whereas by contrast, severe early-onset IUGR is typically characterized by reduced branching of the peripheral villi and the underlying vasculature. On the basis of recent observations using mouse models, it has become apparent that these human placental pathologies may be the result of distinct molecular abnormalities in the differentiation of the trophoblast lineage.
1.6.1 Molecular Basis of Placental Development in Mice
The trophoblast origins of pre-eclampsia and IUGR are difficult to investigate as these diseases represent investigative endpoints, and hence, very early gestational samples are not available. However, the last ten years has seen a substantial increase in our knowledge regarding trophoblast development due primarily to transgenic and knockout studies in the mouse (167). Although they differ in their overall structure (Figure 1-8) their basic developmental plan is the same and as such, they are thought to be quite similar in terms of their molecular regulation (168). Because some genes known to be involved in murine placental development are expressed in an analogous manner in humans, this indicates that key molecular determinants of placental development in mice are also likely to be involved in humans (for full review see (169)). This data, albeit limited, suggests that trophoblast subtype-specific function is conserved between the two species (reviewed in (168)), and have highlighted the importance of the trophoblast and its differentiation into the different lineages in directing the different stages of placental development.
Figure 1-8 Comparative anatomy of the murine and human placenta
(a) Structure of the mouse placenta. The inset details the fetal-maternal interface in the labyrinth (b) Structure of the human placenta. The inset image shows a cross-section through a chorionic villus;
trophoblast-derived structures (blue) and mesoderm-derived tissues (orange). The inset images illustrate the number and type of cell layers between maternal and foetal blood (169).
1.6.2 Trophoblast Stem Cell Maintenance and Differentiation
Trophoblast stem cells (TS) arise in the polar trophectoderm (170) and are distributed throughout the extra-embryonic ectoderm (chorion) (171). These cells are characterized by their ability to 1) retain an undifferentiated proliferative phenotype and 2) differentiate into the various trophoblast cell-types in the mouse (i.e. spongiotrophoblast (SpT), trophoblast giant cells (TG), or syncytiotrophoblast (SynT) of the labyrinth).
Differentiation into these various cell-types is characterized by arrest of mitosis and the promotion of transcription factor pathways that ultimately determine cell fate decisions (Figure 1-9) (172).
Maintenance of the proliferative TS phenotype is dependent on stimulation by fibroblast growth factor (FGF) signalling through its ligand (FGF4) (173), (expressed in the early embryo, the ICM and the epiblast of the post-implantation embryo (169)) and its receptor, FGFR2 (expressed in the trophectoderm and chorion (174)), and the downstream transcription factors Cdx2 (175) and Eomes (176) (all are promoted by Nodal (170)). Removal of FGF4 from cultured trophoblast stem cells leads to the rapid down-regulation of Cdx2 and Eomes and proliferation ceases owing to their exit from the cell cycle (173) and subsequent differentiation into the various labyrinth trophoblast
cell types via expression of transcription factors (Hand1, Stra13 (TG), Gcm1 (SynT)) (172) (For a full review of murine trophoblast genetics, see (169; 170; 177)).
Figure 1-9: Trophoblast stem cell fate in the mouse
Segregation of the murine trophoblast lineage first occurs at the blastocyst stage prior to implantation.
Blastomeres on the outside of the conceptus form the trophectoderm destined to become trophoblast, whilst the inner cells become part of the ICM; these cells are prevented from differentiating into trophectoderm by the expression of Oct4. Subsequent differentiation of trophoblast stem cells into syncytiotrophoblast, spongiotrophoblast, trophoblast giant cells and glycogen cells is associated with down regulation of Cdx2 and Eomes and upregulation of lineage-specific transcription factors.
Differentiation into trophoblast giant cells is induced by up-regulation of two basic helix-loop-helix (BHLH) transcription factors, Hand 1 and Stra13, whilst differentiation into and maintenance of the spongiotrophoblast layer is dependent upon the BHLH Mash 2, a subpopulation of which differentiates into trophoblast glycogen cell promoted by the Igf2 gene (adapted from 170).
Significantly however, the developmental potential of TS cells is lost from the murine placenta coinciding with the development of the labyrinth layer at E8.5, since TS cells can only be derived from the chorion up until E8-8.75 (171; 178). Because the placenta
trophoblast cells are detected in the labyrinth layer, and may well represent labyrinth trophoblast progenitors (179; 180). Such progenitor cells are also predicted to exist in the human placenta as these cells act as the germinal layer of the functional syncytial layer of the chorionic villi throughout gestation.
1.6.3 Branching Morphogenesis and Labyrinth Development in Mice
The villous part of the murine placenta, the labyrinth layer- functionally analogous to the chorionic villi in humans, begins to form after embryonic day (E) 8.5 and is dependent upon attachment of the chorioallantois to the allantoic mesoderm (derived from the embryoblast) (181). Here, the mesoderm gives rise to mesenchymal cells and blood vessels within the labyrinth layer, whereas the different trophoblast layers within the chorion give rise to the three layers of differentiated trophoblast in the labyrinth (post-mitotic mononuclear trophoblast cells, and two syncytial layers formed by cell-cell fusion).
Branching morphogenesis and syncytiotrophoblast formation in the labyrinth layer depends upon the trophoblast transcription factor, glial cell missing 1 (Gcm1), the expression of which depends upon signalling from the allantois (182). Gcm1 mutant mice fail to initiate branching morphogenesis and syncytiotrophoblast formation demonstrating the fundamental role of this transcription factor for labyrinth development in the mouse (181). Initially, Gcm1 is expressed in small clusters in post-mitotic cuboidal trophoblast cells (182) in the flat basal surface of the chorion as early as E7.5 (183) at sites where the chorion begins to fold to form the primary villi; GCM1 positive cells become elongated at the onset of branching morphogenesis (181). Because of its restricted chorion expression to labyrinth-committed cells, it suggests that GCM1 may be critical in maintaining the multi-potent stem cell pool (173) in the early placenta.
Gcm1 expression is subsequently restricted to the tips of the primary branches and differentiated syncytiotrophoblast begin to form (181) where Gcm1 is localized to the SynT-II cells (182). Syncytiotrophoblast formation requires cell-cycle exit and cell-cell fusion to form a syncytium, although the underlying fusion mechanism(s) or whether this is mediated by Gcm1 is currently not known in the mouse (170). By E9, primary
villous branches are apparent which undergo further branching and elongation to form the mature labyrinth at around E10.5 (182). However, the cellular processes regulating subsequent growth and expansion of the labyrinth layer are largely unknown, since (in contrast to the human placenta) proliferating trophoblast cells become rare in the labyrinth layer towards the end of gestation after ~E12.5 (182). Instead, mitotically active cells are restricted to the basal part of the labyrinth suggesting that growth may be seeded from the basal layer to the tips, and given that the labyrinth continues to expand in volume towards term (184), growth maybe achieved in part by an increase in cell volume (182).
1.6.4 Trophoblast Stem Cells in the Human Placenta
Despite the knowledge regarding trophoblast stem cell maintenance in murine placentas, the localisation, regulation and fate of human trophoblast stem cells still remains unclear. However, it is generally accepted that in the human placenta, the cytotrophoblast population represents the proliferative stem cell pool. In contrast to the mouse, proliferating cytotrophoblast cells can be found in the base of the anchoring villi and in the chorionic villi as they are needed to regenerate the syncytium throughout gestation.
Studies suggest that cytotrophoblast cells may be a heterogeneous pool, and that these different cell phenotypes arise by lineage specific progenitors. It was originally thought that the villous cytotrophoblast represents a single stem cell pool, in that they produce both syncytiotrophoblast and extravillous trophoblasts during the first trimester. This statement is supported by in vitro cell culture studies (185), and the establishment of in vitro 3D floating explant models in which villi remain in their native environment. In syncytium-denuded models, cytotrophoblast cells adopt an extravillous phenotype (upon addition of FGF4) at distal portions of cytotrophoblast outgrowths, whilst in the absence of growth factors, cytotrophoblast spontaneously differentiate into syncytiotrophoblast. FGF4 initiates cytotrophoblast proliferation (173), and results in smooth cytotrophoblast outgrowths, the more proximal cells of which adopt an
physically separating the outer layers of cells from the underlying basal lamina and mesenchymal cells.
Hence what would normally constrain cytotrophoblasts to make syncytiotrophoblast is lost and subsequently cells adopt an extravillous phenotype. Interestingly, cytotrophoblast cells do not enter the extravillous invasive pathway upon addition of FGF4 to syncytium-intact models, suggesting that syncytiotrophoblast may itself be necessary to maintain cells in the villous phenotype and to prevent their differentiation into an extravillous phenotype (for a full review see (185)).
A counter-argument against cytotrophoblast bi-potentiality is presented by James et al (187). These authors indicate that villous and extravillous trophoblast populations arise from two separate pools of cytotrophoblast stem cells in the first trimester, in comparison to the generally accepted single pool, and that these cells are lineage-restricted.
The differentiation and/or maintenance of the cytotrophoblast columns, analogous to the spongiotrophoblast layer in mice, is dependent upon an upstream assembly of transcription factors including Hash2 (188), Stra13 , Id2 and E-factor (189). As cytotrophoblast cells adopt an invasive phenotype (trophoblast giant cells in mice), Hash2, Id2 and E-factor are down-regulated and Stra13 is up-regulated in vitro (189).
These expression patterns are analogous to those seen in the mouse, reinforcing the idea that these are homologous cell types. However, some species disparity may exist as differentiation into invasive extravillous trophoblast in humans is not dependent upon Hand1 (190), a BHLH transcription factor essential for trophoblast giant cell differentiation in the mouse. Although Hand1 mRNA is not detectable in both first and third trimester placenta, expression of Hand1 is detectable in JEG-3 and JAR trophoblast cell lines (190).
As in the mouse, recent studies in humans have found that villous morphogenesis and syncytiotrophoblast formation is completely dependent upon the trophoblast-specific transcription factor, Gcm1 (181; 183; 191). Originally thought of as a homogenous population of cells, these studies have shown that in fact villous cytotrophoblast represents a heterogeneous stem cell population in which a subset of cells display
(likely asymmetrical) expression of GCM1 protein; presumably the post-mitotic daughter cells destined for syncytial fusion, and it is speculated that those negative for GCM1 may retain the stem cell phenotype. The factor(s) behind this asymmetrical expression of GCM1 are at present unknown. Up-regulation of GCM1 in this subset drives the cell to exit the cell cycle and subsequently results in de novo formation of syncytiotrophoblast, which is supported by numerous observations that Gcm1 up-regulates Syncytin1 (192), a gene involved in trophoblast fusion (193). Furthermore, GCM1 expression is associated with villous sprouts which represent the sites of primary villous formation, suggesting GCM1 initiates villous morphogenesis in the human placenta (191).