Transcriptomic and functional analyses of 3D placental extravillous trophoblast spheroids

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 Δ 9 -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.

Histological changes in placental rat apoptosis via FasL and cytochrome c by the nano-herbal Zanthoxylum acanthopodium

“…Moreover, such cells can increase the destruction of cytotrophoblasts ( Yang et al, 2019 ). The reduced number of cytotrophoblasts that were invaded cannot sufficiently modify the SA ( Stenqvist et al, 2013 , Wong et al, 2019 ). Based on placental histology, FasL expression was lower than cytochrome c expression.…”

Devices and techniques used to obtain and analyze three‐dimensional cell cultures

“…This gives them an edge over 2D monolayers and helps them better mimic the heterogenous environment found in vivo. 76,77 Recent studies support the use of microsensors in the standardization of growth patterns and investigation of various pathways that occur in vivo using 3D cultures. Microsensors are advantageous in gauging the transient effects which do not require cellular markers to evaluate thus providing the experimenter information beyond what one gathers from an array of optical imaging tools and microscopy.…”

“…The potential to maintain different types of cells within the same population can be attributed to the design of the 3D spheroid, owing to the varying depths at which the cells are located from oxygen and nutrient sources. This gives them an edge over 2D monolayers and helps them better mimic the heterogenous environment found in vivo 76,77 …”

The Role of the 3Rs for Understanding and Modeling the Human Placenta

“…White et al showed a placental spheroid system using JAr cells in 1988 [ 65 ]. Also, Wong et al used the HTR-8/SVneo extravillous trophoblast cell line to create a spheroid model to show placental invasion [ 66 ]. Spheroid systems are the simplest of 3D in vitro systems to set up and can be useful for drug discovery, as they provide a more realistic and physiologically relevant model than 2D in vitro assays.…”