For differentiation into endoderm, following culture in pre-differentiation media cells were further cultured in differentiation media for three days. Briefly, day one media was removed and replaced with fresh CDM-PVA media supplemented with recombinant Activin-A (100 ng/ml; CSCR, University of Cambridge), zebrafish FGF2 (80 ng/ml; CSCR, University of Cambridge), BMP4 (10 ng/ml R&D Systems Inc.), LY29004 (10 mM), and CHIR99021 (3 mM). Day two media was removed and replaced with fresh CDM-PVA supplemented with recombinant Activin-A (100 ng/ml), zebrafish FGF2 (80 ng/ml), BMP4 (10 ng/ml), and LY29004 (10 mM). Day three media was removed and replaced with RPMI media supplemented with B27 (1x, Life Technologies UK), recombinant Activin-A (100 ng/ml), zebrafish FGF2 (80 ng/ml), and Non-Essential Amino Acids (1x, Life Technologies UK).
