Efficient Induction of Syncytiotrophoblast Layer II Cells from Trophoblast Stem Cells by Canonical Wnt Signaling Activation

Abstract

SummaryThe syncytiotrophoblast layer is the most critical and prominent tissue in placenta. SynT cells are differentiated from trophoblast stem cells (TSCs) during early embryogenesis. Mouse TSCs can spontaneously differentiate into cells of mixed lineages in vitro upon withdrawal of stemness-maintaining factors. However, differentiation into defined placental cell lineages remains challenging. We report here that canonical Wnt signaling activation robustly induces expression of SynT-II lineage-specific genes Gcm1 and SynB and suppresses markers of other placental lineages. In contrast to mouse TSCs, the induced SynT-II cells are migratory. More importantly, the migration depends on hepatocyte growth factor (HGF) and the c-MET signaling axis. Furthermore, HGF-expressing cells lie adjacent to SynT-II cells in developing murine placenta, suggesting that HGF/c-MET signaling plays a critical role in SynT-II cell morphogenesis during the labyrinth branching process. The availability of SynT-II cells in vitro will facilitate molecular understanding of labyrinth layer development.

ARHGEF18/p114RhoGEF coordinates PKA/CREB signaling and actomyosin remodeling to drive trophoblast cell-cell fusion during placenta morphogenesis

“…We next asked whether p114RhoGEF-depleted TSCs can differentiate into labyrinthspecific trophoblast lineages. Induction of syncytiotrophoblast II cells (SynT-II) was achieved by GSK3β inhibition with CHIR9902 (Zhu et al, 2017). As expected, induction of differentiation resulted in downregulation of the TSC marker Cdx2 in control and p114RhoGEF-depleted cells; however, induction of SynB, a marker for SynT-II cells, was attenuated by p114RhoGEF depletion (Fig.…”

The Transcription Factor OVOL2 Represses ID2 and Drives Differentiation of Trophoblast Stem Cells and Placental Development in Mice

“…Since culture conditions that favor mouse TS cell self-renewal or differentiation have been known for decades, these cells have provided the best studied in vitro models to investigate development of trophoblast lineages. Using two separate differentiation protocols, we induced mouse TS cells to either undergo conventional differentiation [4], or differentiate to preferentially form cells representative of the syncytiotrophoblast layer II lineage [21]. We found that Ovol2 mRNA levels robustly increased during both TS cell differentiation protocols.…”

“…TS cells were induced to differentiate by removal of MEF preconditioned media, FGF4, heparin, and activin A (differentiation media) [20]. To stimulate TS cell differentiation toward the syncytiotrophoblast II lineage (a sublineage of the labyrinth zone), cells were cultured in differentiation media, along with a GSK3β inhibitor, CHIR99021 (3 µM, Stemgent, Cambridge, MA, USA) [21]. Controls for TS cells exposed to CHIR99021 consisted of cells exposed to dimethyl sulfoxide (DMSO, 1:1000), which was the vehicle used to prepare CHIR99021.…”

Impaired plasma membrane localization of ubiquitin ligase complex underlies 3-M syndrome development

“…To determine the function of 3-M genes in trophoblast migration, we utilized a previously established primary trophoblast stem cell (TSC) culture system (37). The TSCs can be maintained in TSC medium in the presence of FGF and heparin and induced to differentiate into trophoblast giant cells or syncytiotrophoblast layer II (SynT-II) cells by removing FGF and supplementing with retinoid acid (RA) or GSK inhibitor (CHIR) (38,39). Ccdc8 expression was undetectable in TSCs, but they started to express Ccdc8 upon differentiation ( Figure 6E).…”