FIG. 1.

Comparison of seven published protocols for definitive endoderm (DE) differentiation. (A) Summary of published DE differentiation protocols [6–11]. In the modified D'Amour (Mod-D'Amour), Chetty, Teo, Tahamtani 1, and Tahamtani 2 protocols, growth factors, small molecules, and supplements were added to R medium (comprising 1% nonessential amino acids, 2 mM l-glutamine, 50 units/mL of penicillin, and 50 μg/mL of streptomycin in RPMI-1640 medium) in a two-step manner. In the Pauklin and Hannan protocols, chemically defined medium containing polyvinyl alcohol was used as the basal medium. FBS, fetal bovine serum; BMP4, bone morphogenetic protein 4; FGF2, fibroblast growth factor 2. (B) The percentage of eGFP⁺ cells after 4-day DE differentiation of S17d5 cells. (C) Total live cell numbers at the end of 4-day DE differentiation of S17d5 cells. Independent runs were normalized to 1.0×10⁶ starting undifferentiated cells (dotted line). *P<0.05 compared with the Mod-D'Amour protocol by Student's unpaired t-test. Data are presented as mean±standard error of the mean (SEM), n=3–5. (D) Representative flow cytometric histograms of SOX17-enhanced green fluorescent protein (eGFP) induction through published DE differentiation protocols.