ERK1/2 signaling is necessary for definitive endoderm formation

Purpose: To compare gene expressions of undifferentiated cells and differentiated cells treated with basal media, basal media + FGF2, basal media + SU5402 and bsal media + ERKiII. Methods:For differentiation, hPSCs was set up 2 days before initiation of DE at Day 0 (D0). On D0, media was replaced with using RPMI/2%B27, Activin, CHIR99021, LY294002 +/- FGF2, SU5402 or TCS ERKi. The cells were harvested on D3. Cells were cultured in triplicates for each condition, total RNA extraction was performed and 1000 ng of purified RNA was sent for RNA-seq. Genes that are differentially expressed were analyzed by comparing RNA expression at different conditions. Results: Principal component analysis confirmed that our replicate samples clustered together, indicating good reproducibility. ACLY and ACLYF30 samples clustered close to each other but were non-overlapping, suggesting hish similarity in their cellular identities. Contrastingly, ACLYSU and ACLYERKi samples are found isolated from each other and also from undifferentiated hPSCs (UD), ACLY and ACLYF30 samples, indicating that the inhibition of FGFR or ERK1/2 had a huge impact on redirecting DE cell fate despite the presence of DE-inducing conditions. Conclusions: Blocking of FGFR with SU5402 and ERK2 signalling with ERKiII perturb cell cycle and cell fate specification even in the presence of definitive endoderm-inducing conditions. Overall design: 3 replicates per condition - undifferentiated hESCs, D3 ACLY, D3 ACLYF30, D3 ACLYSU, D3 ACLYERKi