Three-dimensional cultures of trophoblast stem cells autonomously develop vascular-like spaces lined by trophoblast giant cells

Abstract

The maternal blood space in the mouse placenta is lined not by endothelial cells but rather by various subtypes of trophoblast giant cells (TGCs), defined by their location and different patterns of gene expression. While TGCs invade the spiral arteries to displace the maternal endothelium, the rest of the vascular space is created de novo but the mechanisms are not well understood. We cultured mouse trophoblast stem (TS) cells in suspension and found that they readily form spheroids (trophospheres). Compared to cells grown in monolayer, differentiating trophospheres showed accelerated expression of TGC-specific genes. Morphological and gene expression studies showed that cavities form within the trophospheres that are primarily lined by Prl3d1/Pl1α-positive cells analogous to parietal-TGCs (P-TGCs) which line the maternal venous blood within the placenta. Lumen formation in trophospheres and in vivo was associated with cell polarization including CD34 sialomucin deposition on the apical side and cytoskeletal rearrangement. While P-TGCs preferentially formed in trophospheres at atmospheric oxygen levels (19%), decreasing oxygen to 3% shifted differentiation towards Ctsq-positive sinusoidal and/or channel TGCs. These studies show that trophoblast cells have the intrinsic ability to form vascular channels in ways analogous to endothelial cells. The trophosphere system will be valuable for assessing mechanisms that regulate specification of different TGC subtypes and their morphogenesis into vascular spaces.

From the stem of the placental tree: trophoblast stem cells and their progeny

“…These endeavours will involve optimising the culture conditions for known placental stem and progenitor cells and deriving novel self-renewing trophoblast progenitor sub-populations. This could be accomplished by establishing placental organoids that rely on intrinsic signals driving self-organisation coupled to self-renewal (Rai and Cross, 2015). This strategy has proved to be successful for a number of mouse and human organoid systems (Huch and Koo, 2015).…”

The developmental expression of the CDK inhibitor p57kip2 (Cdkn1c) in the early mouse placenta

“…). Recent work using a three‐dimensional culture system has shown that trophoblast stem cells differentiate into a variety of TGC subtypes, and create microcavities lined by TGCs that express a variety of markers (Rai and Cross, ); it will be of interest to investigate the expression of p57 kip2 during their differentiation in such a system.…”

Trophectoderm stem cells to model the effect of altered periconceptional diet on embryos

“…3D culture of so-called "trophospheres" is a new technique used to form differentiated trophectodermal cells from TS cell aggregates (Rai and Cross, 2015), and is described as being more reproducible than simple monolayer differentiation. This technique results in cavitated structures lined by parietal TGCs which surround maternal blood sinuses in the placenta; such forced and defined differentiation may make interpretation of endocytosis assays more robust, and could be applied to our TS cells.…”

“…taken from Rai and Cross, 2015 This method was attempted for our TS clones, using hanging drops to culture TSCs. After trypsinisation, drops (30-50µl) of~500 TSCs were placed in a non-adhesive culture dish, then hung upside down to culture.…”