Extraction of ImPSC cell lines

ImPSC #1 cells were a gift from Jurre Kamphorst(Auciello et al., 2019). Briefly, healthy pancreas tissue extracted from C57Bl6/J mice was minced and digested for 20mins at 37°C with 0.1% DNase (Roche), 0.05% Collagenase P (Roche) and 0.02% Pronase (Roche) in Gey’s balanced salt solution (GBSS; Sigma Aldrich). The tissue was then titurated until the large pieces were no longer visible, passed through a 100μm filter and washed with GBSS. The cells were then pelleted and resuspended in 9.5ml GBSS with 0.3% BSA and 8ml Nycodenz solution (Sigma Aldrich). The cell suspension was layered beneath GBSS containing 0.3% BSA, and centrifuged at 1400 x g for 20 min at 4°C. Stellate cells were harvested from the interface of the Nycodenz solution at the bottom and the aqueous solution at the top. The PSCs isolated were then washed with GBSS and resuspended in DMEM with 10% characterized FBS (HyClone), 100 U/ml penicillin and 100μ/ml streptomycin (Invitrogen). The cells were immortalized with the pRetro. Super.shARF retroviral plasmid (provided by the Karen Vousden lab) and selected with blasticidin (4μM).

ImPSC #2 & #3 lines were isolated using a very similar protocol as ImPSC #1 with some minor differences detailed below. Pancreas tissue was extracted from Pdgfra^{tm11(EGFP)Sor} mice(Hamilton et al., 2003) (JAX stock #007669), minced with a scalpel and digested with 0.1% DNase I (Thermo Fisher Scientific, 90083) and 0.05% collagenase P (Roche, 11 213 865 001) in GBSS for 30mins at 37°C. The solution was then passed through a 100μm filter, washed with GBSS, pelleted and resuspended in 6ml GBSS containing 0.3% BSA. The cell suspension was then mixed with 8ml Histodenz solution (43.75% in GBSS), layered beneath GBSS containing 0.3% BSA, and centrifuged at 1400 x g for 20mins at 4°C. Stellate cells were harvested from the interface of the Histodenz solution at the bottom and the aqueous solution at the top. The PSCs were washed in PBS containing 3% FBS and resuspended in DMEM containing 10% FBS, 1% penicillin- streptomycin, 0.2% amphotericin B and glutamine (2mM). After culture was established, fibroblasts expressing GFP were isolated via FACS and immortalized spontaneously.

ImPSC #1 cells stably expressing GFP (ImPSC-GFP cells) were generated by the PiggyBac transposon system. Briefly, 5 × 10⁴ ImPSC #1 cells were seeded in a 6-well plate. 24h after seeding, cells were transfected using Lipofectamine 3000 (Thermo Fisher Scientific, LS3000008) with 1.5μg Super piggyBac Transposase expression vector (Cambridge Biosciences, PB210PA-1) and 0.6μg PB-GFP PB-CMV-MCS-EF1-GreenPuro (Cambridge Biosciences, PB513B-1). 24h after transfection, cells were selected in 5μg/ml puromycin for 48h, until puromycin sensitive control cells treated in parallel were dead.