Saracatinib Promotes Definitive Endoderm Differentiation by Inhibition of FAK-YAP Signaling Axis

Optimizing the efficiency of definitive endoderm differentiation is significant for the generation of diverse organ-like structures. In this study, we utilized saracatinib to enhance definitive endoderm differentiation in pluripotent stem cells. We found saracatinib significantly improved the definitive endoderm differentiation at low concentrations. To investigate the impact of 0.5 μM saracatinib on definitive endoderm differentiation of ESC H1 cells, we conducted RNA-seq analysis with differentiated cells with or without 0.5 μM saracatinib treatment. To investigate the role of saracatinib in definitive endoderm (DE) differentiation, we treated differentiating embryonic stem cells. Initially, the cells were cultured for three days to induce DE differentiation. Embryonic stem cells were seeded at a density of 1×10^6 cells per well on Matrigel-coated 6-well plates. The differentiation medium used was DMEM supplemented with B27, Glutamax, 50 ng/mL Activin A, and 2.5 μM CHIR99021 for 24 hours. Subsequently, CHIR99021 was removed from the medium for the following two days. The experimental group comprised the treatment group, where 0.5 μM saracatinib was added to the cells throughout the differentiation period (3 days). As a control group, an equal volume of DMSO was added to the cells to serve as a volume control.