Bulk RNAseq NIEHS P30 CURES Pilot Environmental hormonal and mutagenic stress in fluorescent-ubiquitinated cell cycle indicator (FUCCI) mESC 2021

This experiment examines how toxicants can override normal stemness (NS) conditions, which LIF maintains, causing lineage imbalance and otherwise affecting cells in the early embryo. It also compares NS to normal differentiation (ND) (without LIF/ LIF-). Sorbitol is a positive control for stress overriding LIF+. Retinoic acid (RA) modified LIF- is a positive control for 1st lineage differentiation. But RA also activates Hox gene in anterior posterior patterning at gastrulation. The third control is NS itself, and all stimuli are compared to NS. Other toxicants are Benzo(a)pyrene (BP) - a mutagen, Cortisol - a stress hormone, and two endocrine disruptors Diethyl phthalate (DEP), and Perfluorooctanoic acid (PFOA). Overall design: Bulk RNAseq of FUCCI mESC cultured for 72hr with different stimuli. Triplicate biological experiments. Baseline maintenance media was LIF at 1,000U/mL and 15% serum which enables heterogeneous normal stemness, putatively at naïve pluripotency like the day E4.5 inner cell mass. 72hr of LIF removal for ND putatively yields formative pluripotency, a restriction of pluripotency compared with naive, but also preparing cells for the start of gastrulation. Toxicant dose responses (1,10,100nM. 1, 10, 100uM) were performed in a live imager to determine lowest observable adverse effect level (LOAEL) for growth and IC75 level (concentration with 75% growth suppression) and these two doses were used as dose-dependent test exposures in the 72hr culture prior to cell lysis and RNAseq. FUCCI ESC were 129/sv strain ESC with FUCCI transgenes, a gift from Dr Savatier: from Coronado, D., Godet, M., Bourillot, P.Y., Tapponnier, Y., Bernat, A., Petit, M., Afanassieff, M., Markossian, S., Malashicheva, A., Iacone, R., Anastassiadis, K., Savatier, P., 2013. A short G1 phase is an intrinsic determinant of naive embryonic stem cell pluripotency. Stem Cell Res 10, 118-131.