Development of an in vitro explant model for syncytiotrophoblast regeneration in first trimester human placentas

Introduction: The placenta is a temporary organ that develops during pregnancy to support fetal development. The syncytiotrophoblast (ST) is a multinucleated cell layer that covers the maternal-fetal interface. This cell performs barrier and immune functions, mediates nutrient and waste exchange, and secretes hormones like human chorionic gonadotropin (hCG). Unlike other epithelial layers, the ST must be maintained through continuous fusion of underlying proliferative progenitor cytotrophoblasts (vCT). Poor fusion leading to ST dysfunction is a feature of pregnancy complications like preeclampsia and intrauterine growth restriction. It is appreciated that these conditions start developing in the first trimester, but few studies have examined the mechanisms governing first trimester vCT differentiation. Multiple methods for the removal and spontaneous regeneration of the ST layer in term explants exist to study term vCT to ST differentiation. Therefore, our goal was to optimize a ST regeneration model for use with first trimester tissue. Methods: First trimester human placental tissues of gestational age nine to twelve weeks were cut into approximately 2mm3 explants. Explants were digested with 0.25% trypsin-EDTA for seven minutes and then cultured in medium (IMDM, 5% heat-inactivated FBS, 50µg/mL gentamicin, 1X ITS-X supplement) either at the liquid-gas interface or as floating explant culture. Cultured explants were fixed at 24h, 48h, 72h, 96h and stained with anti-E-cadherin antibody (vCT marker), phalloidin (F-actin marker), and Hoechst (nuclei marker), to identify the vCT and ST using immunofluorescence (IF). The proportion of vCT with apically localized ST, identified by nuclei and F-actin staining above E-cadherin positive vCT, was quantified using Volocity Imaging software and analyzed using unpaired student’s t-test (n=3). Results: Multinucleated ST structures above E-cadherin positive vCT were 96.5% removed after 7 minutes of trypsinization and 24h in culture medium. Explants cultured in floating explant culture showed a mean 50.4% ST structure recovery above CT nuclei at 72h post-trypsinization. Comparatively, explants cultured at the liquid-air interface showed a mean 93.4% ST structure loss after 24h and a mean 49.1% ST structure recovery at 72h. Additionally, high numbers of cells in the stroma displayed highly condensed or apoptotic nuclei in explants cultured at the liquid-gas interface. Conclusion: Our data shows that the ST can be stripped from first trimester explants 24h post-trypsinization and can regenerate within 72h post-trypsinization, optimally using floating explant culture. Explants cultured at the liquid-gas interface were generally more unhealthy, evidenced by more apoptotic nuclei, less consistent ST regeneration, and less metabolic activity compared to those cultured in floating culture. Future directions are to assess functional differentiation via hCG secretion in this model and to identify the efficacy of siRNA knockdown on this model. Our current model will serve as an additional method to observe vCT differentiation in first trimester tissue and help identify mechanisms by which fusion occurs, thereby contributing to studies concerning aberrant ST differentiation and dysfunction.