Differentiation of human pluripotent stem cells into pancreatic progenitors

hPSCs were differentiated to pancreatic progenitors (PPs) using two previously published protocols with slight modifications in media composition [19]. hPSCs differentiated using the two protocols were either dissociated and re-plated following formation of endoderm or left unmanipulated (Fig. 1a). Differentiation was initiated when hPSCs reached 60–75% confluency. mTesR1 media was replaced with MCDB 131 (ThermoFisher Scientific, USA) composed of 2 mM glutamax, 0.5% fatty acid free bovine serum albumin (BSA; Sigma, USA), 1.5 g/L NaHCO₃ (VWR, USA) and 1% penicillin/streptomycin as basal media and supplemented with 100 ng/ml hActivin A (R&D Systems, MN, USA), 2 μM CHIR99021 (Stemgent, USA), 0.25 mM vitamin C (Sigma, USA), and 10 μM Y-27632 (Rock inhibitor; Stemgent, USA) for day 1 of differentiation (stage 1 day 1). For days 2–3, the basal media were supplemented with 100 ng/ml hActivin A, 0.25 mM vitamin C, and 5 ng/ml basic fibroblast growth factor (bFGF; Stem Cell Technologies, USA). On day 4 (stage 2 day 1), the cells for each protocol were either dissociated using TrypLE and re-plated on fresh Matrigel (1:50) coated dishes or left adherent. Basal media as prepared in stage 1 were supplemented with 0.25 mM vitamin C, 50 ng/ml hFGF10 (R&D Systems, USA), 0.25 μM CHIR99021, and 50 ng/ml NOGGIN (R&D Systems, USA) for 2 days of stage 2 (days 4–5). For all experiments, except when stated otherwise, the dissociated cells were re-plated at densities within the range 2.5–3.5 × 10⁵ cells/cm², which was about half the endoderm density for the individual experiments. For Protocol 1 (P1; both dissociated (D) and non-dissociated (ND)), stage 3 treatment was performed for 2 days, while for Protocol 2 (P2; both dissociated (D) and non-dissociated (ND)) this was performed for 4 days. For stage 3 differentiation, DMEM (ThermoFisher Scientific, USA) was supplemented with 1% penicillin/streptomycin, 1% vol/vol B27 supplement without vitamin A (ThermoFisher Scientific, USA), 0.25 mM vitamin C, 50 ng/ml hFGF10, 50 ng/ml hNOGGIN, 2 μM retinoic acid (Sigma, USA), and 0.25 μM SANT-1 (Sigma, USA) for the specified number of days for each protocol. At the end of stage 3, media were changed to DMEM supplemented with 1% vol/vol B27, 0.25 mM vitamin C, 50 ng/ml hFGF10, 50 ng/ml hNOGGIN, 100 ng/ml hEGF, and 10 mM nicotinamide (Sigma, USA) for 4 days of stage 4 treatment for each protocol (Fig. 1a). For generation of endocrine progenitors (stage 5), P2-D pancreatic progenitors were washed twice with DPBS at the end of stage 4 and supplemented with stage 5 day 1 media as specified by Pagliuca et al. [8] under adherent condition. Stage 5 treatment was performed for 7 days.

a Schematic overview of the protocols used for the differentiation of hPSCs into pancreatic progenitors. hPSCs are differentiated through the stages of definitive endoderm (DE) and posterior foregut to yield pancreatic progenitors through two different protocols differing in their duration of stage 3 treatment. Each of these protocols were either dissociated after generation of DE or left unmanipulated throughout the differentiation stages. Pancreatic progenitors generated using protocol 2 (P2) following dissociation were further differentiated through stage 5 to generate endocrine progenitors in adherent culture. b Representative immunofluorescent images of hESC-derived DE expressing high levels of SOX17 (green) and FOXA2 (red) after stage 1 of differentiation. The differentiated cells lost their pluripotency as indicated by the loss of OCT4 at the end of stage 1 (c). Nuclei are labeled with Hoechst. d Flow cytometry analysis of SOX17 expression at the end of stage 1 of differentiation. All data shown are representative results from at least three independent experiments. Scale bars = 100 μm