Pancreatic Progenitor Cell Differentiation

Initially, the H1 ES cell line was differentiated to PP cells using the STEMdiff Pancreatic Progenitor Kit (STEMCELL Technologies, 05120) according to the manufacturer’s instructions. In short, the hES cell colonies were dissociated into single cells using TrypLE Express (Gibco, 12604-013) and seeded on Matrigel-coated plates as described above at a concentration of 95,000 cells/cm² in mTeSR supplemented with 20 µM ROCKi. The medium was replaced the next day by mTeSR and differentiation was initiated by replacing it with the S1d1 differentiation medium when cells were 60–70% confluent, typically 2 d after the initial seeding. Daily washes with DPBS (Gibco, 14190250) and media changes were done until S4d5 when the cells reached the end of PP stage. The monolayer of PP cells was then dissociated using Accumax (STEMCELL Technologies, 07921), and cells were used for expansion under INI conditions.
Later, H1, H9, and CRTD1 iPS cells were differentiated into PP cells using an adaptation of published procedures (Mahaddalkar et al., 2020 (link); Rezania et al., 2014 (link); Shi et al., 2017 (link); Supplementary file 1d). The monolayer of PP cells was then dissociated using TrypLE Express (Gibco, 12604-013) for 2 min, and cells were used for expansion under C0 to C8 conditions.