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

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. NOTUM was knocked down in human trophoblast stem cells using lentiviral delivery of shRNA. Following selection by puromycin, cells were differentiated for eight days (Okae et al.) into extravillous trophoblast (EVT). On day eight of differentiation, cells treated with either control shRNA or NOTUM-specific shRNA were collected and RNA was isolated.