Abstract
Placental extravillous trophoblast (EVT) invasion is essential in establishing proper blood supply to the fetus during pregnancy. However, traditional 2D in vitro systems do not model the in vivo invasion process in an anatomically-relevant manner. Our objectives were to develop a 3D spheroid model that would allow better emulation of placental invasion in vitro and to characterize the transcriptomic and functional outcomes. HTR8/SVneo EVT cells were self-assembled into 3D spheroids using ultra-low attachment plates. Transcriptomic profiling followed by gene set enrichment and gene ontology analyses revealed major global transcriptomic differences, with significant up-regulations in EVTs cultured as 3D spheroids in canonical pathways and biological processes such as immune response, angiogenesis, response to stimulus, wound healing, and others. These findings were further validated by RT-qPCR, showing significant up-regulations in genes and/or proteins related to epithelial-mesenchymal transition, cell-cell contact, angiogenesis, and invasion/migration. A high-throughput, spheroid invasion assay was applied to reveal the dynamic invasion of EVTs away from the spheroid core into extracellular matrix. Lastly, lipopolysaccharide, dexamethasone, or Δ9-tetrahydrocannabinol exposure was found to impact the invasion of EVT spheroids. Altogether, we present a well-characterized, 3D spheroid model of EVT invasion and demonstrate its potential use in drug and toxin screening during pregnancy.
Highlights
The human placenta is a transient organ that forms the interface between the mother and fetus during pregnancy[1]
HTR8/SVneo extravillous trophoblast (EVT) cells seeded in ultra-low attachment plates at densities of 1,000, 5,000, and 10,000 cells/mL all self-assembled into spheroids within two days with average diameters of 211.1 μm, 323.7 μm, and 411.6 μm, respectively (Fig. 1a)
We further demonstrate that 3D EVT spheroids: (1) express global transcriptomes that are distinct compared to 2D monolayers, (2) exhibit invasive and migratory gene and protein expression profiles alongside dynamic invasive behaviour when embedded into an ECM environment, and (3) may be studied by adapting the innovative spheroid invasion assay, which was originally pioneered by tumour researchers[17,24], revealing responsiveness to exogenous drugs
Summary
The human placenta is a transient organ that forms the interface between the mother and fetus during pregnancy[1]. The primary placental cell type responsible for regulating invasion is the extravillous trophoblast (EVT), which bears the ability to migrate away from the solid trophoblast columns of the anchoring chorionic villi and invade into the maternal decidua[5]. This process is highly regulated by a variety of physiochemical factors (e.g. oxygen, growth factors, nutrients, extracellular matrix proteins), and dysregulated EVT invasion can result in compromised placentation and an inability to properly support both the mother and fetus[6]. We further apply a high-throughput, spheroid invasion assay to measure the actual invasion of the 3D EVT spheroids into extracellular matrix in real-time, and assess the impact of various drugs and compounds
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