Cells were cultured in MCDB-131 medium supplemented with NaHCO3 (2.5 g/liter), 1% GlutaMAX, glucose (5.5 mM), 0.1% FAF-BSA, and ITS:X (1:50,000), and were treated with Wnt3a (20 ng/ml) + AA (100 ng/ml) for 1 d, AA (100 ng/ml) for 3 d, and FGF7 (50 ng/ml; PeproTech) for 2 d. For further endocrine differentiation, cells were cultured in MCDB-131 medium supplemented with NaHCO3 (2.5 g/liter), 1% GlutaMAX, glucose (8 mM), 2% FAF-BSA, and ITS:X (1:200), and treated for 4 d with FGF7 (50 ng/ml) + noggin (100 ng/ml) + retinoic acid (2 µM; Sigma-Aldrich) + SANT-1 (0.25 µM; Sigma-Aldrich) + AA (20 ng/ml); for 3 d with SANT-1 (0.25 µM) + PdBu (200 nM; EMD) + noggin (100 ng/ml); and for 4–6 d with noggin (100 ng/ml) + Alk5 inhibitor (1 µM; Axxora).
Directed differentiations for the scorecard analyses were performed using the following previously published protocols (Gifford et al., 2013 (link)).
Directed differentiations for the scorecard analyses were performed using the following previously published protocols (Gifford et al., 2013 (link)).
