hiPSCs and hESCs were differentiated into pancreatic progenitors via modification of previously described methods.11 (link)–13 (link) In brief, hPSCs were treated with Collagenase IV (Life Technologies, Waltham, Massachusetts) and Dispase (STEMCELL Technologies, Vancouver, BC, Canada) at a 1:1 ratio for 3–5 minutes before being manually passaged and passed through a 70 μm cell strainer to remove the mouse embryonic fibroblasts (MEFs). hPSCs were then differentiated 2 days later in RPMI 2% B27 (without vitamin A) (Gibco, Waltham, Massachusetts) supplemented with 100 ng/mL Activin A (R&D Systems, Minneapolis, Minnesota), 3 μM CHIR99021 (Tocris Bioscience, Bristol, UK), 10 μM LY294002 (LC Labs, Woburn, Massachusetts) for 3 days, 50 ng/mL Activin A for 2 days, 50 ng/mL FGF2, 3 μM all-trans retinoic acid (RA) (WAKO, Osaka, Japan), 10 mM nicotinamide (Nic) (Sigma Aldrich, Singapore) for 5 days, 50 ng/mL FGF2, 3 μM RA, 10 mM Nic, 20 μM DAPT (Abcam, Cambridge, UK) for 4 days and 50 ng/mL FGF2, 10 mM Nic, 20 μM DAPT for 3 days to derive pancreatic progenitors.
hPSCs were also differentiated into SC-β cells following the differentiation protocol by Pagliuca et al7 (link) on low attachment plates with shaking for the duration of the whole differentiation protocol, without transfer to a bioreactor.
hPSCs were also differentiated into SC-β cells following the differentiation protocol by Pagliuca et al7 (link) on low attachment plates with shaking for the duration of the whole differentiation protocol, without transfer to a bioreactor.
