Investigation of human trophoblast invasion in vitro

Abstract

Background: In humans, inadequate trophoblast invasion into the decidua is associated with the 'great obstetrical syndromes' which include pre-eclampsia, foetal growth restriction (FGR) and stillbirth. The mechanisms regulating invasion remain poorly understood, although interactions with the uterine environment are clearly of central importance. Extravillous trophoblast (EVT) cells invade the uterus and transform the spiral arteries. Progress in understanding how they invade has been limited due to the lack of good in vitro models. Firstly, there are no non-malignant cell lines that have an EVT phenotype. Secondly, the invasion assays used are of limited use for the small numbers of primary EVT available from first-trimester placentas. We discuss recent progress in this field with the generation of new EVT lines and invasion assays using microfluidic technology.

Objective and rationale: Our aim is to describe the established models used to study human trophoblast invasion in vivo and in vitro. The difficulties of obtaining primary cells and cell lines that recapitulate the phenotype of EVT are discussed together with the advantages and pitfalls of the different invasion assays. We compare these traditional end point assays to microfluidic assays where the dynamics of migration can be measured.

Search methods: Relevant studies were identified by PubMed search, last updated on February 2020. A search was conducted to determine the number of journal articles published using the cell lines JEG-3, BeWo, JAR, HTR-8/Svneo, Swan-71 and primary human extravillous trophoblast in the last 5 years.

Outcomes: Deep trophoblast invasion into the maternal decidua is a particular feature of human pregnancy. This invasion needs to be finely regulated to allocate resources between mother and baby. A reliable source of EVT is needed to study in vitro how the uterine environment regulates this process. First, we critically discuss the issues with the trophoblast cell lines currently used; for example, most of them lack expression of the defining marker of EVT, HLA-G. Recently, advances in human stem cell and organoid technology have been applied to extraembryonic tissues to develop trophoblast cell lines that can grow in two (2D) and three dimensions (3D) and differentiate to EVT. This means that the 'trophoblast' cell lines currently in use should rapidly become obsolete. Second, we critically discuss the problems with assays to study trophoblast invasion. These lack physiological relevance and have simplified migration dynamics. Microfluidic assays are a powerful tool to study cell invasion because they require only a few cells, which are embedded in 3D in an extracellular matrix. Their major advantage is real-time monitoring of cell movement, enabling detailed analysis of the dynamics of trophoblast migration.

Wider implications: Trophoblast invasion in the first trimester of pregnancy remains poorly understood despite the importance of this process in the pathogenesis of pre-eclampsia, FGR, stillbirth and recurrent miscarriage. The new technologies described here will allow investigation into this critical process.

Keywords: human; invasion assays; microfluidics; organoids; trophoblast.

Figure 1

Human trophoblast invasion. Extravillous trophoblast (EVT) cells migrate from the anchoring villus and invade into the decidua basalis. Their primary function is to remodel the uterine spiral arteries, which results in a four to six times times increase in the diameter of the spiral artery: from high-resistance low-flow vessels into large dilated vessels. In the great obstetrical syndromes of pre-eclampsia, foetal growth restriction (FGR) and still birth, invasion is inadequate and the arteries remain narrow at the opening into the intervillous space.

Figure 2

Regulators of trophoblast invasion. (EVT) cells invade the decidua coming into contact with decidual cells, where certain cell–cell interactions are known to release cytokines that promote invasion. Before the onset of maternal circulation, EVT cells are supported by secretions from the nutrient-rich endometrial glands. There are physical factors that are thought to regulate invasion: the oxygen environment, extracellular matrix (ECM) proteins the EVT have to cleave during migration, and tissue stiffness. Trophoblast invasion is spatially and temporally controlled with varying cell populations and physical factors as EVT migrate into the decidua. Decidua basalis contains abundant EVT by week 10 and at week 15 deep invasion of the myometrial segments is observed.

Figure 3

The range of cells used to study human trophoblast invasion. (A) A PubMed search was conducted for journal articles published over the past 5 years, last updated February 2020 for studies that use JEG-3, BeWo, JAR, HTR-8/Svneo, Swan-71 and primary human EVT. (B) Timeline of the establishment of these cell lines as well as hTSC and organoids.

Figure 4

Conventional in vitro and ex vivo assays used to study trophoblast invasion. (A) The wound healing or scratch assay is simple, economical and 2D. A scratch is made, and the cells migrate to close the gap. The rate of migration is quantified using time-lapse microscopy. (B) The Transwell or Boyden chamber is 3D and cells are seeded in an insert and placed in a well with a cytokine. Cells migrate through a semi-permeable membrane with migration quantified by counting the number of cells that have crossed the membrane. (C) Villous explants from first trimester pregnancies are cultured on an extracellular matrix (ECM) or decidual tissue and extravillous trophoblast (EVT) outgrowth is quantified. (D) Uterine spiral arteries isolated from term pregnancies are embedded in a fibrin gel and cultured with EVT to study invasion under various regulating factors.

Figure 5

Microfluidics assay to study EVT invasion. EVTs are isolated from first-trimester placentas, then stained with a cell tracker dye and embedded in Matrigel® in the central hydrogel channel. A constant flow of medium in the two side channels, either with or without a cytokine, is applied to create a chemical gradient across the hydrogel channel. Individual cell tracks are generated from time-lapse confocal microscopy.