I'm trying to generate definitive endoderm from iPSCs using what I believe to be very standard methods (Activin A alone or combined with other factors in RPMI media and serum). I am the first in my lab to try this so I have been relying on published protocols, but they are not working well for me and I am unsure of the cause.
I am experiencing highly variable results, often very low efficiency (based on SOX17/FOXA2 staining), and a great deal of cell death. Changing the base media to ADEM:F12 prevents the massive cell death I see with RPMI or DMEM:F12. I've also tried various supposedly "synergizing" factors (CHIR99021, WNT3A and BMP4) but the efficiency is still low/variable.
Activin A supplementation has been at 50-100ng/mL. WNT3A and BMP4 at 50-100ng/mL. CHIR99021 at 1-2uM.
Does anyone have any tips or a suggestion for a working protocol?