Directed Differentiation of hESCs to Definitive Endoderm

hESCs were differentiated to definitive endoderm using the STEMdiff Definitive Endoderm Kit (cat. no. 05110). The full protocol is available from STEMCELL Technologies (https://cdn.stemcell.com/media/files/pis/29550-PIS_2_1_0.pdf?_ga=2.73376023.564267965.1597964514-138601152.1597964514). Briefly, hESCs at 80% confluence were harvested using Gentle Cell Dissociation Reagent (STEMCELL Technologies, cat. no. 07174) and reseeded in a single-cell manner on Matrigel plates. This was done daily for 5 d. Every 24 h after a new differentiation, culture was started, and cells were incubated with DE differentiation medium according to the manufacturer’s guidelines. On the fifth day, all five time points were harvested simultaneously using Accutase (STEMCELL Technologies, cat. no. AT104-500). Immunofluorescence was used to confirm differentiation as previously described^{24 (link)} with primary antibodies SOX17 1:250 dilution (R&D Systems, AF1924, lot KGA0916121) and OCT4 1:100 dilution (Abcam, ab109183, lot gr120970-6) and secondary antibodies donkey anti-goat Rhodamine Red 1:1,000 dilution (Jackson ImmunoResearch, cat. no. 705-295-147) and goat anti-rabbit-Cy5 1:1,000 dilution (Jackson ImmunoResearch, cat. no. 111-175-144).