NOTUM-MEDIATED WNT SILENCING DRIVES EXTRAVILLOUS TROPHOBLAST CELL LINEAGE DEVELOPMENT [CHIR99021]

WNT activated signal transduction is a conserved regulator of morphogenesis of many organ systems, including the placenta. In human trophoblast stem (TS) cells, activation of canonical WNT signaling is a key to maintenance of the TS cell stem state. NOTUM, a negative regulator of canonical WNT signaling, was prominently expressed in first trimester EVT cells developing in situ and upregulated in EVT cells derived from human TS cells. Furthermore, NOTUM was required for human TS cell differentiation to EVT cells. Canonical WNT signaling is essential for maintaining human trophoblast cell stemness and prevention of human TS cell differentiation. Downregulation of canonical WNT signaling via the actions of NOTUM is required for EVT cell differentiation. Overall design: Glycogen synthase kinase 3 (GSK3) inhibitor, CHIR99021 was added as a supplement to the standard EVT cell differentiation culture conditions of CT27 human TS cells (Okae et al.). On day eight of differentiation, cells treated with either control or CHIR992021 02µM were collected and RNA was isolated.