gamma secretase inhibitor XX (Millipore) for the first 7 days. On day 27, cells were exposed to MCDB 131 medium supplemented with 1. 5 g/L sodium bicarbonate, 1× Glutamax, 20 mM glucose at final concentration, 2% BSA, 1:200 ITS-X, 1 μM T3, 10 μM ALK5 inhibitor II, 10 μM zinc sulfate heptahydrate, 10 μg/ml of heparin, 1 mM N-acetyl cysteine ​​(Sigma Aldrich), 10 μM Trolox (Millipore), 2 μM R428 (MedchemExpress) for 7-15 days. The medium was subsequently refreshed every day.

Organoid infections were performed at an MOI of 0.1 and harvested at 24 hpi in DMEM supplemented with 0.3% BSA, 4.5 g/L D-glucose, 4 mM L-glutamine and 1 μg/ml TPCKtrypsin.

Total RNA was extracted in TRIzol (Invitrogen) and DNase I treated using Directzol RNA Miniprep kit (Zymo Research) according to the manufacturer’s instructions. cDNA libraries were sequenced using an Illumina NextSeq 500 platform. The resulting single end reads were checked for quality (FastQC v0.11.5) and processed using the Digital Expression Explorer 2 (DEE2) (Ziemann et al., 2019) workflow. Adapter trimming was performed with Skewer (v0.2.2) (Jiang et al., 2014).

Further quality control done with Minion, part of the Kraken package (Davis et al., 2013). The resulting filtered reads were mapped to human reference genome GRCh38 using STAR aligner (Dobin et al., 2013) and gene-wise expression counts generated using the “-quantMode GeneCounts” parameter. BigWig files were generated using the bamCoverage function in deepTools2 (v.3.3.0) (Ramirez et al., 2016).
RNAseq libraries of polyadenylated RNA were prepared using the TruSeq RNA Library Prep Kit v2 (Illumina) or TruSeq Stranded mRNA Library Prep Kit (Illumina) according to the manufacturer’s instructions.