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VOLUME 2 - NUMBER 3 - 2025

Confluency-dependent gene expression in trophoblast cell lines: implications for appropriate physiologic and pathologic models set up

  • Francesca Rossi, Teresa Bulfone, Gabriella Zito, Giuseppe Ricci, Eva Andreuzzi
  • Original Article, 49-54
  • Full text PDF

  • Purpose: This study aimed to determine how culture confluency and intrinsic cell line differences influence the expression of angiogenic mediators and immune markers in commonly used trophoblast models.

    Methods: JEG-3 and JAR cells were cultured at sub-confluent (60%) and fully confluent (100%) states. Immunofluorescence was performed to visualize the actin cytoskeleton and nuclei. Gene expression of EPHA1, FLT4, ANGPT4, and HLA-G was assessed by qPCR using GAPDH as reference. Expression levels were calculated with the 2−ΔΔCt method. Statistical analysis was performed using the unpaired t test to compare cell lines and confluency states.

    Results: A notable distinction between the two models was observed, where JEG-3 cells exhibited higher levels of FLT4 and ANGPT4 compared with JAR. A subsequent analysis of each cell line revealed additional modulations associated with cell density, with JEG-3 demonstrating a heightened susceptibility to culture confluency. In the case of fully confluent JEG-3 cells, there was a significant increase in HLA-G expression, as well as a substantial rise in the expression of FLT4 and ANGPT4. In contrast, EPHA1 remained constant across the various conditions, suggesting that its expression is not influenced by changes in cell density.

    Conclusions: These findings demonstrate that both cell density and model choice critically shape marker expression in trophoblast cell lines. Careful reporting and control of confluency are essential for reproducibility and for selecting the most appropriate in vitro model depending on the biological question.

  • KEY WORDS: Trophoblasts, JEG-3, JAR, cell density, vascular remodeling.