Definitive Endoderm Differentiation Protocol

Definitive endoderm was induced according to Loh et al. (2014) (link). Cells were cultured in CDM2 basal medium that was supplemented with 100 ng/ml activin A (produced in house), 100 nM PI-103 (Tocris Bio-Techne, 2930), 3 µM CHIR99021, 10 ng/ml FGF2 (produced in house), 3 ng/ml BMP4 (PeproTech, 120-05ET), 10 µg/ml heparin (Sigma-Aldrich, H3149) for one day. For the next 2 days the following supplements were applied: 100 ng/ml activin A, 100 nM PI-103, 20 ng/ml FGF2, 250 nM LDN193189, 10 µg/ml heparin. Further induction of foregut progenitors was performed according to Rezania et al. (2014) (link) with analysis at the S4 stage.