Placenta
Volume 29, Supplement , Pages 4-9, March 2008

IFPA Award in Placentology Lecture – Characteristics and Significance of Trophoblast Giant Cells

  • M. Hemberger

      Affiliations

    • Corresponding Author InformationTel.: +44 1223 496 534; fax: +44 1223 496 022.

Laboratory of Developmental Genetics and Imprinting, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK

Accepted 13 November 2007. published online 17 December 2007.

Article Outline

Abstract 

Extraembryonic development in rodents depends on the differentiation and function of trophoblast giant cells. Morphologically striking, giant cells exhibit many extraordinary characteristics adapted to ensure the success of pregnancy. This review summarizes some of the intriguing aspects of giant cell morphology and function. Giant cells are highly polyploid as a result of a switch from a mitotic to an endoreduplicative cell cycle. They further partition their genome content into various fragments which may represent a mechanism to maximize protein synthesis. Similar to metastatic tumour cells, they breach basement membranes and invade deeply into a foreign tissue, the maternal decidualized uterine stroma. Their angiogenic and vasodilatory properties, combined with the ability to remodel arterial walls, enable them to redirect maternal blood flow towards the implantation site. Recent advances have recognized that the giant cell population is more diverse than previously recognized and future studies will have to show how these subtypes differ functionally and how their differentiation is controlled.

Keywords: Trophoblast giant cells, Mouse, Extraembryonic development, Placenta, Endoreduplication, Invasion

 

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1. Trophoblast giant cells – waves of differentiation during development 

During extraembryonic development in the mouse, a variety of trophoblast cell types can be distinguished including trophoblast giant cells that form the border to the maternal decidua, syncytiotrophoblast cells in the labyrinth layer, as well as spongiotrophoblast and glycogen cells in the junctional zone [1]. This review focuses on the characteristics and functions of trophoblast giant cells as they represent a fascinating cell type essential for normal development and exhibit several remarkable features.

Trophoblast giant cells are the first definitive cell type to differentiate after fertilization, emerging from the outer trophectoderm layer of the blastocyst during the peri-implantation period. After implantation, giant cells derived from the mural trophectoderm line the embryonic cavity where they form part of the parietal yolk sac (Fig. 1). Because of the time of their differentiation, they are referred to as ‘primary’ or ‘parietal’ giant cells. A second wave of giant cell differentiation starts after implantation around the margins of the ectoplacental cone. Therefore, these cells are called ‘secondary’ giant cells. The specific functions of secondary giant cells are requisite for the establishment of the feto–maternal blood circulation in the later placenta. In the mouse, the mature placenta is only formed around mid-gestation. Here again, giant cells constitute a discontinuous border towards the maternal decidua. They also provide the lining of canals that funnel maternal blood into the network of blood sinuses in the placental labyrinth (Fig. 1). A late wave of so-called sinusoidal giant cells differentiates around E12.5 when they form within the cytotrophoblast layer (layer I) of the interhaemal membrane in the labyrinth [2], [3].

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  • Fig. 1 

    Schematic diagram of an early mouse post-implantation conceptus (∼E7.5) and a post-midgestation placenta (∼E12.5 onwards), summarizing the localization of trophoblast giant cell subtypes. Parietal or primary giant cells (depicted in dark pink) line the early implantation site. Secondary giant cells can be subdivided into several groups. Giant cells that differentiate at the margins of the ectoplacental cone (depicted in lighter pink and yellow), especially those at its tip, are invasive and make contact with maternal spiral arteries. In the later placenta, giant cells are located at the border towards the maternal decidua. Giant cells are also associated with maternal blood canals (canal giant cells) and with sinusoids that carry maternal blood in the placental labyrinth layer (sinusoidal giant cells). Some of the most prominent functions of specific giant cell subtypes and the overall functions of giant cells are listed. E=embryo.

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2. Morphological characteristics 

On histological sections of mouse implantation sites and placentae, giant cells are immediately obvious because of their striking cell and nuclear size that is correlated with a high degree of polyploidy. Instead of the normal diploid genome, the DNA content of trophoblast giant cells may reach levels up to 1000N [4], [5]. Analogously, extravillous cytotrophoblast cells of the human placenta also become poly- or aneuploid as they differentiate to an invasive phenotype, albeit to a much lesser extent [6], [7]. Polyploidy in murine trophoblast giant cells is achieved by a process called endoreduplication. Their differentiation requires a cell to exit the normal mitotic cell cycle and to undergo repeated rounds of DNA synthesis without intervening cell divisions. Therefore, giant cells contain only one, drastically enlarged, nucleus. Similar to the situation in salivary gland cells in Drosophila, it has also been suggested that giant cell nuclei are polytene, i.e. that sister chromatids remain in contact with each other and are arranged in a parallel orientation [4].

Upon maturation, the polyploid nuclei of giant cells undergo further reorganization processes. These rearrangements can be observed in late stage placentae [8] and upon prolonged differentiation of trophoblast stem cells (Fig. 2). The nuclear envelope invaginates at various sites causing the nucleus to bulge into lobules or even to fragment into multiple parts. Much in contrast to the fragmentation of apoptotic nuclei, this process is not associated with giant cell death but may represent a means to increase the nuclear surface area. To date, it is not clear whether certain genomic regions or chromosomes are preferentially set apart in these lobules and fragments. For example, it could be speculated that particularly active chromosomal domains require such an increase in nuclear surface area to accommodate the extent of protein export. Observation of sex chromosomes in each of the separated areas has suggested, however, that every nuclear fragment may contain at least one genome equivalent [9] although this would argue against a strict polytene chromosomal organization. It also remains elusive how the complex nuclear reorganization is accomplished and how proper transcriptional and translational control is ensured throughout the vastly amplified giant cell genome.

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  • Fig. 2 

    Examples of trophoblast giant cells with nuclear invaginations or fragmentations that differentiated from trophoblast stem cells in vitro. Arrows point towards indentations of the nucleus or nuclear fragments surrounded by nuclear membrane. Note also the doughnut shape of some trophoblast giant cell nuclei in (A). (A), (B), and (D): fluorescent nuclear staining was performed with 4,6-diamidino-2-phenylindole (DAPI). The cellular outline is indicated by the red dotted line. The inset in (B) shows an overexposed image of the same cell visualizing the cytoplasmic perimeter. (C) Differential interference contrast (DIC) image and (D) DIC/DAPI fluorescence overlay of the same area. Magnification bar: 100μm.

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3. Heterogeneity of trophoblast giant cells 

Traditionally, a distinction has been made between ‘primary’ and ‘secondary’ trophoblast giant cells based on the time of their differentiation. These two groups are characterized by expression of the well-established markers placental lactogen I and II, respectively [10]. In most other regards, however, giant cells have been thought to represent a uniform cell population. Some further heterogeneity has been indicated by the expression of several genes such as Thbd [11], Mrj [12] and Cts7/Cts8 [13] in only a subset of primary and/or secondary giant cells. Recently, detailed expression analyses and cell lineage tracing have identified four types of giant cell that can be defined by a combination of gene expression patterns and spatial localization [14]. These include parietal giant cells surrounding the implantation site, giant cells associated with maternal spiral arteries in the uterine wall, those associated with maternal blood canals in the placenta, and sinusoidal giant cells within the labyrinth layer of the placenta (Fig. 1). All four subtypes also differentiate in vitro from trophoblast stem cells. It is not known, however, if and how these types of giant cells differ functionally and what governs their differentiation. From their temporal occurrence in development, some functional divergence is likely while certain physiological (e.g. vasodilator) functions (see below) may be shared by all subtypes. Interestingly, some treatments that are known to enhance giant cell formation promote differentiation of specific subtypes only. For example, retinoic acid specifically induces differentiation of parietal and spiral artery-associated giant cells at the expense of sinusoidal and canal giant cells [14]. With these insights it will be interesting to reassess the extraembryonic tissues of a number of mouse mutants to determine precisely which subtypes of giant cells are affected. Such studies will shed further light on the differentiation pathways and the diverse functions that are attributed to different types of giant cells.

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4. Giant cell differentiation requires a switch in the cell cycle 

The switch from a mitotic to an endoreduplicative cell cycle is controlled by a shift in expression of several cell cycle regulators. The mitotic cyclin B/Cdk1 complexes are down-regulated in favour of replication-associated cyclins A and E [15]. It has been shown that cyclins E1 and E2 are essential for the endoreduplicative cell cycle of trophoblast giant cells [16], [17]. Interestingly though, the absence of E cyclins does not interfere with the differentiation of cells with giant cell-like morphology per se. Instead, cells with enlarged nuclei are formed that contain much less DNA than normal giant cells [17]. These findings indicate that the adoption of giant cell morphology may not be a mere consequence of the accumulation of DNA. The necessity of E cyclins for the endoreduplicative cell cycle is further demonstrated by mutations that result in increased or constitutive cyclin E levels. Thus, lack of the F-box protein Fbxw7 and the ubiquitin ligase complex component Cul1, both factors that mediate degradation of cyclin E, results in more and bigger giant cells [18], [19]. In addition to cyclin/Cdk-mediated control, the cell cycle regulator Geminin normally prevents re-replication in mitotic cells. In the absence of Geminin, all cells of the early embryo start to endoreduplicate, become polyploid and then incapable of further cell division, leading to preimplantation lethality [20]. Furthermore, degradation of the cyclin-dependent kinase Cdkn1c is required to initiate each round of endoreduplication in trophoblast giant cells [21]. Interestingly, trophoblast giant cells acquire higher levels of ploidy in the absence of the DNA methyltransferase-like protein Dnmt3L [22]. Hence, endoreduplication is negatively regulated on the epigenetic level by a locus that is controlled by DNA methylation, and this effect may be due, in part, to loss of imprinted Cdkn1c expression in the absence of maternal Dnmt3L [23].

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5. Physiological role of trophoblast giant cells 

Upon dissection of early post-implantation mouse conceptuses, the presence of trophoblast giant cells is strikingly correlated with the occurrence of maternal blood pools. Thus, the implantation site is outlined by red blood cells with particularly large accumulations in the area surrounding the ectoplacental cone. This correlation is not limited to the environment of the uterine bed as haemorrhaging has been associated with the ectopic differentiation of trophoblast giant cells underneath the kidney capsule or in teratocarcinoma-like tumours [24], [25], [26]. This unusual capacity of trophoblast giant cells is based on their production of several angiogenic and vasodilatory factors such as VEGF, proliferin, adrenomedullin, thrombomodulin, and nitric oxide [27]. In situations where hormone production by giant cells is disturbed, vascularization of the decidua is compromised [28]. Thus, the angiogenic and vasodilatory functions of trophoblast giant cells are essential to modify the maternal uterine vasculature in order to promote maternal blood flow towards the implantation site.

In addition to the synthesis of vasoactive factors, giant cells also have the capacity to directly target maternal arteries. They are able to breach the vascular basement membrane and to displace the vessels' endothelial cell lining, a behaviour that contributes to the haemorrhaging that is linked with the ectopic occurrence of giant cells. Thereby, parietal giant cells of the early conceptus create an anastomosing network of blood spaces surrounding the implantation site [29]. Secretion of a number of proteases that mediate the availability of metabolites, as well as the phagocytic activity of trophoblast giant cells, further emphasizes their pivotal role in ensuring nutrient and gas transport across the yolk sac membrane [30], [31].

Soon after implantation, the artery remodelling properties of giant cells are coupled with an extraordinary invasive capacity. From about E7.0 onwards, ‘secondary’ giant cells at the margins of the ectoplacental cone start to penetrate deeply into the decidualized uterine stroma. In serial sections, giant cells have been found as far as 300μm away from the main trophoblast/placental border [32]. Invasion is spatially restricted and directed towards the mesometrial pole of the decidua where the uterine artery has branched into a network of spiral arteries. Giant cells invade into this region and make contact to spiral arteries. Through this coordinated invasion, these spiral artery-associated giant cells create entirely trophoblast-lined sinuses within the decidual stroma that carry maternal blood towards the embryo [32], [33]. Thus, the invasive and vessel remodelling capacities of trophoblast giant cells complement their angiogenic and vasodilatory functions and allow them to directly tap into the maternal blood circulation.

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6. Defects in trophoblast giant cell differentiation cause embryonic lethality 

From the outlined characteristics and functions of trophoblast giant cells it is obvious that these cells are essential for placental and, consequently, embryonic development. Defects in giant cell formation cause embryonic lethality. Interestingly, genetic studies in the mouse showed that inhibition of giant cell formation and an overabundance of giant cells are equally detrimental. Reduced numbers of giant cells are observed, for example, in mutants of Hand1, Mdfi, Dp1, Eed and Mfn2 that die before or around mid-gestation [34], [35], [36], [37], [38]. Similarly, development does not proceed beyond this stage in cases of increased giant cell differentiation such as in conceptuses deficient in Ascl2, Ccne1/2, Fbxw7, Chm, and Krt8/Krt19 [16], [17], [18], [39], [40], [41]. While often, though not always, the number and size of giant cells are positively correlated, recent insights into the diversity of giant cells necessitate a thorough analysis of the precise giant cell subtypes that are formed in these mutants. Morphologically, non-invasive parietal giant cells appear in general much larger than invasive, spiral artery-associated giant cells. Thus, the extent of endoreduplication is carefully orchestrated in each of the giant cell subtypes and may determine their functions. The process of giant cell differentiation is also associated with a massive reorganization of the cytoskeleton. Fully differentiated, large giant cells contain prominent stress fibres and a multitude of internal and peripheral focal adhesions, and become immotile in culture [42]. Therefore, overexpansion and terminal differentiation of giant cells likely interferes with their ability to penetrate into the decidua and to remodel the maternal vasculature. Consequently, the underlying reasons for embryonic lethality may be the same in cases of reduced and increased giant cell differentiation (both in terms of numbers and size) in that both types of mutants suffer from inadequate access to the maternal blood supply.

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7. Why giant cells? Possible reasons for their differentiation 

What are the advantages of forming a giant cell instead of multiple diploid cells? Speculations on why giant cell differentiation may have been favoured include that a massively enlarged genome and cell size allow the maximization of protein output to produce the vast amounts of hormones required for uterine homeostasis and the continuation of pregnancy. Further, giant cell formation enables large-scale protein synthesis without the need for cell proliferation and tissue remodelling. Finally, from the mother's point of view, decidual invasion by a polyploid cell is much more favourable than uterine infiltration by diploid fetal cells. Polyploidy renders giant cells incapable of mitotic cell divisions and thereby reduces the chance of tumours arising from invaded trophoblasts left behind after parturition. Similar mechanisms are also in place in the human placenta as cytotrophoblasts acquire aneuploidies upon differentiation into an invasive phenotype [7]. Thereby, invasive trophoblasts can gain access to the maternal blood supply deep inside the uterine environment without putting the health of the mother at risk.

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8. Concluding remarks 

Trophoblast giant cells are unusual cells of utmost importance for normal intrauterine development. Gaining insights into the factors, both intrinsic and extrinsic, that regulate differentiation of giant cells and underlie their diverse functions will further our understanding of the requirements for reproductive success. Since trophoblast giant cell differentiation and function involve a multifaceted array of disciplines, their study may at the same time serve to advance a number of biological fields.

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9. Conflict of interest 

The authors do not have any potential or actual personal, political, or financial interest in the material, information, or techniques described in this paper.

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Acknowledgements 

M. Hemberger is supported by an MRC Career Development Award.

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PII: S0143-4004(07)00271-8

doi:10.1016/j.placenta.2007.11.007

Placenta
Volume 29, Supplement , Pages 4-9, March 2008

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