Department of Anatomy, Cell Biology and Physiological Sciences, Faculty of Medicine, American University of Beirut, Beirut, 1107-2020, Lebanon.
Department of Anatomy, Cell Biology and Physiological Sciences, Faculty of Medicine, American University of Beirut, Beirut, 1107-2020, Lebanon.
Placenta. 2020 Jan 15;90:90-97. doi: 10.1016/j.placenta.2019.12.013. Epub 2019 Dec 13.
The placenta is a transitory organ essential for proper fetal maturation and growth. Trophoblasts, the main cell type of the placenta, differentiate along the villous or extravillous pathways. The ability of villous cytotrophoblasts to undergo an epithelial-to-mesenchymal transition to form the invasive extravillous trophoblasts is vital for a successful pregnancy outcome. Many trophoblastic cell lines, including HTR-8/SVneo, have been widely used to investigate extravillous trophoblast biology and functions. We have previously reported that HTR-8/SVneo cell line contains a mixed populations of epithelial and mesenchymal cells. Uncovering the mechanisms underlying this heterogeneity is essential for the proper study of normal and pathological placental function.
HTR-8/SVneo was subjected to monoclonal isolation, spheroid formation assay and cell sorting to isolate pure epithelial and mesenchymal populations. These fractions were maintained in culture and assessed for expression of epithelial and mesenchymal markers using quantitative real-time PCR and immunofluorescence. In addition, the implication of TGFβ in the EMT process was investigated using a selective inhibitor of TGF-βR1 (A83-01).
Passaging of the pure epithelial population maintained under normal culture condition resulted in a shift to a mesenchymal phenotype. This transition was reduced upon inhibiting TGF-βR1. Similarly, E-cadherin positive HTR-8/SVneo spheroids plated in 2D culture resulted in the emergence of streams of invading mesenchymal cells.
HTR-8/SVneo cell line is undergoing EMT under normal culture condition and TGFβ is a key mediator of this process. Our results raise the possibility of using HTR-8/SVneo cell line as a model to investigate EMT in extravillous trophoblast cells.
胎盘是一种对于胎儿正常成熟和生长至关重要的过渡性器官。滋养细胞是胎盘的主要细胞类型，沿着绒毛或绒毛外途径分化。绒毛滋养细胞能够发生上皮间质转化，形成侵袭性绒毛外滋养细胞，这对于成功的妊娠结局至关重要。许多滋养层细胞系，包括 HTR-8/SVneo，已被广泛用于研究绒毛外滋养层的生物学和功能。我们之前曾报道 HTR-8/SVneo 细胞系含有混合的上皮和间充质细胞群体。揭示这种异质性的机制对于正确研究正常和病理性胎盘功能至关重要。
对 HTR-8/SVneo 进行单克隆分离、球体形成测定和细胞分选，以分离出纯上皮和间充质群体。这些部分在培养中维持，并使用定量实时 PCR 和免疫荧光法评估上皮和间充质标记物的表达。此外，还使用 TGF-βR1 的选择性抑制剂（A83-01）研究了 TGFβ 在 EMT 过程中的作用。
在正常培养条件下传代的纯上皮群体导致向间充质表型的转变。抑制 TGF-βR1 可减少这种转变。同样，在 2D 培养中种植 E-钙粘蛋白阳性 HTR-8/SVneo 球体导致侵袭性间充质细胞流的出现。
HTR-8/SVneo 细胞系在正常培养条件下发生 EMT，TGFβ 是该过程的关键介质。我们的结果提出了使用 HTR-8/SVneo 细胞系作为研究绒毛外滋养细胞 EMT 的模型的可能性。
