1.Introduction
Oxidative stress (OxS) is implicated in pregnancy pathologies; however, its effects on trophoblast development remain unclear. Difficulties mimicking the in vitro placental villi environment remain an ongoing challenge in placental research [1,2]. Human pluripotent stem cells (PSCs) have been used to differentiate trophoblast; however, cultures are only viable for 9 days [3], limiting the use of this system as a tractable platform. This study aimed to investigate how induced OxS influences trophoblast populations in an extended placentoid culture system.
2.Methods
Human PSCs were cultivated on polydimethylsiloxane scaffolds with engineered-synthetic villi (~300µM x 400µM) under varying oxygen tensions in hypoxic incubators, prior to trophoblast differentiation (BMP4, activin/nodal and FGF inhibitors[4]). Media was collected every two days for hGCβ ELISA assay (n=3). Organoids were fixed and stained with Ki67, hCGβ, SDC-1 and HLA-G (n=3), then imaged using an Olympus FV3000 confocal microscope. Proteins were extracted from cultures on days 4, 7, 9 and 12 (n=3) for western blotting (ITGA6, SDC1, Oct4, Trx). Statistical analysis across all groups performed using one-way ANOVA.
3.Results
Placentoids from all oxygen scenarios stained positive for hCGβ, SDC1 and HLA-G, confirming syncytiotrophoblast (STB) and extravillous trophoblast formation. A subset of cells in placentoids at each timepoint in each oxygen scenario were proliferative (Ki67+). Western blotting showed that ITGA6 was abundant across all time points, suggesting cytotrophoblast (CTB)-like cells were maintained in placentoids, while Oct4 was restricted to pre-differentiation days, confirming pluripotency loss. Trx (OxS marker) was not significantly increased in organoids grown at higher oxygen tensions. Concentrations hCGβ were detected in all placentoids cultures and were unaffected by oxygen conditions. Day 12 placentoids released significantly greater hCGβ than early stages (Day 8, p<0.0001,student t-test).
4.Conclusion
These findings support the use of placentoids as a tractable platform to investigate mechanisms underpinning placental development and pathology.