LGR4 is a progenitor cell marker in high surface area tissues including the colon. It functions as a WNT pathway enhancer by binding its ligand R-spondin 1 (RSPO1); enhancing downstream signalling. In the colon, LGR4+ cells direct rapid proliferation (‘transit amplification’) and differentiation into terminal epithelial cell phenotypes. The placenta generates rapidly during pregnancy to form a complex high surface area organ dedicated to maternal-fetal exchange. The epithelial placental surface (syncytiotrophoblast) forms via underlying cytotrophoblast fusion and undergoes constant turnover. We hypothesise LGR4+ progenitor cells may be present in placenta and dysregulated in placental insufficiency diseases including preeclampsia. This study sought to characterise LGR4 in preeclampsia and assess roles in isolated human (cyto)trophoblast (placental) stem cells (hTSCs).
LGR4 mRNA was measured in early-onset (<34-week gestation, n=81 vs n=19 controls) and late-onset preeclamptic placentas (≥34-weeks, n=33 vs n=20 controls). It was significantly increased in early-onset preeclamptic placentas only (p=0.0148). In situ hybridisation localised LGR4 to proliferating (MKI67+) villous cytotrophoblasts, and ligand RSPO1 to endothelial cells (PECAM1+) (n=3 preeclamptic and n=3 control).
Preeclampsia is associated with placental hypoxia and inflammation. Thus, we tested whether these increased LGR4 in hTSCs. However, neither pro-inflammatory cytokines interleukin-6, tumour necrosis-α, nor hypoxia significantly altered LGR4 expression. We assessed changes in LGR4 with cellular differentiation by differentiating hTSCs into extravillous trophoblast (EVT) or syncytiotrophoblasts for 96h. Differentiation was confirmed by increased HLA-G (EVTs) and reduced cadherin-2/increased syndecan-1 (syncytiotrophoblasts). Intriguingly, LGR4 increased with EVT differentiation (p<0.0001) and peaked at 48h of syncytiotrophoblast differentiation (p=0.002). Finally, LGR4 siRNA-mediated knockdown did not affect hTSC proliferation (MTS assay) nor differentiation into EVTs or syncytiotrophoblasts relative to control siRNA.
Placental LGR4 is elevated in preeclampsia, and hypoxia nor inflammation alter its expression. Localisation to actively proliferating cytotrophoblasts and increases across differentiation allude to a transit amplifier role in placental development and disease.