Characterising the Subpopulations of the HTR8/SVneo Cell Line: A Step Toward Understanding Placenta-Associated Pregnancy Complications

Abstract

Background: Trophoblast differentiation and invasion are crucial processes in placental development, with aberrations leading to pregnancy complications such as preeclampsia and intrauterine growth restriction (IUGR). The HTR8/SVneo cell line, an immortalized trophoblast model, consists of heterogeneous subpopulations exhibiting epithelial and mesenchymal characteristics. Understanding the functional and molecular differences between these subpopulations is essential for deciphering trophoblast lineage commitment and invasive behavior. This study aims to characterize the epithelial and mesenchymal subpopulations of the HTR8/SVneo cell line by assessing their phenotypic, proliferative, and migratory properties using two-dimensional (2D) cultures, as well as sphere-forming ability through three-dimensional (3D) in vitro models.

Methods: HTR8/SVneo cells were sorted into freshly sorted E-cadherin positive (E-cad⁺) and negative (E-cad⁻) subpopulations using Fluorescence-Associated Cell Sorting. The two subpopulations were maintained in culture and different stages along Epithelial to Mesenchymal transition (EMT) and Mesenchymal to Epithelial transition (MET) were derived from the E-cadherin positive cells. Accordingly, freshly sorted E-cadherin positive, E-cadherin negative, post-EMT intermediary cells, and post-MET polygonal cells were subjected to functional assays. To assess the proliferation rate of the different subpopulations, cells were subjected to MTT assay. Furthermore, the scratch assay was used to investigate the migration ability of these cells. In addition, to assess the stem- like properties of the distinct trophoblast subpopulations, sphere-formation assay was performed.

Results: Our functional assays revealed marked heterogeneity in the proliferative and migratory capacities of the four HTR8/SVneo trophoblast subpopulations. The MTT assay demonstrated that freshly sorted E-cad⁺ epithelial cells exhibited minimal proliferation. In contrast, freshly sorted E-cad⁻, post-EMT mesenchymal-like, and post-MET polygonal cells all displayed robust proliferative profiles, suggesting that the mesenchymal-like and re-epithelialized phenotypes share enhanced growth potential. The Wound healing assay further underscored these functional differences: E-cad⁺ cells consistently maintained the largest residual wound area at all time points, indicating the lowest migratory and invasive capacity. Post-MET polygonal cells achieved complete closure by 48 hours, representing the most invasive phenotype, while E-cad⁻ and mesenchymal-like cells exhibited intermediate closure rates. Consistent with these trends, our preliminary sphere-formation assays data revealed that post-EMT intermediary and post-MET polygonal cells generated significantly more spheres than either E-cad⁺ or E-cad⁻ populations (p < 0.01), whereas no difference was observed within each epithelial-like or transitional phenotype group, suggesting enhanced stem-like potential in transitional states. Together, these results link distinct phenotypes to specific in vitro functional signatures low-proliferation, low- migration epithelial-like cells versus high-proliferation, high-migration post-MET derivatives, mirroring the in vivo diversity of trophoblast roles in placental development.

Conclusion/Future Perspectives: This functional stratification of HTR8/SVneo subpopulations provides a framework for linking in vitro behavior to key in vivo processes: epithelial-like E-cad⁺ cells resemble less invasive villous cytotrophoblasts involved in placental barrier maintenance, while post-EMT intermediary cells align with the highly migratory extravillous trophoblasts that physiologically invade the maternal endometrium during early gestation. Notably, our findings demonstrate that post-MET polygonal cells exhibited the highest invasive capacity in vitro, underscoring the remarkable plasticity of trophoblasts, where invasive behavior can persist or re-emerge even after re-epithelialization, potentially in response to environmental demands. Clinically, impaired invasive function of trophoblasts underlies pathological mechanisms in preeclampsia and fetal growth restriction, whereas dysregulated or prolonged invasion could contribute to abnormal placental attachment disorders. These findings underscore the clinical relevance of trophoblast plasticity, with distinct phenotypes in healthy in vitro models reflecting functional states that in vivo support normal placentation. Future work should examine how these signatures are altered in pathological pregnancies, integrating in vivo models and clinical samples to uncover biomarkers and therapeutic targets for conditions such as preeclampsia and IUGR