Oral Presentation ESA-SRB 2023 in conjunction with ENSA

Investigating side-population marker expression in placental insufficiency (#37)

Georgia P Wong 1 2 , Natalie J Hannan 1 2 , Ping Cannon 1 2 , Manju Kandel 1 2 , Anna Nguyen 1 2 , Tuong-Vi Nguyen 1 2 , Natasha Pritchard 1 2 , David G Simmons 3 , Tu'uhevaha J Kaitu'u-Lino 1 2
  1. Mercy Perinatal, Mercy Hospital for Women, Heidelberg, Victoria, Australia
  2. Department of Obstetrics & Gynaecology, University of Melbourne/Mercy Hospital for Women, Heidelberg, Victoria, Australia
  3. School of Biomedical Sciences, University of Queensland, Brisbane, Queensland, Australia

An emerging concept suggests dysregulated progenitor cell populations may contribute to placental insufficiency pathogenesis. Side-population cells are identified by their ability to efflux DNA dye, (Hoechst33342) and demonstrate progenitor properties. Recently, side-population cells were isolated from human placenta and identified as having enriched expression of 8 specific genes, relative to other cells (Gamage et al 2020, Stem Cell Rev Rep). We characterised these side-population markers in placentas from patients with preeclampsia or fetal growth restriction (FGR), and in differentiating human trophoblast stem cells (hTSCs).

mRNA expression of placental side-population markers ELL2, GATA6, HK2, HLA-DPB1, CXCL8, INTS6, SERPINE3 and UPP1 were measured in <34-week human placenta (n=78 preeclampsia, n=30 FGR, n=18 gestation-matched controls). ELL2, HK2 and CXCL8 mRNA were elevated in preeclamptic (p=0.0006,p<0.0001,p=0.0335 respectively) and FGR placentas (p=0.0065, p<0.0001,p=0.0001 respectively) versus controls. Conversely, GATA6 was reduced in placentas from both preeclampsia and FGR (p=0.0014,p=0.0146 respectively). 

To identify the cells expressing side-population markers, multiplex immunofluorescence was used in human placental sections (n=3 each group, 3 proteins/serial section). We next sought to identify whether the 4 side-population markers most dysregulated in preeclampsia/FGR were altered as hTSCs underwent differentiation into extra-villous trophoblast (EVTs) or fusion into syncytiotrophoblasts across 96h (n=5). EVT differentiation was confirmed by reduced hTSC marker, TEAD4 (p<0.007); and elevated EVT marker HLA-G (p<0.026). ELL2, GATA6 and HK2 all increased with EVT differentiation (p=0.0015,p=0.0018,p<0.0257), whereas CXCL8 was unaltered. Syncytiotrophoblast differentiation was confirmed at 96h by TEAD4 loss (p<0.018) and elevated SDC1 (p<0.0223). As cells sycnytialised, CXCL8 and GATA6 expression were unchanged while ELL2 and HK2 increased (p<0.0407,p<0.0414).

We have demonstrated that some of the 8 side-population marker genes are dysregulated with placental insufficiency, and how they change with placental cell differentiation. Our ongoing analysis of multiplex immunofluorescence is likely to provide insight into the location and frequency of this progenitor population.