© 2019. Published by The Company of Biologists Ltd | Development (2019) 146, dev163428. doi:10.1242/dev.

163428

REVIEW

Development of the human placenta

Margherita Y. Turco1,2,3,* and Ashley Moffett1,2

ABSTRACT Anatomy of human placental development

The placenta is essential for normal in utero development in The human placenta develops from the trophectoderm (TE), the
mammals. In humans, defective placental formation underpins outer layer of the pre-implantation embryo, which forms at ∼5 days
common pregnancy disorders such as pre-eclampsia and fetal post fertilisation (dpf ). At this stage, the pre-implantation embryo
growth restriction. The great variation in placental types across (termed a blastocyst) is segregated into two lineages: the inner cell
mammals means that animal models have been of limited use in mass (ICM) and the TE. The polar TE (the part of the TE that is
understanding human placental development. However, new tools contiguous with the underlying ICM) attaches to the surface
for studying human placental development, including 3D organoids, epithelium of the uterine mucosa: the endometrium (Fig. 1A).
stem cell culture systems and single cell RNA sequencing, have Although the earliest stages of implantation have not been
brought new insights into this field. Here, we review the morphological, visualised in humans, morphological observations of early
molecular and functional aspects of human placental formation, with a pregnant hysterectomy specimens and higher primates suggest
focus on the defining cell of the placenta – the trophoblast. that, following attachment to the uterine surface epithelium at ∼6-
7 dpf, the TE fuses to form a primary syncytium. This is the
KEY WORDS: Development, Maternal, Placenta, Trophoblast prelacunar phase of placental development (Boyd and Hamilton,
1970).
Introduction Following implantation, the primary syncytium quickly invades
The placenta is the largest fetal organ and the first to develop. It through the surface epithelium into the underlying endometrium,
plays a central role in the health of both the fetus and its mother, and which is transformed during pregnancy into a specialised tissue
has a lifelong impact on their future wellbeing. Indeed, disordered known as decidua (Fig. 1B) (Schlafke and Enders, 1975). By the
placental development is the primary defect in major diseases of time of the first missed menstrual period (∼14 dpf ), the blastocyst
pregnancy, such as pre-eclampsia, fetal growth restriction, recurrent is completely embedded in the decidua and is covered by the
miscarriage and still-birth (Brosens et al., 2011). The links between surface epithelium (Fig. 1C) (Hertig et al., 1956). Fluid-filled
the in utero environment and susceptibility to chronic disease in spaces (lacunae) then appear within the syncytial mass that enlarge
adults are also well recognised (Barker, 1995). However, despite and merge, partitioning it into a system of trabeculae. This is the
its importance in reproductive success, there is still limited lacunar stage. The syncytium also erodes into decidual
understanding of how the human placenta develops. The obvious glands, allowing secretions to bathe the syncytial mass (Hertig
ethical and logistical obstacles in investigating early human et al., 1956).
pregnancy, and the lack of long-term physiologically relevant The trophoblast cells beneath the syncytium (termed
in vitro models, make experimentation problematic. This is further cytotrophoblast cells) are initially not in direct contact with
compounded by the great diversity of strategies used by different maternal tissue but rapidly proliferate to form projections that
eutherian mammals to build a placenta, which make extrapolating push through the primary syncytium to form primary villi
data from other species to humans difficult (Carter and Enders, (a cytotrophoblast core with an outer layer of syncytiotrophoblast,
2004). SCT); this is the villous stage of development (Fig. 1D). The villous
Here, we provide a broad overview of current knowledge of trees are formed by further proliferation and branching, and the
human placental development, based on morphological studies of lacunae become the intervillous space. Cytotrophoblast cells
archival specimens, immunohistochemical and bulk transcriptome eventually penetrate through the primary syncytium and merge
analyses, and in vitro studies using primary cells, cell lines and laterally to surround the conceptus in a continuous cytotrophoblast
villous explants. We also highlight recent technological advances, shell between the villi and the decidua (Fig. 1D). The blastocyst is
such as comparative genomic and transcriptomic studies at the now covered by three layers: the inner chorionic plate in contact
single cell level, organoid culture systems, and systematic analyses with the original cavity; the villi separated by the intervillous space;
of biomarkers for placental function, that now allow us to study the and the cytotrophoblast shell in contact with the decidua.
early stages of human placental development in more detail. Soon afterwards, around day 17-18, extraembryonic
mesenchymal cells penetrate through the villous core to form
secondary villi. By day 18 dpf, fetal capillaries appear within the
DEVELOPMENT

1
Centre for Trophoblast Research, University of Cambridge, Cambridge CB2 3EG, core, marking the development of tertiary villi. The villous tree
UK. 2Department of Pathology, University of Cambridge, Cambridge CB2 1QP, UK. continues to rapidly enlarge by progressive branching from the
3
Department of Physiology, Neuroscience and Development, University of
Cambridge, Cambridge CB2 3EG, UK.
chorionic plate to form a system of villous trees. Where the
cytotrophoblast shell is in contact with the decidua (the maternal-
*Author for correspondence (myt25@cam.ac.uk) fetal interface), individual cytotrophoblast cells leave the shell to
M.Y.T., 0000-0002-3380-7375; A.M., 0000-0002-8388-9073
invade into decidua as extravillous trophoblast (EVT) in a process
closely resembling epithelial-mesenchymal transition (EMT). In
This is an Open Access article distributed under the terms of the Creative Commons Attribution this way, by the end of the first trimester, the blueprint of the
License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use,
distribution and reproduction in any medium provided that the original work is properly attributed. placenta is established.

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REVIEW Development (2019) 146, dev163428. doi:10.1242/dev.163428

A B
LE TE

Uterine
GE lumen
mn. tr

vs
ICM

pr. syn

ac

Endometrium Decidua

C D

eec

1◦ ys

exm
cs

lac

Fig. 1. The early stages of human placental development. Diagram depicting the early steps in placenta formation following blastocyst implantation.
(A,B) The pre-lacunar stages. (C) The lacunar stage. (D) The primary villous stage. 1° ys, primary yolk sac; ac, amniotic cavity; cs, cytotrophoblastic shell;
eec, extra-embryonic coelom; exm, extra-embryonic mesoderm; GE, glandular epithelium; ICM, inner cell mass; lac, lacunae; LE, luminal epithelium;
mn. tr, mononuclear trophoblast; pr. syn, primary syncytium; TE, trophectoderm; vs, blood vessels.

Cell types of the human placenta surface area five- to seven-fold (Teasdale and Jean-Jacques, 1985).
Trophoblast cells Microinjection of fluorescein-labelled dextrans into the SCT
The major functions of the placenta are performed by trophoblast confirms that it is multinucleated with no cell borders,
cells. The appearance of the trophoblast is an important presumably to facilitate diffusion across its entire structure and
evolutionary advance that defines placental mammals. The term protect the fetus from pathogens (Gaunt and Ockleford, 1986).
‘trophoblast’ was first used by the Dutch embryologist Ambrosius Proteomic analyses of SCT membranes show that the microvilli are
Arnold Willem Hubrecht in 1889 to describe cells that transport rich in receptors for growth factors and hormones (Robinson et al.,
nutrients and form the protective barrier between mother and fetus 2009). Both the apical and basal membranes of the SCT are packed
(Pijnenborg and Vercruysse, 2013). He also observed that the with transporter proteins for amino acids and glucose, as well as
trophoblast is inherently highly invasive or ‘corrosive’, and those that efflux xenobiotics. The SCT is also a major endocrine
dependent on decidua to support its development (Hubrecht, organ, secreting hormones and proteins into the maternal circulation
DEVELOPMENT

1908). Since these early studies, a variety of human trophoblast to drive the physiological and metabolic adaptations to pregnancy.
subtypes have been identified. These include the SCT, the villous Furthermore, SCT functions as a protective immunological barrier
cytotrophoblast (VCT) and subtypes of the EVT. because it never expresses any human leukocyte antigen (HLA)
The SCT is the outer lining of the placental villi that is in direct molecules, meaning that, despite the presence of the allogeneic
contact with maternal glandular secretions and, later, with maternal fetus, circulating immune cells will not detect the SCT as ‘non-self’
blood flowing into the intervillous space (Fig. 2). It is the main site (Moffett and Loke, 2006). The SCT also expresses the neonatal Fc
of maternal/fetal exchange of gases and nutrients necessary for the receptor (FcRn) that allows transport of maternal IgG antibodies to
growth of the feto-placental unit. The SCT has a highly polarised the fetal circulation (Roopenian and Akilesh, 2007). The antibodies
epithelial layer densely covered with microvilli, which increases its that preferentially bind to FcRn on the SCT are digalactosylated

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Fig. 2. The maternal-fetal interface and

trophoblast subtypes. Cells contained
within the villi of the early first trimester
placenta and the major trophoblast
subtypes in relation to the decidua are
represented. The decidual region has
been illustrated to include the
myometrium. Synctiotrophoblast (SCT,
grey), villous cytotrophoblast (VCT,
pink), the cytotrophoblast cell column
(CCC) and extravillous trophoblast (EVT)
populations (endovascular and
interstitial EVT, orange) are indicated.
The endpoint of EVT differentiation,
placental bed giant cells, are also
indicated.

IgG1 molecules that are effective at activating fetal natural and conversion into a vessel that is capable of high conductance at
killer (NK) cells to protect the neonate before birth (Jennewein low pressure, an essential adaptation for normal pregnancy (Brosens
et al., 2019). et al., 1967). The iEVT invade as far as the inner third of the
The mononuclear VCT lies beneath the SCT on a basement myometrium (the muscular layer of the uterine wall), where it is
membrane (Fig. 2). Historically, the VCT have been considered the thought they fuse to form placental bed giant cells (Pijnenborg et al.,
‘germinative’ layer of trophoblast because they are mitotic and 1980). After the arterial transformation occurs, the eEVT move in a
express proliferative markers (Simpson et al., 1992). In early retrograde manner down the artery to form a plug that prevents
pregnancy, when they form a continuous layer, the VCT are blood entering the intervillous space until towards the end of the
cuboidal cells with a large nucleus:cytoplasm ratio. As the villous first trimester, when the full haemochorial circulation is established
trees expand, the VCT layer becomes discontinuous and covers only (Boyd and Hamilton, 1970; Burton et al., 1999; Hustin and
25% of the villous surface by term, when only a thin syncytial layer Schaaps, 1987).
separates most of the villous core from maternal blood (Benirschke
et al., 2012). Other placental cells
As the placenta enlarges, the cytotrophoblast shell becomes Besides trophoblast cells, the placenta contains a range of cells
discontinuous and cytotrophoblast cell columns (CCCs) emerge present within the stromal core of the villi, including fibroblasts,
from the distal tips of the anchoring villi in contact with the decidua immune and vascular cells (Fig. 2). These cells are all thought to be
(Fig. 2). The cells in the columns are rounded, cohesive and rich in generated from the extra-embryonic mesenchyme, the origin of
glycogen. From the shell, and later the anchoring villi, the EVT which in humans is uncertain. Suggestions are that it arises from the
migrate into the decidua along two differentiation pathways: the VCT that undergoes EMT, or that it originates from the hypoblast,
interstitial EVT (iEVT) migrate through the decidual stroma the endodermal derivative of the ICM, with contribution from the
towards the maternal spiral arteries, while the endovascular embryonic mesoderm after gastrulation (Boss et al., 2018).
trophoblast EVT (eEVT) moves down the inside of the spiral Single cell RNA-sequencing (scRNA-seq) of first-trimester villi
DEVELOPMENT

arteries (Pijnenborg et al., 1980). In the decidua, the iEVT have a shows there are at least two fibroblast populations distinguished by
pleomorphic and fusiform morphology, tetraploid nuclei, and are the presence or absence of the imprinted gene DLK1 (Kagami et al.,
non-cycling and show changes in senescence (Velicky et al., 2018). 2012; Vento-Tormo et al., 2018). DLK1+ cells have similarities to
They home towards the spiral arteries to form a cuff of surrounding pericytes and may be involved in vascular development (Liu et al.,
cells. This is associated with loss of actin in smooth muscle cells of 2018; Suryawanshi et al., 2018). The fibroblasts link up to form a
the arterial media, which is replaced by amorphous eosinophilic network of canals that feed into the extra-embryonic coelom. These
material, resulting in the histological appearance known as canals contain placental macrophages termed Hofbauer cells
‘fibrinoid’ change (Pijnenborg et al., 2006). This trophoblast- (Burton, 1987; Castellucci et al., 2000; Kaufmann et al., 1977).
mediated transformation of the artery results in loss of vasoactivity Because Hofbauer cells are the only immune cells in the placenta

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and appear in the villous core from around day 18 postcoitum, attributable to placental abnormalities and several of the genes
before there is any vascular connection to the embryo itself, they are identified using this approach are known to play a role in EMT
likely to be generated from haemangioblastic cells in extra- (Perez-Garcia et al., 2018). A better rodent model to study arterial
embryonic tissue (Boyd and Hamilton, 1970; Castellucci et al., transformation is the laboratory rat as, in this system, the trophoblast
1987). Their functions have been little investigated but they are extends deeply into the uterine wall to remodel the artery feeding the
likely to play roles in protecting the fetus from vertical infections, in placentation site (Soares et al., 2012).
influencing trophoblast and placental vascular development, and in
transferring nutrients to the extra-embryonic coelom. The vascular In vitro models
system also develops from haemangioblastic populations in the Until recently, the study of human placental development and
mesenchyme and connects to the fetus via the umbilical cord by the trophoblast specification and differentiation had been hampered by
end of the first trimester (Dempsey, 1972; Robin et al., 2009). These the lack of reliable, physiologically relevant and reproducible
immature endothelial cells have a specific pattern of gene in vitro models. While various cell lines and models are available,
expression, e.g. they express EGFL7, which is known to regulate they do not all retain key features of the equivalent cells in vivo.
vascular morphogenesis (Parker et al., 2004). However, recent advances have led to the development of more
robust in vitro systems that can be used to model human
Models for the study of human placental development placentation.
Animal models
Mammals exhibit an enormous diversity of placental strategies, Trophoblast cell lines
particularly with regard to the degree of trophoblast invasion into While a number of trophoblast cell lines exist, a major problem with
uterine tissues and the number of cell layers between the maternal using these has been the lack of consensus about how to define the
and fetal circulations (Roberts et al., 2016). Both the laboratory human trophoblast in vitro, with a wide range of features being used
mouse and primates have a haemochorial type of placenta, where in each case (King et al., 2000a). This is the issue for the various cell
the trophoblast invades through the uterine epithelium, stroma and lines that have been derived from first-trimester or term placentas,
maternal arterial walls to come into direct contact with maternal so their identity is still not clear (Feng et al., 2005; Genbacev et al.,
blood (Georgiades et al., 2002). Lesser degrees of invasion are seen 2011, 2016; Graham et al., 1993; James et al., 2007, 2015;
in carnivores with an endotheliochorial placenta, where trophoblast Perez-Garcia et al., 2018; Straszewski-Chavez et al., 2009; Takao
cells contact maternal endothelial cells. In the least invasive form, et al., 2011; Zdravkovic et al., 2015). Care must be taken, as
an epitheliochorial placenta, which is characteristic of ruminants contaminating maternal epithelial and fetal mesenchymal cells can
and ungulates, the trophoblast remains superimposed on the uterine outgrow trophoblast isolated from the placenta (Heazlewood et al.,
epithelium (Carter and Enders, 2013). 2014; Turco et al., 2018). We recently proposed that four criteria
There is no perfect experimental model to investigate human could together be used to characterise human first-trimester ( post-
placentation, even in species with haemochorial placentas (e.g. the implantation) trophoblast: the expression of genes highly expressed
laboratory mouse and non-human primates) (Carter and Pijnenborg, in trophoblast, such as TFAP2C, GATA3 and cytokeratin (KRT7);
2011). An additional problem is that, from the vantage point of an a unique pattern of HLA expression, either HLA null (VCT and
obstetrician, disorders such as pre-eclampsia in which the primary SCT) or HLA-G+, HLA-C+ but HLA-A− and HLA-B− (EVT);
defect is failure of placentation are found only in humans and very high expression of the C19MC microRNA complex; and
possibly in great apes (Carter, 2011). The differences between hypomethylation of the ELF5 promoter (Lee et al., 2016a). Indeed,
mouse and human placentation are considerable. In humans, the the analysis of ELF5 methylation and HLA class I expression
polar TE attaches to the uterus, whereas, in mice, it is the mural TE indicates that most trophoblast cell lines do not share the profile of
(which lies opposite the ICM) that implants into the uterus first, in vivo first trimester trophoblast and may instead be mesenchymal
followed by the polar TE that will give rise to the ectoplacental cone cells (Abou-Kheir et al., 2017; Hemberger et al., 2010; King et al.,
(EPC) (Georgiades et al., 2002). Moreover, EPC cells are 2000a). Undoubtedly, further improvements in defining and
mononuclear, and their invasion into the decidua occurs almost characterising human trophoblast in vitro will be made in the future.
halfway through murine gestation, unlike the initial invasion by
primary syncytium in humans. The EPC in mice forms the Choriocarcinoma cell lines
spongiotrophoblast and, although this is viewed as equivalent to Although trophoblast cell lines derived from choriocarcinomas
the EVT in humans, there is no deep interstitial invasion of the (malignant tumours of trophoblast), such as JEG3, JAR and BeWo
decidua. This type of invasion is characteristic only of humans and cells, do fulfil the four criteria defining trophoblast and have been
great apes (gorillas and chimpanzees), and is not seen in other widely used as in vitro models, their genetic signatures are quite
primates (Pijnenborg et al., 2011). Unlike the human villous unlike that of normal trophoblast (Apps et al., 2009; Poaty et al.,
placenta, the site of placental exchange in rodents is a labyrinth that 2012). BeWo cells are derived from metastatic deposits of a
has a complex tightly packed arrangement of maternal and vascular gestational choriocarcinoma that have been cultured by passaging
channels. These substantial differences in trophoblast development within the hamster cheek pouch more than 300 times. The JEG-3
DEVELOPMENT

between mouse and humans mean that findings obtained in line was subcloned from BeWo; both are hypertriploid with ∼70
studies of the mouse must be treated with a certain degree of chromosomes (Hertz, 1959; Kohler and Bridson, 1971; Pattillo and
healthy scepticism. Indeed, genome-wide expression profiling Gey, 1968). JAR cells are HLA null, like VCT; however, although
of mouse and human placentae across gestation has revealed JEG3 cells, like EVT, do express HLA-G and HLA-C, the HLA-G
clusters of genes with very different co-expression patterns (Soncin dimers characteristic of normal trophoblast are absent and only
et al., 2018). HLA-G monomers are expressed (Apps et al., 2007). This limits the
Despite these caveats, important insights can be made from experimental use of JEG3 cells as targets for maternal uterine
studying murine genetic knockout models of in utero fetal loss immune cells (NK cells and myeloid cells) that express the LILRB1
(Rossant and Cross, 2001). In ∼70% cases, the phenotype is receptor for the dimeric form of HLA-G dimers (Shiroishi et al.,

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2006). Moreover, genome-wide DNA methylation studies problems with using primary cells for experimentation are obvious;
comparing primary trophoblast to many of these cell lines show ethical permission is essential, limiting use to certain countries and
significant variability in their profiles that are likely to contribute to requiring good liaison with clinical staff. There are also several
differential expression profiles (Apps et al., 2011; Novakovic et al., difficulties relating to cell viability, degeneration of SCT and
2011). This highlights the caution needed for interpreting results reproducibility. Indeed, the innate variability between samples,
using such cell lines and the importance of validating findings by which depends on factors such as gestational age, parity,
using primary cells. Indeed, much of the controversy in the literature intercurrent disease and area of placenta sampled, means that
has arisen from using cell lines that are not representative of bona multiple experiments are needed to ensure validity of results.
fide trophoblast cells in vivo. The recent development of human embryo culture has allowed
the visualisation of human embryo reorganisation and development
Human embryonic stem cell-derived trophoblast cells beyond the stage of implantation, although this is only possible to
Another approach has been to differentiate human embryonic stem observe up to day 13 (owing to ethical constraints). The trophoblast
cells (hESCs) into trophoblast cells by culturing them in the lineages that emerge in this culture system and how well they
presence of BMP4 and inhibitors of FGF2 and TGFβ signalling recapitulate in vivo events remain still to be understood.
(Amita et al., 2013; Horii et al., 2016; Xu et al., 2002). A
comprehensive analysis of all reports using this method, and a Trophoblast stem cells and organoids
discussion of how the culture conditions (including the source of Progress has been now made in generating bona fide human
suppliers for BMP4) and different hESC lines can affect trophoblast trophoblast stem cells (hTSCs) from both trophectoderm and first-
differentiation, is available (Roberts et al., 2018). Characterisation trimester placentas (Okae et al., 2018). hTSCs are grown on
of such hESC-derived trophoblast cells using our four putative collagen IV and can be cultured long term in a medium that
trophoblast criteria is incomplete, but it has been shown that these stimulates WNT and MAPK (through EGF) signalling, and inhibits
cells exhibit partial (but not complete) hypomethylation of the ELF5 TGFβ/activin A, HDAC and Rho kinase. These cells are genetically
promoter and downregulate some of the C19MC complex stable and fulfil all four characteristics of first-trimester trophoblast.
microRNAs (Lee et al., 2016a). The formation of SCT that They cannot be derived from term placentas, in keeping with the
secretes human chorionic gonadotropin (hCG) and expresses the dramatic loss of proliferative potential of the VCT after ∼10 weeks
EVT marker, HLA-G, as well as other genes expressed by gestation and the scarcity of the VCT in term villi (Mayhew, 2014).
trophoblast, such as KRT7, GATA2/3 and TCFAP2A/C, has also By modifying the culture conditions, hTSCs can be induced to
been reported (Amita et al., 2013; Horii et al., 2016; Krendl et al., differentiate along either syncytial or extravillous lineages. They
2017). However, transcriptomic analysis of such hESC-derived also have clonal ability and a clone can generate both the SCT and
trophoblast reveals that these cells are different from both term EVT, providing the first solid evidence of the bipotentiality of
trophoblast and mesoderm lineages; this has been a subject of hTSCs, a subject of ongoing debate (Baczyk et al., 2006; Haider
discussion as some markers are expressed in both cell types et al., 2016; James et al., 2005; Lee et al., 2018). Comparison of the
(Bernardo et al., 2011; Jain et al., 2017). A defining feature of transcriptomes of hTSCs and the first-trimester trophoblast shows
trophoblast – the absence of HLA-A and HLA-B – has not yet been close similarity to the VCT. However, the exact anatomical location
analysed in these cells. Thus, although this approach seems to of hTSCs in vivo in both the early placenta and the TE is still
promote some transition towards the trophoblast lineage, the unclear. Likewise, while mouse TSCs can be derived from both
identity of the trophoblast cells generated and the cells they are the TE and the chorion prior to E11.5, and can be differentiated
most similar to in vivo is still unclear. One possibility is that hESC- to all trophoblast populations, further investigation is needed to
derived trophoblast cells represent an early post-implantation understand which murine trophoblast population they resemble
trophoblast population (Roberts et al., 2018). in vivo and how they relate to human TSCs (Natale et al., 2017;
Tanaka et al., 1998). Single cell RNA-sequencing has now been
Placental explants or primary trophoblast cells performed on the mouse mid-gestation placenta and on human term
Many labs have used explants of placenta or isolates of primary placenta, as well as on these placentas early in their development
placental cells. Explants are prepared from the villous placenta and (Liu et al., 2018; Nelson et al., 2016; Pavličev et al., 2017;
adhere to plastic or a defined matrix. Extravillous HLA-G+ cells Suryawanshi et al., 2018; Vento-Tormo et al., 2018). These studies
then move away from the tips of the villi. Preparations of primary may help elucidate the identities of the various trophoblast cultures
isolates of trophoblast and other placental cells can be obtained from and their relation to trophoblast in vivo.
early gestation or at term. At term, because the VCT is not easily hTSCs lines grow as a monolayer and therefore cannot
visible by light microscopy and only a thin layer of the SCT covers recapitulate the complex branching morphology of early placental
the villi, any placental isolates may potentially contain other cells villi. Recently, 3D cultures of human trophoblast cells, termed
from the villous core (Hofbauer cells, fibroblast and endothelial trophoblast organoids, that closely model the villous placenta and
cells) as well as maternal and fetal blood cells, together with can be differentiated into EVT have been established (Haider et al.,
attached decidual cells. In the first trimester, the villi are covered by 2018; Turco et al., 2018). Like other organoid culture systems, these
DEVELOPMENT

the SCT and an inner layer of the VCT; the latter can be isolated by organoids are likely to be transformative in advancing our
enzymatic digestion and density gradient sedimentation (Male et al., understanding of the physiology and disease of human tissues
2012). However, the cells need to be plated onto an ECM matrix as (Clevers, 2016; Huch and Koo, 2015). Trophoblast organoids grow
they adhere poorly to plastic; after overnight culture they are all for >1 year, are genetically stable and fulfil all trophoblast criteria
HLA-G+. The cells can be used within 3-4 days but within 1 week (Turco et al., 2018). They also secrete placental hormones and
become overgrown by mesenchymal contaminants, as the proteins that alter maternal metabolism, appetite and preparation for
trophoblast cells do not proliferate in vitro. Other placental cells lactation. The ability to biobank hTSC cells/organoids from
from the villous core can also be isolated, and phenotypic markers individuals with a range of possible pregnancy problems also
such as CD34 for endothelial cells can be used to verify purity. The offers great potential. We anticipate these trophoblast culture

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systems can be used to dissect the genetic and epigenetic hypomethylation and focal hypermethylation at CpG islands,
mechanisms underlying the identity of trophoblast lineages, the including at promoters of tumour suppressor genes (Nordor et al., 2017).
differentiation of SCT and EVT, and the regulation of HLA Studies of the molecular changes occurring during human
expression. Furthermore, it will be possible to test the role of many placental development have been mainly descriptive due to the
genes directly in hTSCs using knockdown or knockout approaches lack of, until recently, long-term, genetically normal human
(e.g. using siRNAs or gene editing with CRISPR/Cas9). trophoblast cell lines with which to perform functional studies.
Nonetheless, these studies have provided insights into the molecular
Bioengineering-based approaches factors that contribute to trophoblast development and placentation.
Although hTSCs and organoids represent a major step forward in Below, we discuss these studies, highlighting relevant mouse data
studying human trophoblast in vitro, there are several limitations; and, when available, data in humans, with a focus on papers that
they model only the trophoblast component of the placenta and are have validated the work using primary trophoblast cells. If other
heterogenous. Furthermore, because other cellular components of in vitro tools were used, this is specified.
the placenta play an important role in regulating their development
and function, future work is necessary to build more complex Establishment of the trophoblast lineage
models. Microfluidics approaches that have been previously used to In mice, a hierarchy of transcription factors (TFs) drives TE lineage
model human placental functions, such as transport and invasion, commitment. This process begins when, at the morula stage of
have also shown promise (Abbas et al., 2017; Lee et al., 2016b). preimplantation development, Hippo signalling upregulates Tead4
More recently, a variety of bioengineering systems such as the in a subset of cells, which then drives Cdx2 expression in the future
use of biomimetic scaffolds and synthetic hydrogels are being TE (Nishioka et al., 2009; Yagi et al., 2007). Tcfap2c is already
implemented for other organoid systems to generate improved expressed at the eight-cell stage and also regulates Cdx2 expression
tissue-like models (Takebe and Wells, 2019). These approaches by binding to its enhancer (Cao et al., 2015). Cdx2 represses Oct4 to
have the added advantage of providing mechanical and signalling maintain its own expression while Gata2/Gata3 act in parallel to
cues that further control cell behaviour and function while ensure trophoblast development (Home et al., 2017; Ralston et al.,
improving reproducibility (Yin et al., 2016). These systems could 2010; Strumpf et al., 2005). The identity of the trophoblast lineage is
be adapted to study the interaction of the trophoblast with placental locked in place through the epigenetic regulation of Elf5, the
stromal components and maternal cells (such as glands or uterine expression of which is reinforced by a positive-feedback loop
NK cells) to investigate the maternal-fetal dialogue of early involving Cdx2 and Eomes (Ng et al., 2008).
pregnancy. For example, our long-term organoid culture system of In human embryos, the timing and expression pattern of these
endometrial glands will provide a tool for studying the effect of TFs is different, suggesting other regulatory mechanisms are
glands on the trophoblast and also provides an in vitro system for already in place from the earliest stage of TE specification. In
studying glandular function in women with implantation failure mice, there is clear differential expression of Oct4 and Cdx2 in the
(Turco et al., 2017). ICM and TE, respectively, while in humans, lineage segregation
between the ICM and TE occurs later in development; OCT4 is still
Molecular mechanisms underpinning human placental expressed in the TE until day 6, whereas CDX2 is only expressed at
development the blastocyst stage (Blakeley et al., 2015; Boroviak et al., 2018;
Trophoblast cells have many unique features that set them apart from Niakan and Eggan, 2013). Some ICM cells even retain TE identity
all other cell types, including global hypomethylation, differential at day 7 (Petropoulos et al., 2016; Stirparo et al., 2018). Thus, there
expression of microRNAs, unusual patterns of HLA molecule seems to be more plasticity in the human TE lineage leading up to
expression and expression of endogenous retroviral products implantation. In a pioneering study of gene editing of human
(Macaulay et al., 2017; Sadovsky et al., 2015). Imprinted genes, embryos by CRISPR/Cas9, deletion of OCT4 at the diploid zygote
preferentially expressed from one parental allele, are also important in stage compromised the development of both the ICM and the TE,
placental development and, out of the 92 human imprinted genes, 75 highlighting the molecular differences between humans and mice
are expressed in the placenta (Monk, 2015). The C19MC cluster, during these early fate decisions (Fogarty et al., 2017).
which encodes microRNAs, is paternally expressed in the trophoblast.
Although these miRNAs are expressed in hESCs, expression is much VCT and CCC formation
higher in the trophoblast (Lee et al., 2016a; Malnou et al., 2019; The molecular events occurring between implantation and villous
Noguer-Dance et al., 2010). Apart from the C19MC cluster, several formation in humans are obviously still a black box. Presumably,
other genes that are involved in reproduction are located on trophoblast populations arise from the polar TE as these are the cells
chromosome 19q13.4 (Moffett and Colucci, 2015). These include that initially implant. VCT is the proliferative compartment of the
genes encoding choriogonadotropin subunit β (CGB), pregnancy- placenta and expresses the trophoblast TFs found in mouse TSCs –
specific glycoproteins (PSGs), killer-cell immunoglobulin-like GATA2, GATA3, TFAP2C/A and TEAD4 – but not EOMES
receptors (KIRs), leukocyte immunoglobulin-like receptors (LILRs) (Mirkovic et al., 2015; Soncin et al., 2018). Indeed, four of these
and nucleotide-binding oligomerisation domain, and leucine-rich factors, GATA2, GATA3, TFAP2A and TFAP2C can induce cells
DEVELOPMENT

repeat and pyrin domain-containing family (NLRP) members. purported to be of the trophoblast lineage in human pluripotent stem
Moreover, many X chromosome genes escape X inactivation in the cells (Krendl et al., 2017). In humans, ELF5, the promoter of which
human placenta, leading to transcriptional differences between female is hypomethylated, is expressed in the VCT, similar to mouse
and male pregnancies that may explain the sex-based differences in (Hemberger et al., 2010; Lee et al., 2016a). Although the ELF5
pregnancy disorders (Gong et al., 2018; Moreira de Mello et al., 2010; promoter remains hypomethylated in the EVT, no ELF5 protein is
Vatten and Skjaerven, 2004). There are also striking similarities present so other mechanisms must regulate its expression. TP63, a
between trophoblast and tumour cells. For example, trophoblast TF that is characteristic of epithelial stem cells, is also widely
cells exhibit invasive proclivities and an ability to avoid destructive expressed in the early first-trimester VCT all the way to term (Lee
inflammatory or immune responses, as well as widespread et al., 2007). A proportion of TP63+ cells close to the chorionic

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plate also express CDX2 (Horii et al., 2016; Li et al., 2014). CDX2 et al., 1993). Thus, the SCT itself reinforces the process in a
expression is then downregulated in the VCT throughout the bulk of positive-feedback loop. Activation of PKA signalling through hCG
the placenta during the first trimester, apart from in those cells close is also coupled to phosphorylation of connexin 43 (Cx43) through
to the chorionic plate, where residual expression is seen (Soncin ezrin, which induces the opening of gap junctions and transfer of
et al., 2018). Notably, hTSCs lack CDX2 expression, questioning fusogenic signals between the VCT and SCT (Frendo et al., 2003b;
whether this is a marker of trophoblast stem cells in the human Pidoux et al., 2014). Several other factors may also be involved in
placenta. syncytialisation, such as the MAPK signalling pathway, which acts
The VCT also express a range of surface markers, such as EGFR, through EGF and EGFR, GM-CSF signalling and activin A
MET and specific members of the Wnt family (Jokhi et al., 1994; signalling, which acts through SMAD4 (Garcia-Lloret et al.,
Kauma et al., 1997; Sonderegger et al., 2007), that distinguishes it 1994; Gerbaud et al., 2011; Morrish et al., 1987). On the other
from other trophoblast populations. The importance of these hand, TGFβ produced by the SCT seems to provide a signal that
signalling pathways for VCT proliferation has been confirmed by balances the syncytialisation process by inhibiting fusion (Morrish
the isolation of proliferative trophoblast (Haider et al., 2018; Okae et al., 1991). Notably, interferons induced by infection act to prevent
et al., 2018; Turco et al., 2018). Proliferative cells are also present in infection of other neighbouring cells. One interferon-stimulated
CCCs early in the first trimester, and these gradually decrease in gene, IFITM3, inhibits syncytin-mediated fusion of trophoblast into
number and are mostly localised to the base of CCCs by ∼12 weeks. SCT and this may cause pregnancy failure in infections such as
This ‘generative niche’ was noticed >50 years ago and more CMV or rubella (Buchrieser et al., 2019).
recently trophoblast cells in this location were shown to have a
distinct transcriptome compared with the VCT and EVT, expressing EVT differentiation
Notch1, ITGA2 and CD31 (Haider et al., 2014; Lee et al., 2018; Global transcriptomic studies show clear differences between the
Pattillo et al., 1968). Notch1 expression becomes restricted to the VCT and EVT (Apps et al., 2011; Lee et al., 2018; Tilburgs et al.,
base of the CCC by the end of the first trimester (Haider et al., 2016). 2015). When the EVT migrates out from the CCCs into the decidua,
Whether cells in this niche generate EVT alone or both EVT and features characteristic of EMT are observed. These include: a loss of
VCT is unknown. epithelial characteristics, including downregulation of E-cadherin
and tight junction proteins such as ZO-1; a change in apical-basal
SCT differentiation polarity; a switch in integrin expression, from laminin-binding
SCT covering the villi is terminally differentiated and SCT integrin α6β4 to fibronectin-binding integrins α1β1 and α1β5; an
fragments are continuously shed into the maternal circulation. increase in cell size; and an accumulation of glycogen (Damsky
Placental explants, primary trophoblast cells that can fuse in vitro et al., 1992, 1994; Marzioni et al., 2001). The EVT also upregulates
and the BeWo choriocarcinoma line have been used to study the endothelial markers such as the laminin α4 receptor MCAM, VE-
process of SCT regeneration from the underlying VCT. These cadherin and the metalloproteinases MMP2, MMP3 and MMP9
studies have revealed human endogenous retroviral proteins (Fisher, 1989; Xu et al., 2000; Zhou et al., 1997). A defining feature
(HERVs), such as SYNCYTIN-1 (HERV-W env), as key of EVT differentiation is upregulation of the non-classical MHC
regulators of this process (Mi et al., 2000). Retroviral elements are class I molecule HLA-G, as well as HLA-C (King et al., 1996,
viral gene remnants that have been integrated into the human 2000b; Kovats et al., 1990). The expression of several growth factor
genome and co-opted as either promoters or enhancers and, in the receptors also changes: EGFR, MET (the receptor for HGF),
case of placenta, encode retroviral proteins. Around 8% of the prolactin receptor (PRLR) and the BMP receptor BMPR1A are
human genome contains HERVs (McPherson et al., 2001). The downregulated, while ERBB2 is upregulated (Jokhi et al., 1994;
placenta is a particularly favourable site for HERV expression and Kauma et al., 1999; Ladines-Llave et al., 2013; Stefanoska et al.,
different species have co-opted different retroviral families for the 2013; Tilburgs et al., 2015). All of these changes are accompanied
same function (Frendo et al., 2003a; Harris, 1991). by loss of proliferation, as assessed by Ki67 expression, and a
A key step in syncytialisation is the acquisition of fusion change in expression of cell cycle regulators (Chan et al., 1999;
competence, which requires the VCT to exit the cell cycle. Whether Genbacev et al., 2000). EVT differentiation is also characterised by
the signal that triggers this event originates from the SCT or VCT, endoreduplication, resulting in tetraploid cells (Velicky et al., 2018).
and whether the same process occurs after syncytial damage, is EVT cells themselves produce several proteins and hormones:
unknown. VCT cells upregulate fusion genes, and this is followed TGFβ1, follistatin and hyper-glycolsylated hCG. Activin A has a
by disintegration of cell membranes and incorporation of the stimulatory effect on the differentiation on EVT differentiation in
cytoplasm into the SCT. Using choriocarcinoma lines, a crucial placental explants, in complete contrast to the mouse, where it
event occurring during this process was identified as an increase in maintains trophoblast stem cell proliferation (Caniggia et al., 1997;
cyclic AMP through protein kinase A (PKA) signalling, which Erlebacher et al., 2004).
upregulates GCM1 expression and in turn induces expression of the The endpoint of iEVT differentiation is fusion to placental bed
fusion proteins syncytin 1 and syncytin 2 (Knerr et al., 2005; Liang giant cells. These large multinucleated cells produce human
et al., 2010). The final step in syncytialisation requires fusion of the placental lactogen and are present deep in the decidua basalis and
DEVELOPMENT

cell membranes and intermingling of the cellular contents (Gerbaud the inner one-third of the myometrium – the normal limit of iEVT
and Pidoux, 2015). infiltration (Al-Lamki et al., 1999; Boyd and Hamilton, 1970;
Syncytins use specific receptors, ASCT2 (for syncytin 1) and Brosens et al., 1967; Kurman et al., 1984). The signals and TFs
MFSD2 (for syncytin 2), to allow fusion of the VCT to the SCT driving EVT differentiation into either eEVT or iEVT are unknown.
(Esnault et al., 2008; Hayward et al., 2007). ASCT2 is restricted to The cells in the EVT plugs in the spiral arteries are morphologically
VCT while MFSD2 is expressed on the SCT. Furthermore, SCT very different from iEVT cells (Pijnenborg, 1996). Only two clear
produces hCG, which, after binding to the LH-CG receptor, induces phenotypic differences have been defined so far: eEVT express
cyclic AMP signalling and thereby promotes the upregulation of CD56 (NCAM) and, because occasional CD56+ cells appear within
syncytial genes, as well as that of hCG (Pidoux et al., 2007; Shi the shell overlying the openings of the arteries, it is thought that

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REVIEW Development (2019) 146, dev163428. doi:10.1242/dev.163428

signals from maternal blood may stimulate eEVT differentiation Young, 2017). This is an important issue because evidence to
(Burrows et al., 1994; Kam et al., 1999). In contrast, iEVT suggest that defective decidualisation is an antecedent of pregnancy
specifically express placenta-specific protein 8, PLAC8, which is disorders is growing (Conrad et al., 2017; Garrido-Gomez et al.,
known to be involved in EMT (Chang et al., 2018). 2017). Maternal nutrition, extremes of reproductive life, low or high
Owing to the presence of eEVT plugs in the spiral arteries, the BMI, endocrine disorders (e.g. thyroid disease) and diabetes can all
conceptus is in a physiologically low-oxygen environment before affect the cycling of a healthy endometrium but it is unclear how
∼10 weeks gestation; this is not be confused with hypoxia, which these affect decidualisation and embryo receptivity.
refers to a non-physiological state of insufficient oxygen. The
metabolic consequences of this are a reliance on glycolysis, Uterine leukocytes and other immune cells
preservation of carbon skeletons used for synthesis of cellular The dominant immune cells in the first trimester are a type of innate
components and protection from damage by free radicals (Burton lymphoid cell known as uterine natural killer (uNK) cells (Bulmer
et al., 2017). The subsequent dissolution of the eEVT plugs (at and Lash, 2015; Moffett and Colucci, 2015; Vacca et al., 2019).
∼10 weeks) and the onset of the maternal circulation correlate with These cells make up ∼70% of immune cells in the uterine
changes in oxygen levels, from 2.5% to around 8% (Jauniaux et al., environment, while macrophages account for ∼20% and T cells for
2000; Rodesch et al., 1992). The onset of circulation occurs more in ∼10%. B cells and mast cells are virtually absent, and neutrophils
the centre than at the periphery of the placentation site because of the are also sparse (Vento-Tormo et al., 2018). This is therefore an
more pronounced trophoblast invasion and arterial transformation unusual immune environment where cells of the innate rather than
centrally. The villi regress at the periphery, resulting in formation of the adaptive (T and B cells) immune system predominate. Our
the chorion laeve (fetal membranes composed of amnion and recent scRNA-seq study of first trimester decidual cells defined
chorion superimposed on decidua parietalis) and the definitive three sub-populations of uNK cells, all with specific
placenta (Burton, 2009). Disturbance of this orderly process can immunomodulatory signatures and expressing a range of receptors
result in miscarriage and abnormal placental membranes, for the EVT (Vento-Tormo et al., 2018). Their functional roles await
predisposing to preterm labour and placental abruption (Burton investigation.
and Jauniaux, 2017; Jauniaux et al., 2000). The hypoxic As mentioned, EVT cells have been compared to tumour cells
environment of the first few weeks of gestation might induce due to their invasive properties. However, unlike tumour cells,
EVT differentiation through HIF1A; indeed, more HLA-G+ EVT the behaviour of trophoblast cells within the decidual
are generated in vitro when cultured in 2% O2 with upregulation of microenvironment is controlled. Accordingly, necrosis of the
the imprinted bHLH factor ASCL2, a WNT target involved in EMT decidua is not seen as the EVT migrates deep into the tissue,
(Wakeland et al., 2017). apart from in the enigmatic Nitabuch’s layer, a thin rim of fibrinoid
tissue subjacent to the shell at the maternal-fetal boundary (Boyd
Regulation of placental development by the decidua and Hamilton, 1970). Furthermore, although placentation is often
The human placenta develops within the endometrium, which is considered as an inflammatory process (Chavan et al., 2017), the
transformed into decidua during pregnancy under the influence of defining classical features of inflammation (neutrophil infiltration
progesterone secreted by the corpus luteum (Gellersen and Brosens, followed by granulation tissue, inflammatory cells, capillary
2014). The endometrium is highly dynamic and undergoes cyclical angiogenesis and fibrosis) are always absent. Our scRNA-seq
regeneration, differentiation and shedding during the menstrual study predicts several mechanisms that could explain why
cycle under the control of hormones of the ovarian-pituitary axis. In inflammatory or adaptive immune responses are less likely to
humans, features of decidualisation ( pre-decidual change) are seen occur in this specialised environment (Vento-Tormo et al., 2018).
after the mid-secretory phase of the menstrual cycle and begin
around the spiral arteries (Kurman, 2002). Following implantation, Abnormal placental development and pregnancy
proper decidualisation of the endometrium then plays a key role in complications
the development of the placenta and is likely to involve all the major Many complications of pregnancy have their origins in abnormal
cellular elements of the endometrium: glands, vessels, stromal cells development of the placenta in the first trimester (Smith, 2010).
and immune cells. These include pre-eclampsia, fetal growth restriction (FGR),
unexplained stillbirth, placental abruption and preterm labour;
Endometrial glands and stromal cells these complications are known collectively as the great obstetric
The endometrial glands play a key role in embryo implantation and syndromes (GOSs) (Brosens et al., 2011). These conditions are
development of the placenta in mice and domestic species (Spencer responsible for a high proportion of maternal and neonatal
et al., 2019). Glands become hypersecretory in early pregnancy, morbidity and mortality seen in all populations, but particularly in
exhibiting a characteristic appearance known as the Arias-Stella sub-Saharan Africa (Graham et al., 2016).
reaction (Arias-Stella, 1954). In humans, the conceptus depends on Defective trophoblast invasion is the ultimate cause of the GOSs.
glandular secretions as the source of histotrophic nutrition during Trophoblast cells invade into the decidua to gain access to the
the early weeks of pregnancy, when the endovascular plugs only maternal blood supply and successful EVT transformation of ∼30-
DEVELOPMENT

allow seepage of maternal blood into the intervillous space (Burton 40 spiral arteries deep into the myometrium is essential for normal
et al., 2002, 2007). fetal growth and development (Burton et al., 2009; Collins et al.,
The stromal cells of the endometrium also produce a wide range 2012). If the arteries are not sufficiently converted and retain their
of growth factors that stimulate the glands. As they decidualise, the contractile media, there is disordered perfusion of blood flow into
stromal cells secrete a rim of basement membrane proteins, the intervillous space. This, together with an inadequate supply of
fibronectin and laminin, which provides a scaffold for the EVT to nutrients and oxygen, reduces the progressive branching of the
move through (Aplin et al., 1988). Exactly what defines the villous tree as gestation proceeds, reducing the surface area available
characteristics of a receptive decidualised endometrium that will for exchange, with the possible outcome of FGR and stillbirth. In
support the developing placenta is still unclear (Koot et al., 2016; addition, if the process of regression of the chorion frondiosum to

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REVIEW Development (2019) 146, dev163428. doi:10.1242/dev.163428

Fig. 3. Emerging technologies for the study of

Cell culture technologies Omics technologies human trophoblast biology and placental
development. New techniques that can be applied
Human embryo culture Single cell RNA profiling to investigate the fundamental biology of human
Human trophoblast stem cells Spatial transcriptomics placentation are shown. These include various cell
Trophoblast organoids Methylome culture technologies, omics techniques, advanced
Endometrial organoids imaging approaches and analyses of biomarkers for
CyTOF
Microfluidics clinical use.
Proteomics
Crispr/Cas9 genome editing
Metabolomics
Synthetic matrices

Advanced imaging Pregnancy biomarkers

techniques for clinical use

MRI
3D Doppler Maternal circulating markers
Computational modelling Exosomes
Imaging mass cytometry microRNAs

form the chorion laeve does not occur correctly, the chorionic ‘fibrinoid’ appearance to that seen when the trophoblast transforms
membranes can separate prematurely, resulting in placental the spiral arterial media. Furthermore, fusion to placental bed giant
abruption or preterm labour. Pre-eclampsia results from the cells, which is normally observed at the end of EVT migration, is
release of products from the poorly perfused and stressed placenta drastically reduced (Hannon et al., 2012). Together, these findings
into the maternal circulation, triggering a systemic endothelial highlight that the territorial boundary between placenta and mother
disorder (Burton et al., 2019; Roberts and Redman, 1993). Thus, the needs to be finely controlled and that this balancing act is mediated
exact clinical outcome of defective trophoblast invasion depends on by the decidua.
the extent of arterial invasion and the number of arteries invaded. Memory and specificity are features of immune responses. These
Because deep trophoblast invasion into the uterus is a feature seen have resonance in pre-eclampsia and point to a role of the immune
only in humans and great apes, defective transformation of arteries system in its pathogenesis (Wikström et al., 2012). Pre-eclampsia
has been hard to characterise and hence diagnose early in exhibit some level of memory, whereby immune cells ‘remember’
pregnancy. A number of clinical measurements are being encounters with specific pathogens and subsequently respond
developed to overcome this, such as uterine artery Doppler differently to the same pathogen (Sun et al., 2014). Indeed, the
velocimetry, which measures the resistance to blood flow and is incidence of pre-eclampsia is highest in first pregnancies and then
thus an indirect readout of the degree of spiral artery remodelling falls if the second pregnancy is with the same partner. There are also
(O’Gorman et al., 2017). As the EVT moves deeper, expression of partner-specific effects on pre-eclampsia, as seen in pregnancies in
pregnancy-associated plasma protein-A (PAPPA-A) increases, and women who change partners (Wikström et al., 2012). These
measurement of this protein in maternal serum in the first trimester epidemiological findings led us to explore the interactions between
is a useful predictor of a GOS (Gaccioli et al., 2018). The ratio of KIR and their HLA-C ligands in uNKs. This study revealed that
soluble fms-like tyrosine kinase 1 (Sflt1) to placental growth factor both the maternal KIR and fetal HLA-C ligands are highly
(PlGF) is also elevated in women before they develop the clinical polymorphic, and particular KIR/HLA-C combinations are found
symptoms of pre-eclampsia (Zeisler et al., 2016); as such, a low ratio in pregnancies complicated by pre-eclampsia and FGR (Moffett and
can be used to predict the women who will not go on to develop the Colucci, 2015). Exactly how these genetic findings translate into
syndrome in the following week. Another approach is to screen functions of uNK cells is still unknown; the uNK cells might affect
maternal blood for biomarkers that reflect placental function. These EVT invasion directly or act indirectly on the arteries or glands.
include miRNAs, exosomes, free DNA, proteins and short non-
coding RNAs (Barchitta et al., 2017; Gaccioli et al., 2018; Rolnik Conclusions and future directions
et al., 2018; Tsang et al., 2017; Yoffe et al., 2018). The benefits of Although much has been learnt recently, there are still many
these screening tests in low risk populations are still not obvious, questions to answer about human placental development: the
particularly as the only intervention possible at present is early specification of the TE lineage, the identity of TSCs, differentiation
delivery with obvious risk to the neonate; moreover, some reports into the two main lineages and the impact of the maternal
suggest that these screening tests may actually be harmful (Monier environment on both the VCT and EVT. Although recent studies
et al., 2015). have revealed specific and unique features of human trophoblast and
DEVELOPMENT

As defective trophoblast invasion is the ultimate cause of the the decidual microenvironment, it is clear that the development of
GOS, it is important to understand how EVT invasion into the the human placenta is complex and challenging to study. However,
uterus is regulated. A role for decidua in preventing placental cells new techniques are now being used to study human placental
from invading too far is clear from clinical reports where the development to overcome these challenges. These include scRNA-
placenta implants on a site where decidua is deficient or absent seq, spatial transcriptomics, epigenetics, miRNA expression,
(Jauniaux et al., 2018). This can occur in the lower segment of the advanced imaging techniques and organoid cultures (Fig. 3). This
uterus close to the cervix or over a caesarean section scar from a is therefore an exciting time for placental research as we begin to
previous pregnancy. In these situations, the EVT penetrates into the address the fundamental issue of how the human placenta develops
myometrium and destroys the smooth muscle cells with a similar in normal and abnormal pregnancies.

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REVIEW Development (2019) 146, dev163428. doi:10.1242/dev.163428

Blakeley, P., Fogarty, N. M. E., del Valle, I., Wamaitha, S. E., Hu, T. X., Elder, K.,
Snell, P., Christie, L., Robson, P. and Niakan, K. K. (2015). Defining the three
Further resources cell lineages of the human blastocyst by single-cell RNA-seq. Development 142,
Relevant human placental specimens are held in the Centre for 3613-3613. doi:10.1242/dev.131235
Trophoblast Research (CTR) at the University of Cambridge (www. Boroviak, T., Stirparo, G. G., Dietmann, S., Hernando-Herraez, I., Mohammed,
trophoblast.cam.ac.uk). Applications to view these in person can be made H., Reik, W., Smith, A., Sasaki, E., Nichols, J. and Bertone, P. (2018). Single
to the CTR. The Human Placenta Project (HPP) of the Eunice Kennedy cell transcriptome analysis of human, marmoset and mouse embryos reveals
Shriver National Institute of Child Health and Human Development is a common and divergent features of preimplantation development. Development
collaborative research effort towards understanding the role of the 145, dev167833. doi:10.1242/dev.167833
Boss, A. L., Chamley, L. W. and James, J. L. (2018). Placental formation in early
placenta in health and disease (www.nichd.nih.gov/research/supported/
pregnancy: how is the centre of the placenta made? Hum. Reprod. Update 24,
HPP/default).
750-760. doi:10.1093/humupd/dmy030
Boyd, J. D. and Hamilton, W. J. (1970). The Human Placenta. w. Heffer & Sons Ltd.
Brosens, I., Robertson, W. B. and Dixon, H. G. (1967). The physiological response
of the vessels of the placental bed to normal pregnancy. J. Pathol. Bacteriol. 93,
569-579. doi:10.1002/path.1700930218
Acknowledgements
Brosens, I., Pijnenborg, R., Vercruysse, L. and Romero, R. (2011). The “Great
We thank Graham Burton, Charlie Loke and Jenny Nichols for helpful discussions,
Obstetrical Syndromes” are associated with disorders of deep placentation.
and Philip M. Ball for the figures. We apologise to colleagues whose work we could Am. J. Obstet. Gynecol. 204, 193-201. doi:10.1016/j.ajog.2010.08.009
not cite due to space limitations. Buchrieser, J., Degrelle, S. A., Couderc, T., Nevers, Q., Disson, O., Manet, C.,
Donahue, D. A., Porrot, F., Hillion, K.-H., Perthame, E. et al. (2019). IFITM
Competing interests proteins inhibit placental syncytiotrophoblast formation and promote fetal demise.
The authors declare no competing or financial interests. Science 365, 176-180. doi:10.3410/f.736185701.793563844
Bulmer, J. N. and Lash, G. E. (2015). The Role of Uterine NK Cells in Normal
Funding Reproduction and Reproductive Disorders, pp. 95-126. Cham: Springer.
The authors’ research is funded by the Wellcome Trust (200841/Z/16/Z), the Medical Burrows, T. D., King, A. and Loke, Y. W. (1994). Expression of adhesion molecules
by endovascular trophoblast and decidual endothelial cells: implications for
Research Council (MR/P001092/1), the Centre for Trophoblast Research and the
vascular invasion during implantation. Placenta 15, 21-33. doi:10.1016/S0143-
Royal Society (RGF\R1\180028). M.Y.T. is a Royal Society Dorothy Hodgkin Fellow
4004(05)80233-4
(DH160216).
Burton, G. J. (1987). The fine structure of the human placental villus as revealed by
scanning electron microscopy. Scanning Microsc. 1, 1811-1828.
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