Determining the role of GREB1L in trophoblast cells

KO cells self-replicate and have the same survival rate as controls. However, we find downregulation of the differentiation marker Integrin-α-1. Loss of GREB1L impairs syncytiotrophoblast formation according to our immunofluorescence staining and hCG ELISA data.

Differentiation into extra villous trophoblasts is also impaired in GREB1L KO cells as determined by flow cytometry. These observations were confirmed by RNAseq analysis, which also shows perturbed expression of molecules involved in cell adhesion. We later obtained embryonic day 9.5 placental sections from a Greb1l KO mouse model that dies on or before embryonic day 11.5.

Analysis of these sections revealed a severely underdeveloped placental labyrinth layer that could be causing early embryonic lethality. We did not observe evidence of impaired retinoic acid signaling in GREB1L KO trophoblast stem cells. However, GREB1L is indeed predicted to have the same protein domains as GREB1, a known O-GlcNAcylase, according to AlphaFold and Dali server analyses.

This study therefore shows that GREB1L is a putative glycosylase that mediates human trophoblast stem cell differentiation and successful placentation in mice and humans.