Wild-type and TP53 genetic knockout BOBSC hiPS cells were maintained in the undifferentiated state or differentiated to definitive endoderm over 72 hours, with or without 5 ppm MMS treatment at 24 hours.
Expression profiling by high throughput sequencing
Summary
The ability of human induced pluripotent stem cells (hiPSCs) to differentiate in vitro to each of the three germ layer lineages has made them an important developmental model and tool for tissue engineering. Here we explore the impact of inducing DNA damage at different points during differentiation. We show that, during a critical window in this program, DNA damage diverts cells from an endodermal to mesodermal fate without significant upregulation of an apoptotic program. This change in transcriptional program is driven by p53: cells lacking p53 continue to differentiate towards endoderm with high efficiency despite significant genomic damage and G2/M cell cycle arrest. Thus, DNA damage-induced upregulation of p53 decreases the efficiency of hiPSC differentiation to definitive endoderm by diverting cells towards a mesodermal fate.
Overall design
On day -1 wild type and TP53-/- cells were passaged and on day 0 the sequencing experiment was started. Undifferentiated (UD) cells were cultured, in parallel to the differentiating (ENDO) cells, in Essential 8 Flex medium. RNA was extracted from both cell lines in the undifferentiated state and during differentiation every 24 hours. In parallel, both undifferentiated and differentiated cells were treated with 5 ppm MMS at 24 hours and the RNA was extracted at day 2 (48 hours) and day 3 (72 hours). This gives 20 conditions and there were three biological replicates of each condition.
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