Redifferentiation of Human Islet Cells

Human islets (Table 1) were received 2–6 days following isolation. Islets were dissociated into single cells. Cells were cultured as previously described [1 (link)] in CMRL 1066 medium containing 5.6 mM glucose and supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT); 100 units/ml penicillin, 100 mg/ml streptomycin, and 100 mg/ml gentamycin (PSG); 5 mg/ml amphotericin B; and 3.5 mg/ml L-glutamine. The cells were refed twice a week and split 1:2 once a week. 293T cells were cultured in DMEM supplemented with 10% FBS, PSG, and 3.5 mg/ml L-glutamine. Redifferentiation cocktail (RC), consisting of 1% BSA fraction V (Sigma), 1X insulin/transferrin/selenium (ITS, Invitrogen), D-Glucose (final concentration 25 mM), 8 nM exendin-4 (Acris), 8 nM activin A (Cytolab/PreproTech), 1X B27 supplement (Stem Cell Technologies), and 10 mM nicotinamide (Sigma), in CMRL 1066 medium supplemented with PSG, was prepared and applied to cells as previously described [27 (link)]. ALK5 inhibitor II (Enzo), and FOXO1 inhibitor AS1842856 (Millipore), were applied to cells every 48 hours at a final concentration of 0.1 μM.