Adequate placental function plays an instrumental role during in utero developmental advances. Placental insufficiency or failure of proper placental function are collectively referred to as placental dysfunction and its precise etiology remains unknown. Placental dysfunction gives rise to various pregnancy complications like pre-eclampsia and fetal growth restriction which in turn are related to the circulating angiogenic factor imbalance. Concentration of circulating angiogenic factors like placental growth factor (PlGF) is reduced and soluble Fms-like tyrosine kinase-1 (sFlt-1) is increased. Stress pathways in placental trophoblast cells, including the unfolded protein response (UPR), are thought to be involved in placental dysfunction. Trophoblasts are the key cells of the placenta and knowledge of trophoblast function is still limited due to scarce availability of placental tissue because of ethical constrains and unavailability of robust in vitro model depicting placental development. Thus, an in vitro trophoblast model is crucial to understand placental function/dysfunction. Trophoblast organoids (T-orgs) are a novel in vitro model to study trophoblast function. They are self-organising structures consisting of cytotrophoblast (CT) and CT-derived multinuclear syncytiotrophoblasts (ST). T-orgs formed from human trophoblast stem cells produced the pregnancy hormones of interest like PlFG, sFlt-1 and human chorionic gonadotropin, which were detected through the use of respective enzyme linked immunosorbent assays, from as early as day two of the culture with increase in concentration observed until day 12 of the culture, suggesting early gestation placental development. T-orgs were cultured for up to one month to model mature placental villi of later gestation in vitro. Immunofluorescence microscopy revealed distinct organoid regions consisting of proliferative CT-like cells and multinucleated syncytium-like cells observed until day 29 of the culture. Pharmacological induction of the unfolded protein response (UPR) in T-orgs culture using tunicamycin was verified by detection of X-box binding protein-1 (XBP1) mRNA splicing and led to significant reduction in the production of all the three hormones. Immunofluorescence microscopic analysis showed fragmented nuclei with almost no proliferation in the tunicamycin treated T-orgs as compared to untreated controls, suggesting tunicamycin treated reduced organoid viability. Thus, this study has shown that T-orgs can be used to study placental pathophysiology. Overall, T-orgs can prove a better in vitro trophoblast model to study the angiogenic profile of the placenta and can further be explored for similar experiments.
Human trophoblast stem cell derived trophoblast organoids: a potential model for studying angiogenic marker-defined trophoblast dysfunction
Patil, S. (Author). 1 Aug 2023
Student thesis: Master of Philosophy