08 September 2021 1 10K Report

We are experiencing a significant amount of cell death upon differentiation of h9 hESCs to definitive endoderm cells. We grow h9 cells in feeder free conditions and induce differentiation to endoderm using RPMI 1640 supplemented with 2mM glutamine, 1X NEAA, 100ng/ml Activin A, and .1% B27. After 1 day of induction we add .1X ITS, after 2 days induction we add 1x ITS. We are trying to perform an initial RT-qPCR batch assay on day 3 cells, however by that time we have only a few cells left with endodermal morphology - most of our initial stem cell culture has lifted off the plate and died. Does anyone have any successful strategies for increasing cell count at day 3, while maintaining a relatively high degree of differentiation? Would greatly appreciate any insight - Thanks!

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