Differentiation of hESCs into Insulin-Producing Cells

hESCs were cultured and passaged as reported elsewhere [10 (link)]. The differentiation of hESCs into INS⁺ cells was performed using several different protocols. Adherent, flat culture differentiations based on the work of D’Amour et al. [11 (link)] and Kroon et al. [2 (link)] (referred to as ‘flat cultures’). Spin EB differentiations (referred to as ‘spin EBs’) [5 (link)], were set up in APEL medium [6 (link)]. Differentiation of spin EBs under pancreatic-specific conditions was as follows. EBs were formed by the forced aggregation of 2,000 (HES3) or 3,500 (MEL1) hESCs in APEL (the protein-free hybridoma medium component was omitted from this formulation) containing 10 ng/ml bone morphogenetic protein 4 (BMP4) and 150–200 ng/ml activin A (batch dependent) in low-attachment 96-well plates. After 3 days, medium was replaced with APEL containing 200–400 ng/ml noggin (batch dependent). At day 6, medium was replaced with APEL containing 1 × 10⁻⁵ mol/l retinoic acid (RA). At day 9, the medium was changed to APEL without polyvinyl alcohol (AEL) containing 1 × 10⁻⁵ mol/l RA, 100 μmol/l glucagon-like peptide 1 (GLP1), 1 × B27 and 10 mmol/l nicotinamide. At day 15 of differentiation, EBs were transferred to gelatinised, adherent 96-well plates, and insulin production was induced in AEL containing 10 mmol/l nicotinamide and 50 ng/ml IGF-I. With this system, most EBs contained INS-GFP⁺ cells by day 30 of differentiation. In addition, INS-GFP⁺ cells were also differentiated according to a protocol developed by Nostro and colleagues [12 (link)], referred to as the ‘Nostro protocol’. Recombinant human activin A, fibroblast growth factor 10 (FGF10), fibroblast growth factor 7 (KGF), IGF-I and hepatocyte growth factor (HGF) were purchased from R&D Systems (Minneapolis, MN, USA). Basic FGF (FGF2) was purchased from Peprotech (Rocky Hill, NJ, USA). Wingless-type MMTV integration site family, member 3A (WNT3A) and noggin were purchased from R&D Systems or provided by the Australia Stem Cell Centre (Melbourne, VIC, Australia). KAAD-cyclopamine was purchased from Toronto Research Chemicals (North York, ON, Canada); all-trans RA, nicotinamide, SB431542 and GLP1 were purchased from Sigma-Aldrich (St Louis, MO, USA).