Differentiation of mESCs to Mesendoderm

Differentiation to mesendodermal cells was adapted from a published protocol (Thomson et al. 2011 (link)). mESCs were first recovered into 2i media for two days. On day 1 of differentiation, mESCs were seeded on plates coated with 0.1% gelatin (EMD Millipore ES006-B) in N2/B27 differentiation medium, containing a 1:1 mixture of DMEM/F12 (ThermoFisher 11330032) and Neurobasal (ThermoFisher 21103049), supplemented with 0.5% N2 Supplement (ThermoFisher 17502048), 1% B27 Supplement (ThermoFisher 17504044), 1% GlutaMAX (ThermoFisher 35050061), 1% Pen-Strep (ThermoFisher 15140122), 1% MEM nonessential amino acids (ThermoFisher 11140050), 100 μM sodium pyruvate (ThermoFisher 11360070), 0.1 mM 2-mercaptoethanol (Sigma M3148), supplemented with 10 μM ROCK inhibitor Y-27632 (Cell Signaling 13264). On day 2, ROCK inhibitor was removed and differentiation media was supplemented with 3 μM CHIR99021 (R&D Systems 4423) replenished daily for 3 additional days. On day 5, plates were rinsed twice using PBS and cells were collected for further studies.