Hypoxia directs human villous CTB differentiation towards the EVT lineage in a HIF-dependent manner.

We showed that hypoxia directs first trimester primary villous cytotrophoblast (vCTB) differentiation preferentially towards HLAG+ extravillous trophoblast (EVT). Infection of primary vCTB with ARNT-specific shRNA attenuates this effect, suggesting a role of the intact HIF-complex in hypoxia-directed CTB differentiation into EVT. First trimester cytotrophoblast were isolated from five 8-10 week gestational age placentas and plated on fibronectin-coated plates. To assess the role of the HIF-complex in CTB differentiation, we targeted the beta subunit of this complex, ARNT. Cells were infected with either Scramble (control) or ARNT-specific shRNA lentivirus to induce specific ARNT knockdown. Both shScramble and shARNT infected cells were incubated either in 20% oxygen (normoxia) or 2% oxygen (hypoxia) for 4 days. Gene expression profiles were obtained for cultured cells as well as freshly isolated CTB.