2013 Placental Trophoblast Cell Differentiation

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Placental trophoblast cell differentiation:

Physiological regulation and pathological
relevance to preeclampsia
Article in Molecular Aspects of Medicine December 2012
DOI: 10.1016/j.mam.2012.12.008 Source: PubMed
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Lei Ji
Jelena Brkic
Chinese Academy of Sciences
York University
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Guodong Fu
Hui-xia Yang
Mount Sinai Hospital, Toronto
Beijing Medical University
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Molecular Aspects of Medicine 34 (2013) 9811023
Contents lists available at SciVerse ScienceDirect
Molecular Aspects of Medicine

journal homepage: www.elsevier.com/locate/mam
Review
Placental trophoblast cell differentiation: Physiological

regulation and pathological relevance to preeclampsia
Lei Ji a,1, Jelena Brkic b,1, Ming Liu a,1, Guodong Fu b, Chun Peng b,, Yan-Ling Wang a,
a
b
State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, Peoples Republic of China
Department of Biology, York University, Toronto, Ontario, Canada M3J 1P3
a r t i c l e
i n f o
Article history:
Available online 28 December 2012
Keywords:
Placental development
Trophoblast differentiation
Invasive pathway
Preeclampsia
Biomarker
a b s t r a c t
The placenta is a transient organ that forms during pregnancy to support the growth and
development of the fetus. During human placental development, trophoblast cells differentiate through two major pathways. In the villous pathway, cytotrophoblast cells fuse to
form multinucleated syncytiotrophoblast. In the extravillous pathway, cytotrophoblast
cells acquire an invasive phenotype and differentiate into either (1) interstitial extravillous
trophoblasts, which invade the decidua and a portion of the myometrium, or (2) endovascular extravillous trophoblasts, which remodel the maternal vasculature. These differentiation events are tightly controlled by the interplay of oxygen tension, transcription factors,
hormones, growth factors, and other signaling molecules. More recently, microRNAs have
been implicated in this regulatory process. Abnormal placental development, particularly
the limited invasion of trophoblast cells into the uterus and the subsequent failure of
the remodeling of maternal spiral arteries, is believed to cause preeclampsia, a severe pregnancy related disorder characterized by hypertension and proteinuria. Oxidative stress, the
abnormal production and/or function of signaling molecules, as well as aberrant microRNAs expression have been suggested to participate in the pathogenesis of preeclampsia.
Several potential biomarkers for preeclampsia have been identied, creating new opportunities for the development of strategies to diagnose, prevent, and treat this disorder.
Crown Copyright 2012 Published by Elsevier Ltd. All rights reserved.
Contents
1.
2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pathways of human placental trophoblast cell differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
Trophoblast cell fusion differentiation pathway toward multinucleated syncytiotrophoblasts . . . . . . . . . . . . . . . . . . .
2.1.1.
Formation of STBs is downstream of the fusogenic event . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.2.
Balance between tissue renewal and tissue loss in the syncytial layer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Trophoblast cell differentiation along the invasive pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.1.
Interstitial trophoblast cells invasion into uterine stroma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.2.
Endovascular trophoblast cells remodeling of uterine spiral arteries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.3.
Endoglandular trophoblast cells uterine glandular remodeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.
Summary of the characteristics of the various trophoblast subtypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Corresponding authors. Addresses: Department of Biology, York University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3. Tel.: +1 416 736
2100x40558; fax: +1 416 736 5698 (C. Peng), State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen
West Road, Chaoyang District, Beijing 100101, Peoples Republic of China. Tel./fax: +86 10 64807195 (Y.-L. Wang).
E-mail addresses: cpeng@yorku.ca (C. Peng), wangyl@ioz.ac.cn (Y.-L. Wang).
1
Equal contribution.
0098-2997/$ - see front matter Crown Copyright 2012 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.mam.2012.12.008
982
3.
4.
5.
6.
L. Ji et al. / Molecular Aspects of Medicine 34 (2013) 9811023
Model systems for examining placental trophoblast cell functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 989

3.1.
Primary cultures of human trophoblast cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 989
3.2.
Trophoblastic cell lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 989
3.3.
Trophoblast stem cell lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 990
3.4.
Co-culture of trophoblast cells/placental villi and other cell types or explants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 990
3.5.
Animal models for investigating placental development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 991
3.5.1.
Placenta-specific transgene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 991
3.5.2.
Virus-mediated genetic manipulation in rodent placenta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 992
3.5.3.
Transplantation of cells into placenta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 992
Regulatory mechanisms involved in human trophoblast cell differentiation toward the invasive pathway . . . . . . . . . . . . . . . . 992
4.1.
Oxygen tension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 993
4.1.1.
Oxygen tension regulates the balance between trophoblast cell proliferation and invasion. . . . . . . . . . . . . . . . 993
4.1.2.
Hypoxia induces trophoblast cell apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 994
4.1.3.
HIFs are oxygen sensors in placenta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 994
4.1.4.
Role of HIFs in normoxic condition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 995
4.2.
Growth factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 995
4.2.1.
VEGF family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 995
4.2.2.
TGF-b superfamily . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 996
4.2.3.
HGF/c-Met signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 997
4.2.4.
Notch signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 998
4.2.5.
Wnt signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 999
4.3.
Hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 999
4.3.1.
GnRH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 999
4.3.2.
hCG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1000
4.4.
MicroRNAs (miRNAs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1000
4.4.1.
Expression of miRNAs in the human placenta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1000
4.4.2.
Regulation of miRNA expression in trophoblast cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1001
4.4.3.
Modulation of trophoblast cellular activities by miRNAs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1001
4.4.4.
The imprinted H19 gene functions via encoding miR-675 in the placenta. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1003
Implications for the pathogenesis of preeclampsia (PE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1004
5.1.
Overview of Preeclampsia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1004
5.2.
The injury from hypoxiareoxygenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1004
5.3.
Placental factors that contribute to preeclampsia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1005
5.3.1.
Angiogenic and anti-angiogenic factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1005
5.3.2.
Catechol-O-methyltransferase (COMT) and 2-methoxyestradiol (2-ME) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1006
5.3.3.
Corin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1006
5.3.4.
Notch signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1006
5.3.5.
Nodal, Activin A and Inhibin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1007
5.3.6.
HGF/c-Met. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1007
5.3.7.
hCG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1007
5.3.8.
miRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1007
5.4.
Genetic alterations in association with PE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1008
Summary and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1009
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1009
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1010
1. Introduction
The placenta is a transient organ that has a multitude of critical roles in the maintenance and protection of the developing
fetus throughout the entire pregnancy (Anin et al., 2004). The multifaceted nature of the placenta has been demonstrated,
through animal models and studies of human pregnancies, to be vital for the survival and health of the embryo and the
mother (Rossant and Cross, 2001). Serving as the interface between the fetal and maternal environments, the placenta is involved in the exchange of gases, nutrients and waste products between the mother and the growing fetus (Cross, 1998).
Moreover, the placenta serves as an endocrine organ, producing several pregnancy-associated hormones and growth factors
and ensuring the protection of the fetus from maternal immune attack (Regnault et al., 2002).
Embryonic implantation begins with the adherence of the blastocyst to the uterine lumen epithelium. The blastocyst then
invades deep into the uterine wall via an interaction between trophoblasts and the uterine endometrium. In humans, this
initial invasion is characterized by the penetration of the uterine epithelium by the multinucleated syncytiotrophoblast
(STB) (Popek, 1999). The human placenta has been demonstrated to be one of the most invasive placenta types, with trophoblast cells invading into the inner third of the myometrium along with the maternal vasculature (Norwitz, 2007). Fetomaternal contact reduces to one trophoblastic cell layer by the third trimester (Wang and Zhao, 2010).
983
The trophectoderm of the blastocyst is the rst cell lineage that exhibits a highly differentiated function during embryonic development. In humans, highly proliferative, undifferentiated primitive cytotrophoblast (CTB) cells that are derived
from the trophectoderm give rise to differentiated trophoblast cells through two general pathways. Firstly, the mononucleated CTBs fuse into multinucleated STBs that cover the oating villi, which is surrounded by the maternal blood. STBs are
primarily involved in pregnancy-related hormone production and in the exchange of nutrients and waste between the
mother and the fetus. Secondly, CTBs proliferate to form anchoring villi that attach to the uterine wall (Knoer, 2010;
Red-Horse et al., 2004). From the cell columns of the anchoring villi, extravillous trophoblasts (EVTs) can form by detaching
from the placental villi and migrating into the decidua. A subset of these, interstitial trophoblasts (iEVTs), migrate into the
deep layer of the maternal endometrium and even into the inner third of the myometrium, thereby anchoring the fetus to
the mother. Another subset of EVTs acquires endothelial-like characteristics and become endovascular trophoblasts (enEVTs). They penetrate the uterine spiral arteries and replace maternal endothelial cells. In this way, the uterine spiral arteries
are remodeled into low-resistance, high-capacity utero-placental arteries that provide the increased blood ow towards the
placenta that is needed to meet the requirements of the growing fetus (Lyall, 2006).
Trophoblast differentiation during placental development is precisely regulated by environmental factors, such as oxygen
tension within the maternalfetal interface, and by various hormones and growth factors. Recently, microRNAs (miRNAs)
have also been suggested to participate in placental development. The dysregulation of trophoblast activities, especially defects in the invasion of EVTs into the uterus and the subsequent failure in the remodeling of the maternal spiral arteries, are
the key mechanisms that underlie the development of preeclampsia, a pregnancy-associated disorder characterized by
hypertension and proteinurea (Red-Horse et al., 2004; Reynolds and Redmer, 2001; Rossant and Cross, 2001). Elucidating
the regulatory mechanism of trophoblast cell differentiation, especially differentiation toward the invasive pathway, is a
key way to explore the pathogenesis of preeclampsia and to identify reliable biomarkers that can be used as predictive or
therapeutic targets in the context of preeclampsia.
In this review, we summarize the current views on human trophoblast cell differentiation pathways and models available
for examining human trophoblast cell differentiation and functions. We concentrate on recent advances regarding the regulatory roles of oxygen tension, hormones, growth factors, and microRNAs on trophoblast differentiation toward the invasive
pathway. Lastly, the mechanisms by which the deregulation on trophoblast activities can contribute to the onset of preeclampsia is discussed.
2. Pathways of human placental trophoblast cell differentiation
Human cytotrophoblast progenitor cells differentiate toward two general pathways: villous trophoblasts and invasive
extravillous trophoblasts. Interestingly, rst trimester cytotrophoblasts, unlike their term placenta counterparts, favor the
invasive pathway rather than the syncytial pathway, suggesting that trophoblast cell differentiation is dynamic and change
throughout gestation.
2.1. Trophoblast cell fusion differentiation pathway toward multinucleated syncytiotrophoblasts
In the villous pathway, mononucleated CTBs fuse into multinucleated STBs, forming the syncytial layer that covers the
placental villous tree. These cells are intimately involved in the exchange of gases, nutrients and waste across the maternalfetal interface (Kaufmann et al., 2003). The absorptive capabilities of this layer are maximized by the presence of microvilli that considerably increase the surface area (Teasdale and Jean-Jacques, 1985). The syncytial layer also plays a major role
in the maintenance of pregnancy via the production of pregnancy-related hormones, such as human chorionic gonadotropin
(hCG) and human placental lactogen (hPL) (Graham and Lala, 1992). Additionally, STBs are in direct contact with the maternal blood and are therefore required to exhibit a degree of immune tolerance. This tolerance is achieved in large part through
their lack of expression of the classical class I human leucocyte antigens (HLA) (Nakamura, 2009).
Syncytiotrophoblasts are non-proliferative and are therefore continually replenished throughout pregnancy via the fusion
of the underlying CTB cell layer and the shedding of the aged portions of the STBs, i.e., syncytial knots (Kar et al., 2007; Richart, 1961). Interestingly, this cell fusion occurs exclusively between the CTB and the overlaying syncytium, not between
neighboring CTBs. The process of trophoblast cell fusion is poorly understood, although there is unsurpassed evidence that
supports the involvement of a number of hormones, growth factors, cytokines, membrane proteins, protein kinases, intracellular proteases and transcription factors. The roles of these factors have been thoroughly reviewed by Huppertz and Gauster (2011) and Pidoux et al. (2012).
2.1.1. Formation of STBs is downstream of the fusogenic event
It is generally accepted that trophoblast differentiation into endocrine STBs is downstream of the fusogenic event. Specically, the cascade is believed to begin with increased cAMP levels, which occurs via the activation of adenylyl cyclase
and the consequent activation of protein kinase A (PKA). The downstream activation of transcription factors, such as glial
cells missing-1 (GCM-1) and its target genes, such as the well-characterized fusion peptide synctytin-1, induces syncytialization and subsequent increase in hCG production (Baczyk et al., 2009). Syncytins are envelope proteins encoded by human
endogenous retroviral genes (HERVs) that are nearly exclusively expressed in the placenta (Potgens et al., 2004), and
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Syncitin-1 is encoded by a defective HERV, HERV-W. Among the best-characterized placenta specic HERVs, Syncitin-1 is
localized to the syncytiotrophoblast cells with its expression maintained throughout gestation (Mi et al., 2000; Okahara
et al., 2004). Evidence from studies in BeWo cells, which is a well-established human choriocarcinoma cell line and
in vitro model of trophoblast fusion, conclusively demonstrate the role of syncytin-1 in triggering cell fusion (Knerr et al.,
2005; Mi et al., 2000). Another placenta specic HERV from the HERV-FRD family, Syncytin-2, also demonstrates a potential
role as a fusogenic protein. Unlike Syncitin-1, its localization is restricted to the villous cytotrophoblast cells with its expression slowly decreasing throughout gestation (Blaise et al., 2003; Okahara et al., 2004). Similarly to Syncitin-1, transient transfection of Syncytin-2 is also shown to promote cell fusion in a variety of cell types. However, unlike Syncytin-1, its expression
levels signicantly decreases after STB formation (Malassine et al., 2008). Though Syncytin-1 and Syncytin-2 have similar
structure and fusogenic capabilities, their roles in regulating trophoblast differentiation are mediated through different
receptors (Blaise et al., 2003; Blond et al., 2000; Esnault et al., 2008). To date, these two HERVs show a clear evolutionary
selection and consequently the strongest evidence for trophoblast cell differentiation. However, the different localization
of the Syncytins, their receptors, and related proteins in trophoblast cells, as well as the observation that STB is not completely absent in syncytin knockout mice, raise the possibility that Syncytins are not the only fusogens that participate in
syncytialization (Dupressoir et al., 2005, 2009, 2011; Handwerger, 2010; Huppertz and Gauster, 2011). Accumulating evidences reveals that there are several other HERVs believed to be putative fusogens. For example, ERV-3, although not a placenta-specic HERV, is shown to be upregulated in forskolin-treated BeWo cells, while its overexpression is capable of
inducing the differentiated phenotype though to a lesser extent (Lin et al., 1999). The in vivo evidence for ERV-3 is not compelling, mainly due to a premature termination mutation in its transmembrane region. Several other trophoblast-specic
HERVs have been characterized in human placental tissues. HERV-Fb1 expression remains constant throughout gestation,
while HERV-H7/F(XA34) and HERV-HML6-c14 expressions are greatest at term (Okahara et al., 2004). The exact functions
of these HERVs remains unknown, however their expression in the syncytiotrophoblast subpopulation suggests potentially
similar roles to Syncitins in regulating trophoblast cell differentiation.
Recent data indicate that envelope glycoproteins of HERVs have more diverse effects in trophoblast cells than previously
thought. For instance, Syncytin-2 and ERV3 are shown to have additional immunosuppressive abilities. Their strong expression in the placenta may suggest a role in maternal tolerance which is essential at early gestation (Mangeney et al., 2007).
The ubiquitously expressed EnvP(b) and placenta-specic EnvV, two conserved HERV gene-encoded envelope proteins, have
been suggested to intervene in the placenta development, but are not required in trophoblast syncytialization (Blaise et al.,
2005; Vargas et al., 2012). Interestingly, while the other placenta-specic HERVs are localized in the cytoplasm, HERVHML6-c14 expression is localized to the nucleus. Furthermore, HERV-HML6-c14 is the only upregulated placenta-specic
HERV in tissues from women suffering from pregnancy-induced hypertension with the other HERVs being downregulated
(Kudaka et al., 2008). The functional identication of these HERVs will provide novel insights into the mechanics of human
placental development.
Alternatively, there is growing evidence for the hypothesis that the mechanisms that govern the fusogenic and endocrine
properties of STBs, although linked, are independently regulated. As demonstrated by Orendi et al., H-89, a selective inhibitor
of cAMP dependent PKA, prevented cell fusion event in BeWo cells without affecting the production of hCG. This result suggests a second, PKA-independent pathway in hCG synthesis (Orendi et al., 2010). Early studies report a promoting effect of
Activin A in hCG and progesterone secretion. However, studies of human trisomy 21 placentas reveal that Activin A is able to
stimulate the fusogenic event without increasing hormonal markers of differentiation (Pidoux et al., 2012). Leukemia inhibitory factor (LIF), which is actively secreted by the decidua, has been demonstrated to induce trophoblast cell fusion in an
hCG-dependent manner (Hambartsoumian, 1998; Sawai et al., 1995). However, a recent study demonstrated that, although
LIF contributes to forskolin-induced cell fusion in BeWo cells, it reduced hCG hormone production via a signal transducer and
activator of transcription (STAT)1 and STAT3-dependent mechanism (Leduc et al., 2012). Thus, it is possible that morphological and functional differentiation of STBs may be differentially regulated.
2.1.2. Balance between tissue renewal and tissue loss in the syncytial layer
The continual fusion of CTBs into a growing syncytial layer raises questions regarding the balance between tissue renewal
and tissue loss. Whether the syncytium continues to accumulate nuclei throughout pregnancy or if aging nuclei are selectively shed via apoptosis into the maternal circulation is widely debate, and there is a great deal of evidence to support both
claims. On one side of the debate, there is supporting evidence that aging nuclei must be shed to maintain the balance between renewal and loss as new CTBs join the STB layer. Studies that have examined the STB layer have indicated that these
nuclei become packaged into membrane bound vesicles, termed syncytial knots, and released into the maternal circulation
(Nelson, 1996; Yasuda et al., 1995). It has been suggested that it takes approximately 34 weeks following syncytial fusion
for one aging nucleus to be removed from the growing cell (Huppertz and Kingdom, 2004). Alternatively, another argument
states that the nuclei in the STB layer continue to accumulate throughout gestation and that their heterogeneous appearance
is primarily a result of their differential ages (Burton and Jones, 2009).
A large part of this debate focuses on the evidence for the role of apoptosis in trophoblast turnover. One hypothesis
proposes a two-phase apoptotic mechanism. The rst phase is described as the pre-apoptotic phase that commits the CTB
to the syncytium and is involved in the fusion process. This step is followed by a possible brief execution phase that leads
to the shedding of syncytial knots from the STB layer (Huppertz et al., 1998a; Mayhew et al., 1999). The rst phase encompasses the events of syncytial formation that appear to be conned to a subset of the CTB population. Although the precise
985
mechanism by which these cells become committed is still unclear, there is evidence that suggests the transcription factor
GCM-1 plays a key role. In situ studies of GCM-1 mRNA and protein localization in human placentas have reported a very heterogeneous pattern of expression throughout the CTB population. Interestingly, this expression pattern appears to be conserved based on mouse studies, which report no GCM-1 expression in the cells at the base of villous columns that are
destined for the invasive extravillous pathway (Baczyk et al., 2004). Further support is provided with a later study of GCM1 function. This study indicated that the silencing of GCM-1 inhibited de novo syncytialization in human oating villi cultures
(Baczyk et al., 2009). This subset of progenitor cells is believed to undergo biochemical alterations that are representative of
the early, reversible, phase of apoptosis. The activation of the initiator caspase, caspase 8, is demonstrated to be a prerequisite
for CTB fusion and has been used to visualize the syncytialization process (Black et al., 2004; Gauster et al., 2009).
The exact events of the transition from the early apoptotic events that are involved in CTB fusion to the activation of effector caspases and downstream cell death are poorly understood. Several studies have attempted to elucidate this process by
examining the expression patterns of known molecules within the apoptotic cascade. The evidence appears to suggest that
the execution phase is regulated both spatially and temporally within the STBs, resulting in the formation of syncytial knots
(Huppertz et al., 1998a; Nelson, 1996; Ratts et al., 2000; Uckan et al., 1997). However, comparative studies have reported a
higher occurrence of apoptosis in CTBs compared to STBs (Crocker et al., 2001). Most recently, Lontine et al. brought forth a
limitation to previous studies with their ndings that apoptosis in term villi occur in CTBs but not in the STB layer. They
showed that by using a membrane marker, E-cadherin, the apoptotic cells within the STB were in fact deeply integrated CTBs
that account for one third of the entire CTB population (Longtine et al., 2012). Therefore, the exact events that are involved in
trophoblast turnover and differentiation will likely be deciphered in the near future.
2.2. Trophoblast cell differentiation along the invasive pathway
In addition to the formation of oating villi is the establishment of anchoring villi, which serve to attach the placenta to
the uterine wall and to create the degree of placental perfusion that is necessary to sustain the growing fetus. The anchoring
sites are established as early as the second week of gestation and are composed of a heterogeneous population of CTBs (Vicovac et al., 1995).
A subpopulation of rapidly proliferating CTBs, which participate in the creation of the cell column bridge between the
placental villous tip and the maternal decidualized stroma, can be found at the proximal ends of the anchoring sites. Studies
of human placental and decidual explant cultures suggest that decidual contact signals the proliferative burst of CTBs to
break through the STB layer (Vicovac et al., 1995). The stability of this interaction is believed to be mediated by the upregulation of integrin a5b1 and a bronectin-rich matrix on the CTBs that are located in the distal portions of the trophoblast
cell columns (Damsky et al., 1992; Vicovac et al., 1995). There is also evidence that the L-Selectin adhesion system may participate in the formation and maintenance of the anchoring villi early in pregnancy (Prakobphol et al., 2006). At the distal
ends of the columns, CTBs exit the cell cycle and begin to lose their cellcell contacts. These CTBs detach from columns
and, as they come into contact with the decidual extracellular matrix, they differentiate into iEVTs and enEVTs, which have
distinct roles in maternal deciduas (Kemp et al., 2002).
The differentiation process within the heterogeneous trophoblast column population is unclear; however, in vitro studies
suggest that this process may be intrinsic (Knoer and Pollheimer, 2012). A large part of this differentiation process is composed of a switch in adhesion molecule proles and the production and regulation of several proteases.
2.2.1. Interstitial trophoblast cells invasion into uterine stroma
Interstitial EVTs are described as having two distinct phenotypes: large polygonal iEVTs (or X cells) and small spindleshaped iEVTs. The large iEVTs are believed to remain around the placentaldecidua transition, securing the placenta to
the uterus throughout gestation by producing a trophoblast glue that is made of a matrix-type rbrinoid (Huppertz,
2007). In contrast, the small iEVTs have been demonstrated to invade deep into the decidua as far as the inner third of
the myometrium.
Interstitial EVTs have a distinct adhesion molecule and histocompatibility antigen prole. There is a marked downregulation of integrin a6b4 and an upregulation of the bronectin integrin, a5b1, within the distal part of the cell column. The
collegen IV integrin, a1b1, is expressed at higher levels in the iEVTs that invade deeply in the decidua (Damsky and Fisher,
1998; Damsky et al., 1992). The downregulation of E-cadherin is observed in the distal portion of cell column and is believed
to contribute both to the loss of cellcell contact and to the increase in invasiveness in iEVTs (Zhou et al., 1997b). Consistent
with their invasive phenotype, iEVTs have been demonstrated to secrete several proteases that facilitate the breakdown of
the decidual extracellular matrix. Specically, the immunolocalization of urokinase-type plasminogen activator (uPA) and
several matrix metalloproteinases (MMPs), most notably gelatinases MMP-2 and MMP-9, have been reported. Interestingly,
the inhibitors of these enzymes, plasminogen activator inhibitor 1/2 (PAI-1/2) and tissue inhibitor of metalloproteinase-1
(TIMP-1), are observed to be co-localized to iEVTs, indicating a certain degree of ne-tuning with respect to limiting the invasiveness of these cells (Hofmann et al., 1994; Huppertz et al., 1998a). Furthermore, unlike the villous CTBs, iEVTs express HLA
class I major histocompatibility complex (MHC) antigens. Specically, these cells express HLA-E, trophoblast specic HLA-G
and the polymorphic HLA-C (Loke et al., 1997; Sasagawa et al., 1987; Trowsdale and Moffett, 2008).
How the movement of iEVTs through the immune cell-dense decidua during the rst trimester does not elicit a maternal
immune response remains a great topic of discussion (Hammer, 2011). Most notable in this regard is the interaction of iEVTs
986
with decidua-specic natural killer (dNK) cells, which differ from the NK cells that are found in the peripheral blood, and
which make up the largest proportion of leukocytes at the implantation site. The receptor proles on dNK cells allow them
to interact with all three of the HLA antigens that are expressed on iEVTs. Likewise, all of these HLA antigens contribute to
the immune tolerance that is observed at the maternal-fetal interface. HLA-E binds with higher afnity to the more abundant
inhibitor dNK receptor suggesting its role in inhibiting cytotoxicity of dNKs (Hammer, 2011; King et al., 2000a; Moffett-King,
2002). Work by Apps et al. indicates that the inhibitory leukocyte immunoglobulin-like receptor (LILR), LILRB1 has highest
afnity for dimerized HLA-G on iEVTs, suggesting that this placenta-specic process modulates the maternal immune response (Apps et al., 2007). Lastly, studies of the interaction between HLA-C and corresponding dNK killer cell immunoglobulin-like receptors (KIRs) provides important evidence regarding the importance of this interaction in fetal growth and blood
supply (Trowsdale and Moffett, 2008).
The invading iEVTs nally differentiate into placental bed giant cells. Similar to STBs, these cells have the capacity to produce both hPL and hCG, suggesting their role in the maintenance of normal pregnancy. Furthermore, these giant cells produce protease inhibitors that may be involved in limiting EVT invasion past the myometrium (al-Lamki et al., 1999).
2.2.2. Endovascular trophoblast cells remodeling of uterine spiral arteries
Placental circulation is established between 8 and 12 weeks of gestation, with the spiral arteries being fully remodeled by
approximately 2022 weeks. Endovascular EVTs are believed to play key roles in this remodeling process (Lyall, 2006). Decidual biopsies indicate that enEVTs invade maternal vasculature in the decidua and the inner third portion of the myometrium
(Burton et al., 2009). The remodeling of the spiral arteries from high-resistance, low-ow muscular vessels to low-resistance
Fig. 1. A schematic map to illustrate the pathways of human placental trophoblast cell differentiation. (A) The growing fetus is anchored to the uterus (u)
through the formation of the placenta. The umbilical cord (uc) contains fetal arteries and vein that branch into the placental villi (v). The villi are immersed
in maternal blood that is perfused into intervillous spaces (ivs) through the process of uterine spiral artery remodeling. (B) Magnication of chorionic
membrane (chm) where trophoblast progenitor cells (TBPC) are located. The TBPCs are believed to give rise to various trophoblast subtypes throughout
gestation. (C) Magnication of a oating villus (fv). The mononucleated cytotrophoblast cells (CTB) are covered by a layer of multinucleated
syncytiotrophoblasts (STB). Within the villous core, there exist mesenchymal cells (m) and villous capillaries (vc). (D) Magnication of an anchoring villus
(av). The actively proliferating cytotrophoblast cells form column cytotrophoblast. At the distal portion of the column, cytotrophoblast cells detach from the
villus and migrate into the decidua (dec). Interstitial trophoblasts (iEVT) invade into the deep layer of the decidua and stop at the inner third of the
myometrium (myo) by differentiating into placental bed giant cells. Endovascular trophoblasts (enEVT) penetrate the uterine spiral artery (sa) and replace
the endothelial cells (en), remodeling the spiral artery into low-resistance, high-capacity utero-placental artery. Endoglandular trophoblasts (egEVT) invade
into the endometrial gland and may provide histotrophic nutrition to the embryo prior to the establishment of proper placental perfusion. Various immune
cells, including dNK cells (red, round ones), macrophage (purple ones), and T lymphocytes (small blue ones), exist in the decidua. st, decidual stromal cells.
(E) The primary characteristics and the differentiation relationship of different trophoblast subtypes are briey presented.
987

Table 1
The specic biomarkers that have been identied for different trophoblast subtypes.
Markers
CK7
Vimentin
VE-Cadherin
E-Cadherin
PECAM
NCAM (CD56)
CD9
Fibulin-5
Integrin a1b1
Integrin a5b1
Integrin a6b4
Integrin avb5
Integrin avb3
hPL
hCG a
hCG b
EGF/TGF a
EGF-R
TGFb
Endoglin
VEGF
Flt-1
KDR
pro-Renin
Renin-R
IL-1R
IL-1b
HLA class I
HLA-G
HIF-1
pVHL
Galectin-8
PAI-1
PAI-2
uPA
MMP-1
MMP-2
MMP-3
MMP-7
MMP-9
TIMP-1
TIMP-2
Arginase I
Arginase II
Location
STBs
CTBs
Column CTB
iEVT
enEVT
+

+
+

NA

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+/
+
+
+
NA
NA
NA
+
NA
NA
NA

+
+

+
+

+

+
+

+
+/
+
+
+

NA
NA
NA
+

+

+
+
+
+
+
+
NA
NA
NA
+
NA
NA
NA
+
+
+

+
+

+
+

+
+

+
+/

+
+
+
+
NA
NA
NA
+
+
+
+

+
+
+
+
+
+

+
+
+
+
+

NA
NA
+

+

NA
NA
+
+

+
+
+/

+

+
+
+
NA
NA
+
+
+
+

+/
+
+
+
+
+
+
+
+
+
+
NA
NA
NA
NA
+

+
+
NA
NA
+
NA
NA

+
NA
+/

NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Sampling trimester
References
1st
1st
2nd
2nd
2nd
2nd
1st
1st
1st
1st
1st
2nd
2nd
1st
1st
1st
1st
1st
1st
1st
3rd
3rd
3rd
1st
1st
1st
1st
1st
1st
3rd
3rd
1st
1st
1st
1st
1st, 2nd &
1st, 2nd &
1st, 2nd &
1st, 2nd &
1st, 2nd &
1st, 2nd &
1st, 2nd &
1st & 3rd
1st & 3rd
Blaschitz et al. (2000)

Shorter et al. (1993)
Damsky and Fisher (1998)
Blaschitz et al. (2000)
Gauster et al. (2011)
Damsky et al. (1992)
Zhou et al. (1997b)
Zhou et al. (1997b)
Handschuh et al. (2007a)
Handschuh et al. (2007a)
Lysiak et al. (1993)
Muhlhauser et al. (1993)
Selick et al. (1994)
St-Jacques et al. (1994)
Pietro et al. (2010)
Pringle et al. (2011)
Pringle et al. (2011)
Simon et al. (1994)
Simon et al. (1994)
Genbacev et al. (2001)
Genbacev et al. (2001)
Kolundzic et al. (2011)
Hofmann et al. (1994)
Huppertz et al. (1998b)
Vettraino et al. (1996)
Ishikawa et al. (2007)
Ishikawa et al. (2007)
3rd
3rd
3rd
3rd
3rd
3rd
3rd
CTBs, cytotrophoblasts; enEVT: endovascular trophoblast; iEVT, interstitial trophoblast; STBs, syncytiotrophoblasts; +, positively staining; , negatively
staining; NA, data not available.
and high-ow sac-like vessels involves cross-talk between different cell types, with enEVTs as the key players. These events
can be divided into ve stages: (1) decidua-associated early vascular remodeling, (2) iEVT-associated early vascular remodeling (3) enEVT migration, (4) the incorporation of enEVTs into the vessel wall and (5) the re-endothelization and subintimal
thickening (Cartwright et al., 2010; Kaufmann et al., 2003; Pijnenborg et al., 2006).
During the decidualization process, the maternal vessels are modied to prepare them for EVT invasion. This priming
process appear to be trophoblast-independent based on studies of ectopic pregnancies (Harris, 2010). These alterations include smooth muscle swelling and endothelial cell vacuolation (Pijnenborg et al., 2006). Leukocytes, specically dNKs and
macrophages, have been implicated in this process. MMP-dependent matrix degradation and cell apoptosis are the suggested mechanisms by which trophoblast-independent early vascular remodeling occurs (Smith et al., 2009).
Two possible origins of the enEVTs, iEVTs and enEVT plugs have been suggested. During early pregnancy, spiral arteries in
the supercial decidua are surrounded by iEVTs (Pijnenborg et al., 1980). These iEVTs position themselves along the arteries
and begin to disrupt and disorganize the vascular smooth muscle cell layer. As these iEVTs invade the lumen of the arteries,
they are believed to switch to the endovascular phenotype. However, it has been suggested that the switch from iEVTs to
enEVTs occurs only in regions of the spiral arteries in the supercial zone of the decidua and that deeper regions of the arteries are remodeled by enEVTs from a second origin, i.e., enEVT plugs (Pijnenborg et al., 2006). Endovascular EVTs from the
initial plugs are believed to retrogradely travel down the vessel lumen (Cartwright et al., 2010; Kaufmann et al., 2003). This
988
inltration by the EVTs results in maternal vascular apoptosis, the detachment of these cells from the surrounding extracellular matrix and their migration away from the vessels (Cartwright et al., 2010). These enEVTs go on to replace the endothelial cells of the maternal vessels through a process that is referred to as pseudovasculogenesis or vascular mimicry (Damsky
and Fisher, 1998; Khankin et al., 2010; Zhou et al., 1997b). Endovascular EVTs are able to exist within the maternal
vasculature in a manner that is similar to endothelial cells primarily due to their switch from an epithelial to an endothelial
adhesion molecule phenotype (Anin et al., 2004). These cells downregulate the epithelial-type markers E-cadherin and a6b4
and upregulate the expression of the adhesion molecules VE-Cadherin, PECAM, and NCAM (CD56) as well as integrins a5b1,
a1b1 and aVb3 (Damsky and Fisher, 1998; Zhou et al., 1997b). Lastly, studies in mice have provided evidence for maternal
vascular repair. This process encompasses re-endothelization and by the appearance of subintimal thickening between the
restored endothelium and the brinoid layer that surrounds the enEVTs (Pijnenborg et al., 2006; Zhou et al., 1997a).
The remodeling of maternal spiral arteries continues until the mid-second trimester, and can be observed as deeply as the
inner third of the myometrium (Cartwright et al., 2010). This process does not occur uniformly throughout the entire placenta; it is most prominent in the central placental bed and less prominent at the periphery (Kaufmann et al., 2003). The
remodeled spiral arteries are increased in length, exhibit a several-fold increase in lumen diameter and are unresponsive
to vaso-constrictive agents (Anin et al., 2004; Kam et al., 1999). This low resistant and high capacity phenotype provides
for the necessary ow of maternal blood into the intervillous spaces, which sustains the growing demands of the fetus
throughout gestation.
2.2.3. Endoglandular trophoblast cells uterine glandular remodeling
Evidence has recently emerged to suggest the existence of another subpopulation of EVTs, termed the endoglandular EVTs
(egEVTs). In vitro rst trimester decidua parietalis and placental villous explant co-cultures provided the rst evidence of
glandular remodeling by egEVTs, a process that is similar to the enEVT remodeling of spiral arteries (Moser et al., 2010).
It is suggested that the replacement of glandular cells by egEVTs may provide a mechanism both for the opening of the uterine gland to the intravillous space and for histotrophic nutrition to the embryo prior to the establishment of proper placental
perfusion (Fitzgerald et al., 2010). More studies are required to conrm the presence of egEVTs and their role in remodeling
uterine glands.
2.3. Summary of the characteristics of the various trophoblast subtypes
As discussed in the previous section, mammalian placentation represents a remarkable biological process, during which
diverse trophoblast populations are generated. Much work has been performed to identify trophoblast subtypes, especially
at early gestational stages, when trophoblasts are actively differentiating. The morphological characteristics and the specic
biomarkers of different trophoblast populations are summarized in Fig. 1 and Table 1. It is easy to see that enEVTs have been
Table 2
The origin and characterization of the immortalized human trophoblast cell lines.
Cell line
BeWo
JAR
JEG
AC1-1
ACH-3P
HTR-8
HTR-8/ SVneo
NPC
B6Tert-1
ED27
ED31
ED77
HP-W1
HP-A1/A2
SPA-26
TL
HT
TCL-1
IST-1
SGHPL-5
HT-116
Swan 71
HPT-8
Origin
Chc
Chc
Chc
JEG-3
AC1-1
FTrP
HTR-8
FTrP
NPC
FTrP
FTrP
FTrP
FTrP
FTrP
FTrP
TP
TP
TP
FTrP
FTrP
FTrP
FTrP
FTrP
Method of immortalization
Spontaneous
Spontaneous
Spontaneous
Mutation
Fusion of FTrP CTB with AC11
Spontaneous
pSV40neo, large T
Spontaneous
pGRN145-hTERT
Spontaneous
Spontaneous
Spontaneous
SV40
SV40 A209 TSM
tsA255 TSM
Spontaneous
Spontaneous
Retrovirus ZipSV406
Retroviral LXNS16E6E7
pSV40neo, large T
Spontaneous
Retroviral hTERT
Spontaneous
Markers
hCG
hPL
CK-7
HLA-G
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Function
Reference
S
I
I
I& S
I& S
I
I
I
I
I
I
I
Pattillo and Gey (1968)

Pattillo et al. (1971)
Kohler and Bridson (1971)
Funayama et al. (1997)
Hiden et al. (2007)
Graham et al. (1993)
Graham et al. (1993)
Rong-Hao et al. (1996)
Wang et al. (2006)
Morgan et al. (1998)
Morgan et al. (1998)
Diss et al. (1992)
Lei et al. (1992)
Lei et al. (1992)
Chou (1978)
Ho et al. (1987)
Ho et al. (1994)
Lewis et al. (1996)
Shih et al. (1998)
Choy and Manyonda (1998)
Zdravkovic et al. (1999)
Straszewski-Chavez et al. (2009)
Zhang et al. (2011)
+

+
+
+
I
I
I
Chc, choriocarcinoma; FTrP, rst-trimester placenta; hTERT, human telomerase catalytic subunit; I, invasion; S, syncytiolization; TP, term placenta; TSM,
temperature sensitive mutant.
989
poorly characterized compared with other trophoblast subtypes. This relative lack of knowledge is most likely due to the
difculty that is involved in accessing clinical samples that contain the deeper uterine endometrium and the upper one-third
of uterine myometrium, where enEVTs are located. Another issue that must be noted is that the gestational stages of the
placenta tissues that are used to characterize the examined cells. The dynamic gene expression patterns of certain trophoblast subpopulation throughout gestation are coincident with the active differentiation of these cell types during this period.
It therefore becomes very important to distinguish between the markers of trophoblast subtypes at early and late stages of
pregnancy.
3. Model systems for examining placental trophoblast cell functions
The choice of an appropriate in vitro research model is fundamental to our understanding of physiological and pathological placental functions. The commonly used in vitro models include isolated primary cultures, explants, and various trophoblast cell lines that have been derived either from normal placenta specimens or choriocarcinoma tissues. The generation of
trophoblast stem cells is another powerful method that is used to investigate the early differentiation of trophoblasts, but
such investigations are challenging in humans. The co-culture of trophoblast cells with other cell types in either two dimensional (2-D) or three dimensional (3-D) culture systems have been demonstrated to more closely mimic the specialized features of trophoblast cells within the uterus during pregnancy. Methodologies have also been developed for specic genetic
alterations of the rodent placenta, making it possible to understand the regulation of placental development as well as placenta-associated pregnancy complications in vivo.
3.1. Primary cultures of human trophoblast cells
The method that is used to isolate villous CTBs was initially developed by Kilman and was subsequently modied by several groups (Douglas and King, 1989; Fisher et al., 1989; Kliman et al., 1986; Li and Zhuang, 1991; Nelson et al., 1986). Briey,
placental tissues are subjected to trypsinization, Percoll gradient centrifugation and immunopurication. The digestion of
placental tissues results in a mixture of various cell types, including villous trophoblasts, extravillous trophoblasts, placental
broblasts, stromal and endothelial cells, as well as a large amount of blood cells and debris. Density gradient separation
with Percoll reagent can be used to remove cell debris, blood red cells, and a portion of the broblasts and endothelial cells.
Li et al. also reported a method by which a BSA gradient can be used to replace the Percoll gradient in the isolation of CTBs
from the rst trimester placental villi (Li and Zhuang, 1991; Rong-Hao et al., 1996). The resultant cell population following
density gradient separation is generally >95% cytokeratin 7 (CK7) positive, but this population still contains a mixture of trophoblast cell types. Immunopurication using immunomagnetic beads or Flow cytometry can be performed to further purify
certain trophoblast subtypes, and the selection of the specic markers is primarily based on trophoblast cell surface antigens
(Table 1). For example, viable EVT cells can be puried by positive selection with specic antibodies against HLA-G or CD9.
Negative selection with pan-HLA or CD9 antibodies can remove pan-HLA+ or CD9+ cells to purify villous CTBs (Frank et al.,
2000, 2001; Yui et al., 1994). STBs and syncytial fragment contamination can be easily removed by performing a cell attachment step (Frank et al., 2001).
The impact of gestational age must be taken into account when using primary cultured trophoblast cells as an in vitro
model. CTBs that are isolated from term placentas spontaneously fuse and form syncytia within 48 h of culture, whereas rst
trimester CTBs seldom do so. Likewise, EVT cells that are derived from early pregnancy are much more invasive than those
from term placentas. Moreover, the biological identication of the puried trophoblast should be performed. This step consists of the determination of the capacity of the puried cells to fuse into syncytium, their hCG and hPL secretion, as well as
their invasiveness and MMP production.
Although primary trophoblast cultures are useful tools for the examination of trophoblast differentiation and functions,
there are two major limitations to this paradigm. One is the limited ability of trophoblast cells to proliferate in culture. This
limitation has hampered standard gene manipulation experiments, such as gene overexpression or silencing through plasmid DNA transfection. The other limitation is the inconsistent responses of different batches of cells that are derived from
different placentas. Apart from individual variations, the gestational age of the placenta tissues can inuence the differentiation potential of the derived CTBs (Xu et al., 2000) and should be taken into consideration when interpreting the results.
3.2. Trophoblastic cell lines
Three types of human trophoblast cell lines have been developed: (1) CTBs with spontaneously elongated lifespans; (2)
trophoblast cells that have been immortalized by the expression of exogenous genes; and (3) cells lines that have been derived from choriocarcinomas. The trophoblastic nature of these cell lines have been assessed using biomarkers, such as hCG,
hPL, CK7, and HLA-G, and by their invasiveness or syncytia-forming abilities. The origin and characteristics of these cell lines
are summarized in Table 2.
It must be considered, however, that these replicating cell lines may not represent normal trophoblastic functions in all
aspects. For example, HTR-8/SVneo and SGHPL-5 cell lines are commonly used to examine EVT properties, whereas these
cells express vimentin, suggesting the induction of mesenchymal genotypes in these cell lines due to the transformation/
990
immortalization procedure (King et al., 2000b; Manyonda et al., 2001). Li et al. described a normal early placenta-origin cytotrophoblast (NPC) cell line that maintains most endocrine functions of normal CTBs, whereas these cells have limited lifespan
(Li and Zhuang, 1997; Rong-Hao et al., 1996). We introduced telomerase activity in NPC cells and generated an immortalized
normal trophoblast cell line, B6Tert-1. The cells have been identied as acquiring EVT cell properties, but they must be cultured on collagen-coated surface (Wang et al., 2006).
It is most likely that the processes that are involved in extending cellular lifespan, whether spontaneous or via transfection of exogenous factors, may result in unusual patterns of gene expression that are not normally observed in trophoblastic
cells in vivo or in in vitro primary cultures. Recently, Bilban and colleagues compared the gene expression prole of three
choriocarcinoma cell lines (JEG-3, BeWo and ACH-3P), two EVT-derived cell lines (HTR-8/SVneo and SGHPL-5) and primary
trophoblasts (EVTs and CTBs) by performing microarray and bioinformatics analyses (Bilban et al., 2009, 2010). These
authors also analyzed whether the gene expression signatures of these non-primary cell lines were similar to the CTB- or
EVT-specic gene prole. The three groups of cell types were found to be considerably different with respect to their gene
expression proles. Moreover, the gene expression proles of HTR-8/SVneo and SGHPL-5 cells are related but dissimilar from
those of the primary CTBs or EVTs. This fact indicates that tumorigenic and large T-immortalized trophoblast cell lines may
exhibit signicant differences with respect to their trophoblastic characteristics. It is therefore strongly suggested that the
critical experiments that are performed in a given cell line be veried in multiple trophoblast models.
3.3. Trophoblast stem cell lines
Since the establishment of human embryonic stem cells (ESCs) a decade ago (Thomson et al., 1998), evidence has increased that human ESCs are useful models with which to examine the emergence and differentiation of trophoblasts. Thomson et al. rst reported the spontaneous differentiation of human and nonhuman primate ESCs to the trophoblast lineage
(Thomson et al., 1998, 1995, 1996). ESCs can spontaneously differentiate into either villous trophoblasts or EVTs from human
embryonic body (EB) that is cultured in suspension (Gerami-Naini et al., 2004; Harun et al., 2006; Peiffer et al., 2007). The
indispensable role of bone morphogenetic protein 4 (BMP4) signaling in efciently directing hESCs differentiation to trophoblasts has been conclusively demonstrated (Chen et al., 2008; Xu et al., 2002b). Interestingly, the similar event rarely occurred in mouse ESCs without certain genetic or transcriptional modications.
Although ESCs can be induced to the trophoblast lineage, the resulting cells are always mixed populations that contain
cells with various characteristics or are of limited proliferative capability. Trophoblast stem (TS) cells appear to be more reliable models for the examination of the control of trophoblast lineage determination. Accumulating evidence suggests important differences between the TS cell properties of different species. Mouse TS (mTS) cell lines have been successfully
established from the blastocyst or the extraembryonic ectoderm (ExE) at embryonic day 6.5. mTS cells contribute to the tissues of the trophoblast lineage, including the ExE, the ectoplacental cone (EPC) and giant cells, but never to the embryonic
tissues in in vivo chimeras (Tanaka et al., 1998). Stem cells with trophoblast characteristics have been obtained from cows,
pigs (Flechon et al., 1995; Hashizume et al., 2006; Shimada et al., 2001; Talbot et al., 2000), rhesus monkeys (Vandevoort
et al., 2007) and rabbits (Tan et al., 2011). The abilities of these cells to form syncytium, to invade matrices, and to express
trophoblast-specic genes have been characterized.
The establishment of human trophoblast stem cell lines has long been a challenging process. Recently, Genbacev and colleagues report that chorionic mesoderm is a niche for trophoblast progenitor cells (TBPCs) (Genbacev et al., 2011). The culture of chorion cells in medium that contains FGF and an inhibitor of Activin/Nodal signaling yields continuously selfreplicating colonies or monolayers that can further differentiate into the two major human trophoblast lineages multinucleate STBs and invasive EVTs. To date, very little is known regarding the molecular regulation of human placental development at very early stage. Such regulation is key to the growth of this organ as well as to the health of both the fetus and the
mother. These TBPCs will be useful models with which to examine the underlying mechanisms that govern human trophoblast specication and differentiation and to gain a better understanding of the origins of pregnancy complications that involve abnormal trophoblast cell differentiation.
3.4. Co-culture of trophoblast cells/placental villi and other cell types or explants
The microenvironment within the maternalfetal interface changes dynamically during gestation. For instance, early placenta development occurs in relatively hypoxic conditions. The remodeling of uterine spiral arteries by invasive trophoblast
increases uterine blood volume to perfuse the placenta, and the local oxygen tension tends to be normoxic thereafter (Genbacev et al., 1997). Within the invasive differentiation pathway, trophoblast cells come into contact with different cell populations within the uterine wall, such as epithelial, stromal, immune, endothelial, and myometrial cells. The well-regulated
interactions between trophoblast cells and these cells are pivotal for placental development and pregnancy maintenance.
Therefore, various co-culture systems have been developed to better mimic the in vivo environment of these cells.
The 2-D co-culture system is relatively simple to perform, and has yielded a good deal of useful evidence regarding the
interactions between trophoblast cells and other cell types in the uterine wall. For instance, Hu et al. examine the interaction
between dNK cells and trophoblast cells using the collagen-based co-culture of dNK cells and villous explants from concordant rst trimester pregnancies (Hu et al., 2006). These authors reveal that dNK cells exerted contact-independent inhibition
of cytotrophoblast migration via alterations in protease activity and E-cadherin expression; however, these cells do not affect
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trophoblast cell proliferation, apoptosis or differentiation. In a study that is conducted to analyze the effect of trophoblasts
on endometrial stromal cells (Popovici et al., 2006), rst-trimester villous explants are grown on the monolayer of endometrial stromal cells, and a variety of genes that participate in the endometrialtrophoblast interaction are identied. Attempts
have also been made to co-culture trophoblast cells or villous explants with segments of the human spiral arteries (Harris
et al., 2007), immortalized human endometrial stromal cells and epithelial cells (Aldo et al., 2007; Holmberg et al., 2012), and
other cell types. The labeling of cells with enhanced green uorescent protein or cell tracker dyes allows for the tracking of
different cell types during the co-culture experiments.
Paracrine regulation that is mediated by various secreted factors is important for cellcell communications at the fetomaternal interface. The co-culture of two different cell types, with one type at the bottom of culture dish and the other
on the top of a transwell insert, allows for the examination of the interaction between different cell types. The size of the
pores on the transwell insert may be sufciently small to allow for the passage of only solutes or sufciently large to allow
for the movement of cells. Furthermore, each individual cell type may be grown on different extracellular matrix proteins to
mimic thein vivo conditions. Such a system has been used to examine the migration of trophoblasts through a monolayer of
endothelial cells (Gallery et al., 2001).
Three-dimensional culture systems have been well accepted to be more physiologically relevant in vitro models. The suspension spheroid culture for growing trophoblast cells in a 3-D environment has been performed using both primary cytotrophoblasts (Korff et al., 2004) and trophoblast cell line SGHPL-4 (LaMarca et al., 2005). An optimized NASA-engineered
rotating wall vessel (RWV) suspension culture for SGHPL-4 cells resulted in the further differentiation of the cells in the absence of exogenous growth factors or cytokines (LaMarca et al., 2005). The co-culture of placenta and decidua tissues from
the same individual has also been developed and provides an exciting model to examine trophoblast differentiation and
behavior. In this model, placental villi are placed on the patient-matched decidual explants that are grown with the apical
epithelial surface on the uppermost in culture dish inserts (pore size 0.4 lm) that are pre-coated with Matrigel. Following
the parafn-embedding of the tissues at various time points, several events can be observed, including EVT invasion into
arterioles, endothelial cell and smooth muscle cell loss, the relining of arteries by endovascular EVTs, and vascularleukocyte
interactions (Dunk et al., 2003; Hazan et al., 2010; Vicovac et al., 1995). Spiral artery remodeling during human placentation
and the molecular mechanisms that underlie this process remain largely uncharacterized. It is anticipated that the placenta
decidua co-culture model will allow for investigation into the dynamic processes of spiral artery remodeling and the characterization of the genes that are critical for this process. Moreover, the use of trophoblast cell lines in this model will be
invaluable, given that the roles of specic genes in spiral artery remodeling can be identied via their over-expression or
knockdown in these cells prior to co-culture.
3.5. Animal models for investigating placental development
Due to the unique characteristics of human pregnancy, there are no equivalent animal systems by which to examine human placental development. However, the placentas of humans and mice have certain similarities. The villous tree region in
humans and the labyrinth in mice are comparable sites of maternal-fetal exchange, and the remodeling of uterine spiral
arteries by trophoblast cells ensures that maternal blood enters the placenta. The umbilical vessels connect the fetal capillaries of the placental exchange region with the fetal body circulation (Adamson et al., 2002; Georgiades et al., 2002). Therefore, mice can serve as useful tool for studying certain aspects of human pregnancy, especially when the functional
conservation of the relevant genetic control pathways between human and mouse tissues is conrmed. The web-accessible
database that was reported by Cox et al. has provided information that has guided the modeling of human placentation in
the mouse (Cox et al., 2009).
Embryonic lethality that results from conventional gene manipulation is generally a confounding variable in the discrimination of whether a genetic modication affects fetal or placental development. This complication arises given that fetus
survival is largely dependent on the proper functioning of the placenta. Therefore, restricted gene manipulation in the placenta is necessary to clearly dene gene functions in placental development. Given that placental irregularities are associated with various pregnancy complications, the genetic modication of the placenta is particularly valuable for
understanding the possible genetic basis of severe pregnancy complications as well as for developing potential therapeutic
strategies to treat the associated diseases.
To date, the techniques that have been developed for placenta-specic gene manipulation can be placed into the following categories: (1) placental-specic transgene expression; (2) virus-mediated gene manipulation; and (3) transplantation of
cells.
3.5.1. Placenta-specic transgene expression
Placental-specic transgenes are based on the genes exhibiting temporal or cell-specic expression in the placenta. The
endogenous placental lactogen-II (PL-II; Prl3b1) promoter, which is activated in mouse spongiotrophoblast cells at the late
second half of pregnancy, has been used for the specic targeting of genes in this trophoblast subtype (Shah et al., 1998;
Shida et al., 1992). Tpbpa gene exhibits specic expression in the ectoplacental cone beginning at embryonic day 7.5. Expression is subsequently observed in the trophoblast cells that comprise the junctional zone during pregnancy in mice. A promoter region of 340-bp that is approximately 3740-bp upstream of the Tpbpa transcription start site has been used to
drive the exclusive expression of Cre-recombinase and green uorescent protein (GFP) in junctional zone trophoblast cells
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(Calzonetti et al., 1995; Simmons et al., 2007). This Tpbpa-Cre mouse provides a tool for the generation of gene knockouts
specically in junctional zone trophoblast cells in mice (Wenzel and Leone, 2007). The upstream locus control region (LCR) of
human chorionic somatomammotropin (hCS) (Elefant et al., 2000; Jin et al., 2009; Su et al., 2000) and the 501-bp sequence
that is upstream of CYP19 exon I (Kamat et al., 1999, 2005) have both been proven to be sufcient to specically drive the
expression of a reporter gene in mouse STBs despite the lack of rodent orthologs for these genes. Using the 501-bp hCYP19
promoter, Wenzel and Leone (2007) developed a mouse model with Cre recombinase expression in all trophoblast derivatives rather than only in STBs, enabling the generation of trophoblast-specic gene knockout in mice (Wenzel and Leone,
2007).
3.5.2. Virus-mediated genetic manipulation in rodent placenta
Using recombinant viruses to deliver genetic material to the placenta is an effective mean by which to genetically modify
the placenta. Adenoviruses and retroviruses are the most commonly used vectors to mediate gene delivery. Adenoviruses are
capable of infecting both dividing and non-dividing cells at high efciency but cannot integrate their genetic material into
that of the host cells; the carried genes will therefore not be transmitted to the daughter cells. Direct in utero inoculation
with recombinant adenoviruses has been conclusively demonstrated to successfully alter gene expression specically in
the placenta (Katz et al., 2009; Xing et al., 2000). Intra-placental injection can, however, cause injuries that may reduce fetal
viability if performed earlier than E13.5. Recently, a ber-mutant adenovirus vector carrying the Arg-Gly-Asp (RGD) peptide
sequence (Ad-RGD) was observed to exhibit placental tropism at 10100 times that of the wild type adenovirus (Katayama
et al., 2011). The reason for the enrichment of Ad-RGD expression in the placenta remains unknown; however, the high
expression of adhesion receptors that recognize the RGD sequence, e.g., integrin avb3, in the placenta may be a partial explanation. One of the apparent advantages of Ad-RGD is that it can selectively and noninvasively deliver a gene of interest into
the placenta. This can be achieved via maternal intravenous injection.
Unlike adenoviruses, retroviruses are able to stably integrate into the host genome, and their genetic material is transmitted to daughter cells. A subtype of retroviruses, lentiviruses, are commonly used given that they can transduce dividing
and non-dividing cells more efciently than other types of retroviruses. In landmark studies using mice zona pellucida-free
blastocysts that were immersed in media containing lentiviruses, groups led by Ikawa and Rossant report placenta-specic
gene expression during pregnancy (Georgiades et al., 2007; Okada et al., 2007). The application of this method has revealed
that embryonic lethality of mice that are decient in Ets2, Mapk14 or Mapk1 is caused by placenta defects rather than defects in the embryo proper (Okada et al., 2007). Moreover, lentivirus-mediated pol III-driven short hairpin RNA (shRNA) and
lentivirus-mediated pol II-driven Cre recombinase can efciently yield trophoblast-specic gene knockdowns and knockouts, respectively (Morioka et al., 2009). To date, this lentivirus lineage-specic infection system has been effectively performed in several other mammalian species in both gain- and loss-of-function studies (Georgiades et al., 2007; Lee et al.,
2009; Purcell et al., 2009).
3.5.3. Transplantation of cells into placenta
The transplantation of cells with distinct genetic background into the placenta is an effective means by which to introduce genetically modied cells into a placenta. Given that ES cells will give rise to embryonic structures but not trophectoderm derivatives, ES cell-embryo chimeras are useful tools with which to determine lineage-specic outcomes of genetic
mutations (Robertson et al., 1986). A chimera using a pre-implantation tetraploid embryo that is aggregated with ES cells
allows for the analysis of lineage-exclusive cell populations during development. The tetraploid cells give rise wholly to
the extraembryonic entities, and the ES cells produce embryonic structures. Thus, tetraploid complementation is a unique
method that has been used to distinguish whether embryonic lethality results from deciencies in the embryo itself or in
extraembryonic structures. With this method, the lethality of SOCS3-decient embryos was demonstrated to arise from defects in extraembryonic functions (Takahashi et al., 2003). One of the limitations of tetraploid complementation is that it
cannot discriminate between defects in trophoblastic or primitive endodermal derivatives. Takahashi et al. performed microinjections of TS cells into the blastocoel of the SOCS3-defective embryo and partially rescued embryonic lethality (Takahashi et al., 2006). However, this method is less effective than tetraploid complementation.
4. Regulatory mechanisms involved in human trophoblast cell differentiation toward the invasive pathway
The regulation of human placental EVT cell differentiation is very complicated. Various factors act largely in paracrine or
autocrine manner to inuence EVT cell survival and differentiation (Knoer and Pollheimer, 2012). These factors can be derived from trophoblast cells, uterine stromal and glandular cells, myometrial cells, endothelial cells, villous mesenchymal
cells and various immune cells at the maternal-fetal interface. Environmental factors, such as oxygen tension are also important. Likewise, EVT cells can directly or indirectly inuence the activities of these cell types (Wallace et al., 2012). An elaborate regulatory network is thus established at the feto-maternal interface to maintain a successful pregnancy; however,
much remains to be determined before this process is fully understood, with the primary obstacles being ethical restrictions.
Investigation into the regulation of human EVT cell differentiation has been primarily performed in vitro. Apart from the
identication of specic cell markers (Table 1), the acquisition or enhancement of invasive ability is one of the most important criteria for EVT cell differentiation, and the capability of tube formation in Matrigel is occasionally used to measure
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enEVT differentiation. Arterial invasion by trophoblast and placental perfusion in mouse models have been suggested to be
in vivo evidence for enEVT differentiation.
Oxygen tension has been considered to be a key factor in the balance of trophoblast cell proliferation and differentiation
with certain transcriptional factors, Hypoxia-inducible factors (HIFs), acting as oxygen sensors. Hormones, including gonadotropin-releasing hormone (GnRH), hCG, insulin, progesterone, and estrogen etc. are known to participate in the control of
trophoblast behaviors (Chen et al., 2012; Norris et al., 2011). Moreover, numerous growth factors and cytokines, such as
members of the vascular endothelial growth factor (VEGF) family, the transforming growth factor (TGF)-b superfamily, broblast growth factor (FGF), hepatocyte growth factor (HGF), epidermal growth factor (EGF), heparin-binding EGF (HB-EGF),
insulin-like growth factors (IGFs), macrophage-colony stimulating factor (M-CSF), LIF, interleukin (ILs), tumor necrosis factor
(TNF), CXCLs, and others have been reported to actively control trophoblast cell proliferation and migration/invasion. Interestingly, a HERV insertion in the growth factor Pleitrophin gene (PTN) results in a functional HERV-PTN fusion transcript. It is
localized by in situ hybridization to the proliferative and invasive trophoblasts, suggesting a putative role of HERVs in the
differentiation of trophoblast toward the invasive pathway (Schulte et al., 1996, 2000). Various signaling cascades that
are mediated by GTPase RhoA, the protein kinases ROCK, ERK1, ERK2, FAK, PI3K, Akt/protein kinase B and mTOR as well
as Smads, are involved in regulating trophoblast characteristics (Fitzgerald et al., 2005, 2008; Garrido-Gomez et al., 2012;
Maymo et al., 2011; Pollheimer and Knoer, 2005; Wu et al., 2001; Zhao et al., 2012). In recent years, Notch and Wnt signaling, which are important developmental pathways, have also been observed to regulate trophoblast cell invasion.
Although an increasing numbers of regulators are being identied, the downstream transcription factors that control the epithelium-mesenchyme transition (EMT)-like and endothelial-like transformation during the differentiation of iEVTs and enEVTs are largely unknown.
In this section, we briey summarize the roles of oxygen tension and several factors that modulate human trophoblast
cell differentiation toward the invasive pathway. Recent advances that have been made regarding microRNAs expression
and regulation in human placenta will also be discussed.
4.1. Oxygen tension
The importance of the appropriate oxygen concentration for tissue health is well established, with hypoxia or hyperoxia
inducing abnormal reactions in most animal tissues. During pregnancy, the oxygen partial pressure in the intervillous space
varies with the development of maternalplacental circulation (Red-Horse et al., 2004; Tuuli et al., 2011). The mean oxygen
pressures at 810 weeks of gestation are estimated to be as low as 17.9 6.9 and 39.6 12.3 mmHg (Mean SD), respectively. These levels subsequently increase to approximately 80100 mmHg as the result of endovascular invasion, which
facilitates gas exchange between the mother and the fetus (Jauniaux et al., 1999; Rodesch et al., 1992; Rooth et al., 1961;
Schaaps et al., 2005). The gestational-stage-dependent variation in oxygen tension is not simply the result of blood vessel
development in the maternal-fetal interface, but plays important roles in modulating trophoblast cell behaviors to maintain
normal placentation (Adelman et al., 2000; Caniggia et al., 2000a; Genbacev et al., 1996, 1997; Red-Horse et al., 2004).
4.1.1. Oxygen tension regulates the balance between trophoblast cell proliferation and invasion
The striking ndings by Genbacev et al. provide strong evidence that oxygen tension can regulate trophoblast cell proliferation and differentiation along the invasive pathway. Using isolated cytotrophoblasts and anchoring villi explants from
gestational weeks 58, these authors conclusively demonstrate that low oxygen concentration (2% oxygen or 14 mmHg)
stimulates CTBs to enter cell cycle and consequently to actively proliferate, while preventing their differentiate along the
invasive pathway (Genbacev et al., 1996; Genbacev et al., 1997). These ndings have since been supported by several lines
of evidence in trophoblast cells or placental explants (Caniggia et al., 2000a,b; Caniggia and Winter, 2002; Seeho et al., 2008).
Invasion of EVTs to maternal spiral arteries has also been found to be inhibited by low oxygen tension. The co-culture of uorescently labeled EVTs with myometrial spiral arteries under 3% oxygen conditions inhibited EVT invasion compared with
those cultured under 17% oxygen (Crocker et al., 2005). However, the role of oxygen is still controversial. For example, when
using placentas at gestational weeks 818, low oxygen tensions do not affect (Lash et al., 2006a) or even decrease (James
et al., 2006) trophoblast cell proliferation. These data indicate that intrinsic differences may exist in the differentiation
potentials of trophoblasts at various gestational weeks. Studies in HTR8/SVneo cells have produced inconsistent results
on how oxygen levels affect cell invasion. Reports from Graham et al. strongly support the invasion-promoting function
of low oxygen tension (1%) via elevating expression of cell surface uPAR in HTR8/SVneo cells (Graham et al., 1998, 1999,
1993). These authors also suggest that the pro-invasiveness effect of hypoxia is a general property of invasive cells, including
trophoblastic and carcinoma cells (Graham et al., 1999). More recent reports demonstrated decreased invasiveness in HTR8/
SVneo cells that were cultured in low oxygen tension (Kilburn et al., 2000; Lash et al., 2007). Futhermore, a microarray analysis supports the inhibitory effect of hypoxia on cell invasion as HTR8/SVneo cells exposed to 1% oxygen have higher expression levels of many anti-invasive factors (Koklanaris et al., 2006).
These apparently contradictory ndings raise the questions of what variables inuence in vitro experiments and of the
mechanisms by which the in vitro hypoxic conditions reect the physiological and pathological oxygen concentrations that
occur in vivo. Recently, Burton et al. highlighted three major obstacles that must be overcome when working with oxygen
in vitro. These include: (1) the absence of hemoglobin as an oxygen carrier in vitro, (2) the dened measurement of actual
in vivo oxygen concentration for a given gestational age, and (3) the avoidance of oxidative stress that is inadvertently
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introduced during sample collection and culture (Burton et al., 2006). An excellent discussion on this topic was recently provided by Zamudio (2011). One of the important issues in the context of in vitro systems is the infeasibility of replicating the
unique property of hemoglobin, which allows for the delivery of large quantities of oxygen to cells at relatively low oxygen
tensions. Therefore, the oxygen percentage in the atmosphere and in the pericellular region of the culture medium may substantially vary in different culture systems in the absence of an oxygen carrier. Moreover, in case of explant cultures, connective tissue cells are more likely to experience low oxygen levels given that oxygen diffusion from culture medium into
these cells is signicantly hampered by multiple cell layers. Burton et al. (2006) recommend that explant samples should
be trimmed to a <5 mm maximal thickness to limit the potential for the development of unintended tissue hypoxia (Burton
et al., 2006).
Furthermore, there is a great deal of debate over the oxygen levels that should be used to examine trophoblast behaviors
in in vitro models. Tuuli et al. (2011) suggested the pragmatic approach of using a humidied oxygen concentration of
approximately 2% (pO2 approximately 15 mmHg) for specimens from the rst trimester and 8% (pO2 approximately
60 mmHg) for trophoblasts from the second or third trimester to mimic the physiological oxygen condition of gestational
ages (Jauniaux et al., 1999; Rodesch et al., 1992). Taking into account the fact that 80100 mmHg is the physiological oxygen
tension in placenta from 10 to 12 weeks onwards, 510% oxygen has been recommended as the proper normoxic condition
for oxygen experiment in human trophoblast cells (Pringle et al., 2010). The oxygen concentration in standard culture condition is 20%, which by the above criteria is actually hyperoxic. Using cells under this condition as controls for examining
cells that are cultured at low oxygen tension, such as 12%, may not accurately reect in vivo conditions.
4.1.2. Hypoxia induces trophoblast cell apoptosis
A series of studies by Nelsons team have revealed the pro-apoptotic effect of <1% oxygen in human CTBs; this effect
involves the increased internucleosomal cleavage of DNA, the upregulation of pro-apoptotic proteins p53 and Bax and
the decreased expression of the anti-apoptotic Bcl-2 proteins (Chen et al., 2010; Humphrey et al., 2008). Other
in vitro studies have demonstrated similar results (Crocker et al., 2004a,b; Huppertz et al., 2003; Kilani et al., 2003).
It has been considered that hypoxia-induced apoptosis is mediated by the mitochondrial pathway (Levy, 2005; Weinmann et al., 2004) and that p53 is the important downstream effector of HIF-1a that induces apoptosis in vitro (Carmeliet et al., 1998). In STBs, hypoxia results in decreased p53 levels and a weaker induction of apoptosis than in CTBs (Chen
et al., 2010). Based on this evidence, Tuuli et al. speculate that autophagy and apoptosis in human trophoblasts are interrelated pathways, with increases in one process reducing the ux through the other. The reduced expression of p53 in
STBs may promote a greater increase in autophagy compared to CTBs, allowing for the survival of the syncytium and the
maintenance of the interface between the maternal and fetal circulations (Tuuli et al., 2011). However, experimental evidence for this hypothesis is lacking.
Recently, an interesting study provided new insights into this topic by examining amino acid incorporation into proteins
in primary STBs that were exposed to various oxygen concentrations. This study reported that global protein synthesis in
STBs decreased sharply at O2 concentrations lower than 1%, whereas protein synthesis was unchanged at 5% or 3% O2. Global
protein synthesis in conditions of severe hypoxia has been conclusively demonstrated to be supported by glucose metabolism. These data indicate that STBs are resistant to reductions in protein synthesis at O2 concentrations that are greater than
1%. With increasing degrees of chronic hypoxia, a shift is observed from oxidative to glycolytic pathways, allowing for a substantial degree of protein synthesis to be maintained in STBs (Williams et al., 2012). Such hypoxia-tolerance properties of
STBs may, in part, explain their resistance to apoptosis in low oxygen condition; however, further investigation is required.
4.1.3. HIFs are oxygen sensors in placenta
s are a family of basic HelixLoopHelix transcription factors, are key mediators of the cellular response to low oxygen. A wide variety of HIF target genes play critical roles in erythropoiesis, angiogenesis, glucose transport, glycolysis,
etc. Among the mammalian HIF family members, HIF-1 is ubiquitously expressed and plays a general role, whereas
HIF-2 and HIF-3 exhibit more restricted expression patterns and may have specialized functions (Rocha, 2007; Safran
and Kaelin, 2003; Semenza, 2000). HIF-1 is a heterodimer that consists of a HIF-1a subunit with a half-life of <5 min,
and a more stable HIF-1b subunit. During hypoxia, HIF-1a is transcriptionally activated and stabilized via the dehydroxylation of factor inhibiting HIF-1 (FIH-1), which binds to the transcriptional co-activators p300/CBP to facilitate the
interaction of this co-activator with HIF-1a and HIF-2a. Other co-factors, including p300/CBP interacting transactivator
with ED-rich tail 2 (CITED2), are involved in the regulation of HIF activity. CITED2 is also a HIF target gene, and can
compete with HIF-1 to bind to p300/CBP.
The upregulation of HIF-1a or HIF-2a protein and increased HIF-1 DNA binding activity have been observed in in vitro
cultures of human trophoblast cells and in placental villi explants under low oxygen conditions (Caniggia et al., 2000a,b;
Genbacev et al., 2001; Rajakumar and Conrad, 2000). The knockout of the Hif-1a and Hif-2a genes in mice leads to a 17%
reduction in trophoblast invasion (Cowden Dahl et al., 2005), and deciency in the Cited2 gene results in impaired trophoblast differentiation and fetal vascularization of the placenta (Withington et al., 2006). These ndings are indicative of the
critical roles of HIFs as central oxygen sensors during placental development.
Moreover, many invasion-associated factors have been identied as HIF target genes. For instance, increased expression
of uPAR and PAI-2 is observed in early gestational EVT cells cultured in 3% oxygen, which exhibit lower invasiveness (Lash
et al., 2006a). PAI-1 expression in HTR-8/SVneo cells increases upon hypoxia stimulation via the action of HIF-1a or HIF-2a
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as determined by the use of specic siRNAs (Fitzpatrick and Graham, 1998; Meade et al., 2007). TGF-b is a negative regulator
of trophoblast cell invasion, and its expression is induced directly by hypoxia via the action of HIF-1 (Caniggia et al., 2000a,b;
Nishi et al., 2004; Schaffer et al., 2003). On the other hand, TGF-b has been demonstrated to increase HIF-1a protein levels by
inhibiting its degradation (McMahon et al., 2006). The latent form of TGF-b is released by the cleavage of latency-associated
peptide (LAP) by plasmin, an event that is facilitated by binding of uPAR to uPA. Therefore, it is likely that there is feedback
interaction between HIF, uPA and TGF-b signaling; however, more direct evidence for such feedback in human trophoblast
cells is lacking.
The angiogenic factor VEGF is known to act via its receptors VEGFR-1 (Flt-1) and VEGFR-2 to participate in the establishment of the feto-placental circulation. A soluble form of Flt-1 (sFlt-1) that lacks the transmembrane and cytoplasmic domains is generated by alternative splicing. This isoform can bind to VEGF and placental growth factor (PlGF) with high
afnity and hamper their interaction with transmembrane VGFR receptors, resulting in an anti-angiogenic effect. Angiopoietins are also involved in the process of placental vasculogenesis via an interaction with tyrosine kinase receptor Tie-2. The
production of VEGF, angiopoietins, and sFlt-1 has been demonstrated to be inuenced by oxygen concentration via the action
of HIF (Nevo et al., 2006; Rajakumar et al., 2009; Wang and Semenza, 1995; Zhang et al., 2001). A recent impressive study by
Kanasaki et al. provides in vivo evidence that feedback regulation of HIF and angiogenic factors is vital for placental development. Pregnant mice that lack catechol-O-methyltransferase (Comt/ mice), which is the enzyme that is responsible for
the generation of 2-methoxyoestradiol (2-ME) from hydroxyoestradiol, exhibit signicant decidual vascular lesions, elevated
accumulation of nuclear HIF-1a in the placenta, increased circulating sFlt-1 levels and signs of hypoxia in both the decidua
and the placenta as determined by hypoxyprobe incorporation (Kanasaki et al., 2008). These abnormalities can be rescued by
2-ME administration. 2-ME destabilizes microtubules and inhibits HIF-1a (Semenza, 1998). This is also true for human trophoblast, as is demonstrated by the suppressive effect of 2-ME on hypoxia-induced HIF-1a and sFlt-1 expression in HTR-8
cells. Taking into account that hypoxia and placental insufciency can trigger deciencies in placenta-derived estrogens and
hydroxyestradiol (Rosing and Carlstrom, 1984; Takanashi et al., 2000), which may in turn result in decreased 2-ME levels, a
feedback regulatory pathway between Comt, 2-ME, HIF-1, and sFlt-1 is highly possible.
4.1.4. Role of HIFs in normoxic condition
Increasing evidences indicate that HIF is not simply a hypoxia-stimulated transcription factor. Many hormones, growth
factors and cytokines have the potential to regulate the expression, stability or activity of HIFs under normoxic conditions.
These factors include progesterone, estrogen, prostaglandin-E2 (PGE2), angiotensin II, thrombin, EGF, insulin, platelet derived growth factor (PDGF), IGF-I, IGF-II, TGF-b, IL-1b, TNFa, etc. The signaling pathways that are involved in the activation
of MAPK, PI3K, NFjB, or ERK 1/2, ILK/Akt and mTOR also participate in HIF regulation by these factors (Ke and Costa, 2006;
Qian et al., 2004; Zelzer et al., 1998; Zhou et al., 2003). Many of these HIF regulators are also produced and participate in
various cell events during placental development; however, the role of HIFs in the mediation of the functions of these factors
in the placenta has been poorly dened.
The stimulation and stabilization of HIF-1a by inammatory factors under normoxic condition is of great interest. A study
of Blouin et al. in macrophages demonstrates that pro-inammatory stimuli under normoxic conditions can elicit the transcription of a similar set of stress response genes as does stabilized HIF-1a in hypoxic conditions (Blouin et al., 2004). This
stress-response is less rapid than the action of stabilized HIF-1a and involves a different mechanism, namely, the induction
of the transcription of HIF-1a via pathways involving PKC and PI3K. More recently, the interaction between HIF-1a and
NFjB, which is a major transcription factor in the inammatory response, is described. The HIF-1a promoter contains an
active NFjB binding site upstream of the transcription start site, and NFjB is critical for sustaining a basal level of HIF1a production (Rius et al., 2008). Therefore, HIFs are likely not only the mediators of hypoxia, but are also responsive to
inammatory stimuli, particularly those that are mediated by NFjB. A recent study of human placental explant cultures reveals that the pro-inammatory factor AngII, enhances HIF-1a accumulation. This provides evidence that HIFs can also mediate the overlap between the inammatory and hypoxia responses in the placenta (Araki-Taguchi et al., 2008). Such
properties of HIFs have yet to be further explored in the placenta, in which a systemic inammatory response is evident,
and in the context of pregnancy disorders, including preeclampsia, in which inammation is exaggerated (Redman and Sargent, 2009).
4.2. Growth factors
4.2.1. VEGF family
The VEGF family includes ve members: VEGF-A, -B, -C, and -D and PlGF. VEGF functions are mediated via two tyrosine
kinase receptor isoforms, VEGF-R1 (Flt-1) and VEGF-R2 (KDR), both of which are expressed on the cell surface of most blood
endothelial cells (Ferrara et al., 2003). VEGF-A binds to both VEGFR-1 and VEGFR-2, but VEGFR-2 is the major signal transducer of VEGF in endothelial cells. In contrast, VEGF-B interacts only with VEGFR-1, whereas VEGF-C and VEGF-D bind to
VEGFR-2 (Ferrara et al., 2003; Gille et al., 2000). Among VEGF-A isoforms, VEGF165 is the most common one, and an alternative splicing form of VEGF, VEGF(165)b, is capable of inhibiting VEGF165-mediated vasodilation and angiogenesis
(Maynard et al., 2003). VEGF-A is considered to be the foremost factor in vascular development. This protein stimulates cell
proliferation and induces endothelial cell migration, division, and survival. The KDR receptor induces endothelial cell
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proliferation, whereas the Flt-1 receptor stimulates tube formation. PlGF exhibits a 42% similarity in amino acid sequence
with VEGF and binds exclusively to Flt-1.
In the human placenta, both VEGF, PlGF and their receptors are expressed in trophoblast cells (Chung et al., 2004; Clark
et al., 1998; Singh et al., 2012). Placental VEGF is localized in CTB, STB, EVT and perivascular cells. The expression of VEGF
increases during the rst 7 weeks of pregnancy and remains stable as pregnancy progresses (Geva et al., 2002). PlGF is localized to the trophoblast and vascular smooth muscle cells (VSMCs), and its expression rises during the third trimester. It is
suggested that the shift in the balance of placental VEGF and PlGF expression at late gestational stages may contribute to
the switch from the branching villous capillaries to the non-branching vessels during the third trimester (Geva et al.,
2002). Both Flt-1 and KDR receptors are detected in various trophoblast cells, and their expression patterns exhibit specic
temporal change during gestation, whereas Flt-1 is observed at relatively high level in EVTs throughout pregnancy (Clark
et al., 1996). The expression pattern of VEGF and its receptors in the placenta indicates a paracrine or autocrine regulation
of trophoblast cell behaviors by VEGF.
In vitro studies reveal multiple roles of VEGF in human trophoblasts, including the regulations of cell proliferation, migration/invasion, and the production of MMPs, adhesion molecules, nitric oxide (NO), etc. One of the most notable functions of
VEGF is to stimulate the endovascular differentiation of EVT cells. VEGF is capable of inducing EVT cell migration and endothelial-like tube formation on Matrigel and can upregulate VE-cadherin and b-catenin expressions via the activation of MAPKs (p38 MAPK, MEK3/6, and ERK1/2) (Khan et al., 2011; Lala et al., 2012). Recent studies reveal the interaction between VEGF
and decorin, a TGF-b binding, leucine-rich proteoglycan produced by the decidua (Khan et al., 2011; Lala et al., 2012). The
stimulation of endovascular differentiation of EVTs by VEGF can be antagonized by decorin via interfering with p38 MAPK
and ERK1/2 activation. The blocking of VEGF function by decorin in EVTs is independent of TGF-b, but is mediated by binding
to KDR tyrosine kinase receptor (Khan et al., 2011; Lala et al., 2012). Such an interaction may be one of the mechanisms that
ensure uterine blood vessel remodeling at the appropriate level, i.e., not exceeding the inner-third of myometrium.
During pregnancy, the placenta also produces a soluble form of VEGFR-1, sFlt-1. This molecule antagonizes VEGF and PlGF
by competitively binding to these ligands, with a higher afnity to PlGF. This antagonism hampers the activation of VEGF and
PlGF signaling (Clark et al., 1998; Maynard et al., 2003). Trophoblast sFlt-1 is able to antagonize VEGF-stimulated and PlGFinduced trophoblast cell invasiveness.
A novel angiogenic factor, Endocrine gland-derived VEGF (EG-VEGF), is newly identied as being able to stimulate proliferation, migration, and fenestration in capillary endothelial. The expression of human EG-VEGF is restricted to the endocrine glands, including the placenta (LeCouter et al., 2001). Its biological activity is mediated by two G protein-coupled
receptors: prokineticin receptor 1 (PROKR1) and prokineticin receptor 2 (PROKR2). EG-VEGF expression peaks between
the 8th and 11th weeks of gestation, and its expression in trophoblast cells is upregulated by hypoxia and hCG stimulation.
EG-VEGF has been demonstrated to be a negative regulator of trophoblast invasion (Brouillet et al., 2012). The mechanism by
which EG-VEGF regulates trophoblast cell differentiation remains to be elucidated but may involve an interaction between
VEGF and EG-VEGF.
4.2.2. TGF-b superfamily
The TGF-b superfamily consists of over 40 members, including TGF-bs, growth differentiation factors (GDFs), BMPs, Nodal,
Activins, and Inhibins (Chang et al., 2002). In general, these proteins are synthesized as prepropeptide precursors with an Nterminal signal peptide followed by the prodomain and the mature domain. The mature domain is cleaved from the propeptide by a furin-like convertase during secretion (Chang et al., 2002). The mature forms of TGF-b ligands are secreted and function by activating specic transmembrane serine/threonine kinase receptors and downstream mediators, such as Smads
(Attisano and Wrana, 2002; Shi and Massague, 2003). The serine/threonine receptor complexes of the TGF-b superfamily
members generally comprise of type I and type II receptors. The type II receptors phosphorylate the type I receptors, which
then activate Smad proteins. Five type II and seven type I receptors (also known as Activin receptor-like kinase, ALK) have
been characterized in mammals.
Several members of the TGF-b superfamily have been suggested to regulate trophoblast cell functions, and their dysregulation has been implicated in pregnancy-associated diseases. TGF-b is a multifunctional factor in human trophoblast cells.
Both TGF-b1 and TGF-b2 inhibit trophoblast cell proliferation and induces syncytialization (Graham et al., 1992). On the
other hand, TGF-b1, -b2 and -b3 inhibit the differentiation toward the invasive EVT pathway by increasing the expression
of TIMPs, downregulating the production of uPA and MMP-9, and increasing E-cadherin expression (Caniggia et al., 1999;
Graham, 1997; Graham and Lala, 1991; Karmakar and Das, 2002; Lash et al., 2006b). However, the invasion-suppressing effects of TGF-b1 cannot be replicated in choriocarcinoma cell lines, including JEG-3, JAR, and BeWo. This discrepancy may result from the partial loss of Smad3 and abnormal TGF-b signaling in these carcinoma cells (Xu et al., 2002a). More recently,
TGF-b3 is found to enhance the expression of the hypoxia-stimulated matricellular proteins CYR61 (CCN1) and NOV (CCN3)
in JEG-3 cells (Wolf et al., 2010). CCN1 and CCN3 are strongly expressed in invasive iEVTs and are capable of promoting trophoblast cell migration/invasion. The synergistic role of TGF-b3 and hypoxia in upregulating the production of the invasionpromoting factors CCN1 and CCN3 in trophoblast cells appears to conict with the invasion-inhibitory effect of TGF-b3, and
more investigation is needed to clarify this discrepancy.
Nodal plays an important role in regulating trophoblast cell differentiation because a hypomorphic mutation of nodal in
mice results in the expansion of the giant cells and spongiotrophoblast layers and a decrease in labyrinthine development
(Ma et al., 2001). Nodal has also been reported to maintain the trophoblast stem cell microenvironment by inhibiting the
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precocious differentiation directly or indirectly via FGF4 (Guzman-Ayala et al., 2004). However, a more recent study suggests
that Nodal does not act directly on trophoblast stem cells (Natale et al., 2009). The critical role of Nodal during pregnancy is
further supported by the ndings that knockout of Nodal in uterus resulted in defects in placentation (Park et al., 2012). In
addition to severe malformation of the maternal decidua basalis, the fetal layer also displayed moderate disorganization,
such as an expansion of giant cell clustering at the maternalfetal border (Park et al., 2012). It is therefore hypothesized that
Nodal maintains a thin giant cell layer at the maternalfetal interface and a delicate balance of Nodal signaling is critical for
proper placental development (Ma et al., 2001; Park et al., 2012). Nodal and one of its receptors, ALK7, are expressed in CTBs,
EVTs, and STBs of human placenta and their levels vary with gestational stages (Nadeem et al., 2011; Roberts et al., 2003). In
HTR-8/SVneo cells and choriocarcinoma cell lines, overexpression of Nodal inhibited proliferation and induced apoptosis
(Munir et al., 2004). In addition, Nodal inhibited migration and invasion of HTR8/SVneo cells and the outgrowth of EVTs
in rst trimester placental explants (Nadeem et al., 2011). These actions of Nodal appear to be mediated by ALK7 as constitutively active ALK7 has similar effects as Nodal whereas silencing of ALK7 expression or expression of dominant negative
ALK7 blocked the effect of Nodal (Munir et al., 2004; Nadeem et al., 2011). Interestingly, siRNA targeting ALK4, which is
known to mediate Nodal signaling during embryo development, inhibited trophoblast invasion (Nadeem et al., 2011), suggesting that ALK4 has stimulatory effects on trophoblast invasion. It has been reported that Nodal signaling requires a coreceptor, Cripto (Kelber et al., 2008) and is inhibited by Lefty proteins (Nakamura et al., 2006). It will be important to characterize the spatial and temporal expression pattern of these molecules during placentation to better understand the role of
Nodal in human placental development. The molecular mechanisms of how Nodal regulates human trophoblast cell behaviors also await characterizations.
Activin is a dimer of disulde-linked Inhibin b subunits, and Inhibin is a heterodimer composed of a a subunit and a b
subunit. Activin and Inhibin are puried from gonadal uids based on their ability to stimulate or inhibit FSH release from
the pituitary. During pregnancy, the placenta is the predominant source of Activin A and Inhibin A in maternal circulation
(Florio et al., 2001; Muttukrishna et al., 1997). Their receptors are expressed in various types of trophoblast cells (Peng
et al., 1993, 1999; Schneider-Kolsky et al., 2002). In human placental trophoblast cells, Activin A stimulates hCG and progesterone secretion and CTB proliferation and enhances EVT cell migration/invasion, while Inhibin A counteracts these activities
(Caniggia et al., 1997; Florio et al., 1996; Petraglia et al., 1993, 1987, 1989). Recently, Activin A was reported to decrease the
production of cell surface integrin subunits a1, a2, a3, a5, b1, b2 and b4 and to inhibit the binding of cells to ECM ligands
such as bronectin and collagen type I and IV (Stoikos et al., 2010). In the pancreas, kidneys, and lung, Activin A is crucial in
the branching tubulogenesis (Ball and Risbridger, 2001), and it may upregulating VEGF and its receptors to amplify the effect
of VEGF (Maeshima et al., 2004). It is not yet clear whether Activin A can work similarly in placenta to enhance uterine vessel
remodeling. Physiologically, Activin activity can be modulated by follistatin, a 35 KD single-chain glycoprotein, which is able
to prevent the interaction of Activin A to its receptors (Luisi et al., 2001). Furthermore, another binding protein for Activin A
has been identied, namely follistatin-related gene (FLRG). FLRG is structurally similar to follistatin and is also capable of
interfering with the binding of Activin A to ActRs (Tsuchida et al., 2001). Follistatin is mainly localized in CTBs and STBs
(Petraglia et al., 1994), while FLRG is strongly localized to the decidual and placental blood vessels (Ciarmela et al.,
2003a). Therefore, it is most likely that follistatin and FLRG act separately in the placenta to modulate the actions of Activin
A on hormone secretion and the vasculature.
The binding of Inhibin A to ActRII is facilitated by the membrane-anchored proteoglycan betaglycan via the formation of a
ternary complex including Inhibin A, betaglycan and ActRII. Betaglycan can antagonize the Activin A signal by preventing the
binding of Activin A to ActRII (Gray et al., 2002). The localization of betaglycan in the human placenta is restricted to the STBs
(but not the CTBs) and villous endothelial cells (Ciarmela et al., 2003b), and co-localizes with FLRG (Ciarmela et al., 2003a)
and Activin receptors (Schneider-Kolsky et al., 2002). It is therefore possible that the balance between these binding proteins
may modulate the interaction of Activin A and Inhibin A to regulate trophoblast cell endocrine and vascular development.
This hypothesis has yet to be veried.
Activins and BMPs are also important regulators of the inammatory response in various cell types and tissues. The
expression of Activin has been detected in a variety of cell types in the immune system, such as monocytes (Abe et al.,
2002; Eramaa et al., 1992), macrophages (Ebert et al., 2007), dendritic cells (Robson et al., 2008), T and B lymphocytes (Ogawa et al., 2006, 2008) and mast cells (Cho et al., 2003; Funaba et al., 2003). Its expression can be enhanced by immune stimuli
and toll-like receptor agonists. Activin A regulates many of these immunogens. For instance, it negatively regulates the production of cytokines (IL-6, IL-12p70, TNF-a and IL-10) and chemokines (IL-8, IP-10 RANTES, and MCP-1) in blood dendritic
and monocyte-derived dendritic cells (Robson et al., 2008), and induces the directional migration of immature myeloid dendritic cells (iDCs) through the activation of ALK4 and ActRIIA receptor (Salogni et al., 2009). There is increasing evidence that
Activin modulates certain immune responses in an intricate autocrine/paracrine system, but there are few data documenting
Activin-regulated immune responses in the placenta.
4.2.3. HGF/c-Met signaling
Hepatocyte growth factor (HGF), also called scatter factor (SF), is a cytokine that regulates cell growth, differentiation and
morphogenesis in various tissues. It is also a potent angiogenic factor that stimulates the proliferation and migration of
endothelial and smooth muscle cells and promotes the formation of new blood vessels in murine subcutaneous tissue
and rat cornea angiogenesis models (Bussolino et al., 1992; Grant et al., 1993; Walter and Sane, 1999). Its transmembrane
tyrosine kinase receptor, c-Met, is a heterodimer composed of a- and b-chains linked via disulde bonds (Ma et al., 2003).
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The roles of HGF/c-Met signaling have been well documented in cancer development. This pathway is also pivotal in
mammalian placental development, because HGF knockout mice are embryonically lethal in utero due to an extreme reduction in the number of trophoblast cells in the labyrinth layer and have poorly perfused placentas (Uehara et al., 1995). In the
human placenta, HGF is produced by STBs, EVTs, endothelial cells and villous mesenchymal cells, while its receptor, c-Met, is
mainly expressed in trophoblast cells and villous endothelial cells (Kauma et al., 1997; Trovato et al., 2002; Yang et al., 2012).
HGF is a target of HIF-1a in trophoblast cells (Hayashi et al., 2005). The stimulating effects of HGF on trophoblast cell motility, migration, invasion and endothelial cell tubulogenesis have been extensively reported (Dokras et al., 2001; Fitzgerald
et al., 2005; Grant et al., 1993; Kauma et al., 1999; Saito et al., 1995; Walter and Sane, 1999) with the activation of Rho/
Rac, PI3K and ERK/MAPK (Jiang et al., 2005; Rosario and Birchmeier, 2003). Enhanced NO production and activation of associated cGMP signaling in response to activated MAPK are also involved in HGF function in human trophoblast cells (Ayling
et al., 2006; Cartwright et al., 2002). Recently, the homobox gene HLX, which is mainly expressed in EVTs, was shown to be a
novel downstream mediator in promoting HGF-mediated EVT cell invasion (Liu et al., 2012; Rajaraman et al., 2010). Very
recently, HGF was found to be a mediator in dNK cell-enhanced EVT cell migration (Fraser et al., 2012), indicating that it
participates in the immune recognition between dNK and EVT cells.
Studies have revealed that c-Met can be released from the cell membrane to form a soluble, truncated protein (sMet) that
is able to bind HGF and disrupt HGF/c-met signaling and its inuence on trophoblast cell invasion (Wajih et al., 2002; Yang
et al., 2012). We recently report that ADAM10 and ADAM17 are responsible for c-Met proteolysis in human trophoblast cells.
HGF could negatively regulate its effects on trophoblast cell invasion by downregulating c-Met expression and enhancing
receptor proteolysis (Yang et al., 2012). The self-feedback loop of HGF signaling likely reects the subtle control of trophoblast cell invasion. The subsequent question that remains to be elucidated is whether the patterns of HGF, c-Met and sMet in
the human placenta change temporally and spatially during gestation.
4.2.4. Notch signaling
Notch signaling is an evolutionarily conserved pathway from Drosophila to humans that governs the differentiation and
function of many tissues through cellcell contact (Bianchi et al., 2006; Miele, 2006). This pathway plays a particularly
important role in vascular patterning (Roca and Adams, 2007; Swift and Weinstein, 2009). Four Notch receptors (Notch1,
Notch2, Notch3 and Notch4) and ve Notch ligands (DLL1, DLL3, DLL4, Jag1 and Jag2) have been identied in mammals (Kopan and Ilagan, 2009). Each Notch receptor is a large, single-pass transmembrane receptor composed of an extracellular domain (NECD), a transmembrane domain (TM), and an intracellular domain (NICD) with transcriptional activity (Wharton
et al., 1985). The NECD consists of 1036 EGF-like repeats essential for ligand binding and three copies of juxtamembrane
repeat motifs known as Lin-12-Notch Repeats (LNRs) that modulate interactions between the extracellular and intracellular
domains of Notch. The NICD is composed of several functional domains and is subjected to a variety of post-translational
modications, including phosphorylation, ubiquitylation, hydroxylation and acetylation (Zhao and Lin, 2012). Activated
NICD binds to the promoter of NOTCH target genes via interaction with the transcription factor, RBPjk (recombination signal-binding protein 1 for J-kappa) (Dong et al., 2010).
Notch signaling regulates the differentiation of primitive endothelial cells into hierarchical networks by specifying arterial identity. In zebrash, loss of Notch signaling reduces endothelial efnb2 expression (markers of arterial identity) while
simultaneously enhancing ephb4 expression (markers of venous identity). Deciencies in Notch1, Notch2, Jag1, DLL4, RBPjk
or Notch targets, Hey1/Hey2 and Mash2, in mice result in embryonic lethality at mid-gestation, owing to the failed incorporation of the arterial vasculature into the circulation (Fischer et al., 2004; Gale et al., 2004; Guillemot et al., 1995; Krebs et al.,
2000; Roca and Adams, 2007; Swift and Weinstein, 2009; Xue et al., 1999). Therefore, it is plausible that Notch family members play a role in programming EVT-mediated arterial invasion.
The Notch signaling pathway has been implicated in trophoblast differentiation and the invasion of EVT cells based on the
presence of Notch receptors and ligands in different placental trophoblasts. Notch1, Notch2, Notch3, Notch4 and their ligands Jag1, Jag2, DLL1, and DLL4 are all found in the early and late placenta. Villous capillary endothelial cells express
Notch1, DLL4, Jag 1 and DLL1, while STBs and CTBs weakly express Notch1, Notch3 and DLL1 (De Falco et al., 2007; Herr
et al., 2011). A recent report form Hunkapiller et al. gives a more rened picture of the spatial change of Notch molecules
in the trophoblast as they invade the uterine wall (Hunkapiller et al., 2011). The authors show that Notch1 is undetectable
in CTBs, and that Notch2 is weakly and sporadically expressed in the CTB progenitors though strongly expressed in trophoblast column and EVTs. Notch3 is present in CTBs at all stages of differentiation. Notch4 immunoreactivity is high in CTB
progenitors and cell columns and is dramatically reduced in EVT cells, particularly enEVTs. DLL1 expression is observed
in the maternal cells adjacent to CTBs in the interstitial and endovascular compartments. DLL4 is detected in cell columns
but is rapidly lost as CTBs invade the uterine wall. Jag1 is found in CTBs that associate with the maternal spiral arterioles,
and Jag2 is not detected in CTBs. These immunohistochemical results are recapitulated in the in vitro differentiation/invasion
model of CTBs grown on Matrigel, indicating that CTBs can dramatically alter their expression of Notch receptors and ligands
as they differentiate/invade (Hunkapiller et al., 2011).
A role of Notch signaling in promoting vascular invasion of EVTs has been suggested. The conditioned deletion of Notch2
in mice leads to a dramatical reduction in the placental perfusion and arterial invasion by trophoblasts (Gasperowicz and
Otto, 2008). Similarly, when human CTBs are grown on Matrigel, Notch promotes their invasion and the acquisition of an
arterial endothelial cell-like phenotype that is marked by EFNB2 upregulation (Hunkapiller et al., 2011). In addition, one
observation is that the decrease in Notch proteins in the placentas of embryos affected by fetal growth retardation (FGR)
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coincides with a reduction in placental weight (Herr et al., 2011), which may suggest an involvement of Notch signaling in
human trophoblast proliferation. Taken together, these in vitro and in vivo data indicate that Notch signaling appears to be
crucial for TB vascular invasion. However, more studies are required to further elucidate the role and molecular mechanisms
by which members of the Notch pathway exert their roles in trophoblast differentiation and placental angiogenesis.
4.2.5. Wnt signaling
Wnt signaling regulates complex cellular responses through a highly organized network of different ligands, receptors
and downstream mediators. The canonical Wnt signaling pathway functions through FZDLRP heterodimeric receptors,
which regulates the activity and recruitment of b-catenin. In unstimulated cells, b-catenin is localized to the adhere junctions and is mainly involved in maintaining epithelial integrity by binding to E-cadherin and a-catenin. In the absence of
Wnt ligands (off-state), b-catenin forms a complex with the tumor suppressor APC (adenomatous polyposis coli) and the
scaffold protein Axin, both of which facilitate the phosphorylation of the protein by casein kinase Ia (CKIa) and glycogen
synthase kinase 3b (GSK-3b) (Gordon and Nusse, 2006). Upon phosphorylation, b-catenin is ubiquitinated by the b-TrCP
E3-ligase complex and subsequently degraded by the proteasomes. In the presence of Wnt ligands (on state), non-phosphorylated b-catenin is released from the Axin/APC/GSK-3b/CK1a destruction complex, accumulates in the cytoplasm, and is subsequently translocated into the nucleus, where it triggers transcription of Wnt target genes (Sonderegger et al., 2010b). The
non-canonical Wnt signaling is mediated by b-catenin-independent cascades, such as the Ca2+-dependent and the planar cell
polarity (PCP) pathways (Gordon and Nusse, 2006). Signaling through the Wnt/PCP pathway results in the activation of the
RhoA and Rac GTPases and their downstream targets ROCK (Rho-associated kinase) and JNK, respectively. Wnt increases Ca2+
levels by either inhibiting cGMP-dependent protein kinase (PKG) (Ma and Wang, 2006) or activating PLC (phospholipase C)
and elevating inositol 1,4,5-trisphosphate (IP3) levels. Increased intracellular Ca2+ in turn activates PKC, calcium/calmodulindependent kinase II (CamKII) and calcineurin (Kohn and Moon, 2005). Wnts can also signal through the receptor tyrosine
kinase Ryk and the receptor tyrosine kinase-like orphan receptor (Ror) upon binding to their extracellular WIF and CRD domains, respectively (Hendrickx and Leyns, 2008). Furthermore, Wnt ligands such as Wnt3a or Wnt16b may activate other bcatenin-independent cascades such as the ERK or PI3K/AKT signaling pathways (Kim et al., 2007; Yun et al., 2005).
Growing evidence suggests that the canonical Wnt/b-catenin pathway and the activation of Wnt-dependent transcription
factors are also critically involved in placental development and trophoblast differentiation. The expression of Wnt2, Wnt3,
Wnt4, Wnt5a, Wnt7a, and Wnt8b in human endometrial cells suggests that these factors may potentially control invading
trophoblast cells in a paracrine manner (Tulac et al., 2003). Fourteen of the 19 Wnt ligands and 8 out of 10 Fzd receptors are
detected in the human placenta, and the expression of Wnts and Fzds are also observed to be gestation-dependent and trophoblast subtype-specic (Sonderegger et al., 2007). TCF4 is induced in the invasive trophoblast (Pollheimer et al., 2006), and
the co-localization of b-catenin and TCF4 in EVTs suggested that the b-catenin/TCF DNA-binding complexes could be involved in the invasive differentiation process. It has been shown in many cell types that Wnt/TCF signaling promotes cell
proliferation and migration by activating critical target genes associated with either growth, such as cyclin D1 and c-Myc
(He et al., 2008; Tetsu and McCormick, 1999), or invasion, such as MMP-7 and MMT1-MMP (Brabletz et al., 2002; Takahashi
et al., 2002). In the human placenta, Wnt3a stimulates trophoblast proliferation by inducing active b-catenin/TCF transcription factor complexes and the expression of the target gene cyclin D1 (Pollheimer et al., 2006). A more recent study reveals
that canonical Wnt signaling can regulate the degree of trophoblast cell invasion (Sonderegger et al., 2010a). In rst-trimester villous explant cultures and SGHPL-5 cells, canonical Wnt signaling and PI3K/AKT signaling are independently involved in
mediating Wnt3a-stimulated trophoblast cell migration and the release of MMP-2, which has been identied as a novel Wnt
target in human trophoblast cells. An earlier study also indicated that Wnt3a and Wnt5a may act in an antagonistic way in
trophoblast cells (Sonderegger et al., 2007). However, a large gap remains in understanding the role of Wnt signaling in human placental development.
4.3. Hormones
4.3.1. GnRH
Mammalian GnRH (GnRH I) is a well-known hypothalamic hormone that is important in reproduction. The GnRH II subtype is specically identied in humans and differs from GnRH I by three amino acid residues. The roles of GnRH in extrapituitary compartments such as the ovary, placenta, uterus, and immune system are also recognized (Cheng and Leung, 2005;
Ortmann and Diedrich, 1999). In the human placenta, GnRH I is widely expressed in various trophoblast subpopulations
throughout gestation (Chou et al., 2004; Khodr and Siler-Khodr, 1980), whereas GnRH II expression is restricted to villous
CTBs and EVTs in the rst trimester (Chou et al., 2004). The expression of GnRH receptor (GnRHR) is also detected in human
trophoblast cells (Cheng et al., 2000; Lin et al., 1995). In addition to stimulating the productions of hCG (Khodr and SilerKhodr, 1978a,b), prostaglandin (Siler-Khodr et al., 1986), hPL and hCS from the placenta, both subtypes of GnRH can strongly
enhance EVTs invasion with the upregulation of several invasion-associated proteases, such as MMP-2, MMP-9, MMP-26,
TIMP-1, uPA, and PAI (Chou et al., 2003; Chou et al., 2002; Liu et al., 2010). These effects of GnRH are mediated by the activation of PKC, ERK1/2, and c-Jun N-terminal kinase (Liu et al., 2010). Interestingly, GnRH II elicits invasion-promoting action
by transactivating the tyrosine kinase activity of EGF receptor in trophoblast cells (Liu et al., 2009). It is unclear, however,
whether the invasion-promoting ability of GnRH in EVTs is its direct effect or it is through the regulation of other hormones
such as hCG.
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4.3.2. hCG
HCG is among one of the most important hormones produced by the placenta during pregnancy (Gohar et al., 1996;
Handschuh et al., 2007a). The classical function of hCG is to stimulate progesterone production and to participate in the
syncytialization process. New recognitions on the function of hCG in trophoblast cell invasion and angiogenesis emerges
in recent years, primarily based upon its regulation on the production of angiogenic factor VEGF, as well as the discovery
of hyperglycosylated hCG (H-hCG).
HCG has been hypothesized to be a tissue-specic angiogenic factor. It inuences the expression of angiogenic factors
including VEGF and its receptors, angiopoietins and their receptor Tie-2, basic broblast growth factor or PlGF in the ovaries
and testes (Reisinger et al., 2007). This hormone also stimulates capillary formation in endothelial cells in vitro and in an
in vivo chicken chorioallantoic membrane assay (Zygmunt et al., 2002). HCG has been shown to stimulate VEGF production
in human trophoblast cells (Islami et al., 2003), and enhance the migration/invasion properties of trophoblast cells by inuencing the availability of insulin-like growth factor-II (IGF-II), an essential factor for migration of placental and tumor cells
(Zygmunt et al., 1998, 2005). Moreover, the hCG receptor is expressed in the placental vascular tree and it can stimulate the
proliferation of placental microvascular endothelial cell (PMVEC) and vessel formation, demonstrating the role of hCG in placental vascular development (Herr et al., 2007). Recently, a link between hCG and another angiogenic factor, EG-VEGF, has
provided new evidence for the angiogenic role of hCG. HCG was shown to stimulate the productions of EG-VEGF and its two
receptors, PROKR1 and PROKR2, in human placental villus explants and isolated CTBs through the cAMP signaling transduction pathway (Brouillet et al., 2012).
H-hCG, one of the ve isoforms of hCG with more glycol-side chains, is mainly produced by CTBs and EVTs (Handschuh
et al., 2007b). H-hCG is the most acidic glycoprotein currently known, with a peak acidity of 3.1 and 39% sugar content by
molecular weight. It has multiple functions that differ from hCG and may promote trophoblast cell invasion (Cole et al.,
2006a,b; Handschuh et al., 2007b) and MMP activity by antagonizing TGF-b signaling (Cole, 2012). H-hCG may also work
with hCG to promote uterine artery angiogenesis and facilitate umbilical circulation. Therefore, this hormone has been
suggested to be vital for driving hemochorial placentation (Cole, 2012). The functional variation between H-hCG and hCG
is also an interesting topic in the evolution of the hemochorial placentation, as discussed in reviews by Cole LA (Cole,
2010, 2012).
4.4. MicroRNAs (miRNAs)

4.4.1. Expression of miRNAs in the human placenta
MicroRNAs are highly conserved endogenous small (21- to 23-nucleotide) single-stranded RNA molecules. The rst miRNA, lin-4, was discovered in C. elegans in 1993 (Lee et al., 1993). Subsequently, a number of miRNAs have been described in
mammals, including humans (Barad et al., 2004; He and Hannon, 2004). It is now well-accepted that miRNAs play critical
roles in developmental and physiological processes by post-transcriptionally regulating gene expression.
The biogenesis of miRNAs involves several steps. First, the individual miRNA gene is transcribed into a primary transcript
(pri-miRNA) by RNA polymerase II (Bartel, 2004; Cai et al., 2004). The pri-miRNA folds into a distinctive stem-loop structure
and the miRNA portion forms a hairpin. Drosha, a dsRNARNA-specic ribonuclease, and the DiGeorge Syndrome Critical
Region 8 (DGCR8) cleave the pri-miRNA to produce a precursor miRNA (pre-miRNA) with 7075 nucleotides of RNA in length
(Lee et al., 2003). The pre-miRNA is then exported from the nucleus to the cytoplasm by a complex containing Exportin-5
(Exp5). In the cytoplasm, the pre-miRNA is further cleaved by Dicer, an RNase III superfamily member, to form short miRNA
duplexes (Lund et al., 2004). Finally, the duplexes unwind and one of the strands is incorporated into the miRNA-induced
silencing complex (miRISC) (Denli et al., 2004; Gregory et al., 2004; Guo et al., 2010). The miRNA then guides the complex
to its targets through WatsonCrick base-pairing interactions. In general, miRNAs post-transcriptionally regulate gene
expression by partially base pairing to the 30 untranslated regions (30 UTRs) of their target mRNAs, which leads to target
mRNA degradation (Guo et al., 2010) and/or the inhibition of protein translational (Bartel, 2004, 2009; Huang et al., 2010;
Nilsen, 2007; Zhao and Liu, 2009).
In human placenta, miRNAs are initially identied using a MIR-specic oligonucleotide microarray (Barad et al., 2004).
Subsequently, core miRNA biogenesis proteins are detected in human villous trophoblast cells (Donker et al., 2007), demonstrating that trophoblast cells can produce miRNAs. It is currently believed that many miRNAs are expressed abundantly in
the human placenta and that some are specically expressed in placental tissues. A recent report identied the expression of
325 miRNAs in the placenta by analyzing the expression prole of up to 820 miRNAs using placental tissue samples collected
in the rst or third trimester (Mayor-Lynn et al., 2011). To date, many miRNA proling studies have been conducted using
the placentas from normal and compromised pregnancies. For example, the largest gene cluster of human miRNA, the chromosome 19 miRNA cluster (C19MC) miRNAs, have been found to be among the most abundant miRNAs that are exclusively
expressed in human term placental trophoblast cells and in other undifferentiated cells (Bentwich et al., 2005; Bortolin-Cavaille et al., 2009; Donker et al., 2012; Landgraf et al., 2007). The trophoblast-specic C19MC miRNAs can be released into the
maternal circulation in exosomes or bound to plasma proteins (Donker et al., 2012; Kotlabova et al., 2011). They may have
roles in the placentalmaternal communication and possibly direct the maternal adaptation to pregnancy. Other miRNAs
abundant in the placenta are also released into the maternal plasma during pregnancy (Chim et al., 2008), which allows them
to act as a new class of molecular markers for monitoring pregnancy.
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As we emphasized above, placental development is a dynamic process during which the trophoblast cells differentiate
and behave differently throughout gestation. Therefore, the precise expression timing and localization of miRNAs during placental development must be considered to identify the mechanisms of their action. This point is well-reected in a recent
work by Morales-Prieto et al. (Morales-Prieto et al., 2012). They comparatively analyzed the expression level of 762 different
miRNAs in isolated CTBs from healthy term and rst trimester placentas. The expression of the C19MC miRNAs (containing
54 different miRNAs) increased signicantly in the trophoblast cells isolated from the rst to third trimester, while the
expression of the C14MC members (34 miRNAs) decreased and miRNAs within the miR-371-3 cluster were slightly augmented. Another 27 miRNAs were differentially expressed (>100 fold) between the trophoblast cells isolated from term
and rst trimester placentas (Morales-Prieto et al., 2012). We recently observed that miR-378a-5p (Luo et al., 2012) level
varied with gestational stages. The major differences in the miRNA ngerprints between rst trimester and term trophoblast
cells may be due to their different behaviors and characteristics.
4.4.2. Regulation of miRNA expression in trophoblast cells
The regulation of miRNA expression has been extensively studied in various tissues and tumor cells. However, there is
still little evidence describing how miRNA expression is regulated in human placenta. Several studies suggest that hypoxia
is an important regulatory factor. An earlier study revealed that miR-93 is up-regulated and miR-424 is down-regulated in
trophoblast cells under hypoxic conditions (Donker et al., 2007). This nding is conrmed in a recent study that also identifys
additional miRNAs (miR-205, miR-224, miR-335, miR-451, and miR-491) that are upregulated in human primary trophoblast
cells exposed to hypoxic conditions (Mouillet et al., 2010). Among these miRNAs, miR-205 targets a transcriptional coactivator MED1 (Mouillet et al., 2010). Considering the developmental defects observed in the placentas and hearts in Med1decient mice (Crawford et al., 2002; Landles et al., 2003), miR-205 is suggested to function in trophoblast injury. MiR210 is well documented to be induced by hypoxia in both human trophoblast cells (Enquobahrie et al., 2011; Pineles
et al., 2007) and tumor cells (Huang et al., 2009; Zhang et al., 2009). Biochemical and genetic data have revealed that the
miR-210 promoter carries a functional hypoxia-responsive element (HRE) that is recognized by HIF-1a and induces miRNA
transcription upon exposure to hypoxia (Chan and Loscalzo, 2010; Huang et al., 2009; Pulkkinen et al., 2008). In vitro and
in vivo ndings reveal that chronic hypoxia impairs Dicer expression and activity, which affects both HIF-a isoforms and hypoxia-responsive genes in vascular endothelium and result in global consequences in microRNA biogenesis (Ho et al., 2012).
A recent in vitro study in rst trimester placental explants revealed that knocked down Dicer with a specic siRNA resulted in
a global reduction in miRNAs (Forbes et al., 2012). However, it is unclear whether Dicer expression is inuenced by hypoxia
in placental trophoblast cells, as Ho et al. reported in endothelial cells (Ho et al., 2012).
Epigenetic regulation, specically promoter methylation, has been described in several reports and is one of the well-recognized mechanisms of regulating miRNA expression. Some placenta-specic miRNAs have been demonstrated to be epigenetically regulated; one example is the primate placenta-specic gene cluster, C19MC. In most cells, the low expression of
C19MC can be restored by the demethylating agent 5-aza-20 -deoxycytidine (5-Aza-dC) (Tsai et al., 2009). The expression pattern of C19MC is highly correlated with the methylation state of a distal CpG-rich region located approximately 17.6 kb upstream of the miRNA cluster. This CpG-rich region is hypermethylated in gastric cancer cells and HeLa cells that poorly
express C19MC miRNAs, but is hypomethylated in the placenta tissues which express high levels of C19MC miRNAs (Tsai
et al., 2009). Moreover, the work from Noguer-Dance and colleagues adds C19MC to the growing list of imprinted repeated
small RNA gene clusters. These authors reveal a maternal-specic methylation imprint of C19MC that shows DNA methylation in a differentially methylated region (C19MC-DMR1) that overlaps the upstream CpG-rich promoter region. Such genomic imprinting causes the paternally inherited allele being expressed in the placenta (Noguer-Dance et al., 2010).
LIF, a major regulator of embryonic implantation, has been reported to regulate placental miRNA expression. In JEG-3
cells, LIF treatment signicantly stimulates the expression of miR-21 and miR-93 while repressing miR141 expression (Morales-Prieto et al., 2011). The mechanism by which LIF regulate these miRNAs remains unrevealed. Several endocrine disruptors can alter the expression patterns of miRNAs in trophoblast cells. For example, miR-16, miR-21 and miR-146a are
downregulated upon cigarette smoke challenge (Maccani et al., 2010), and nicotine and benzo(a)pyrene treatments can
dose-dependently reduce miR-146a in trophoblastic TCL-1 cells (Maccani et al., 2010). Interestingly, miR-146 can also be
strongly inhibited by bisphenol A exposure in human trophoblast cell lines (TCL-1 and HTR8) (Avissar-Whiting et al.,
2010). Accordingly, these miRNAs may be responsible for regulating cell stress in trophoblast cells.
4.4.3. Modulation of trophoblast cellular activities by miRNAs
Although the placenta expresses a large number of miRNAs, the function of most of the miRNAs in the placenta are largely
unknown. Considering that the abnormal expression of miRNAs in the placenta has been implicated in compromised pregnancies, such as preeclampsia and IUGR (Enquobahrie et al., 2011; Mouillet et al., 2010; Pineles et al., 2007), which is discussed in details in Section 5 of this review, the signicance of miRNAs as major modulators of placental development is
generally emerging. Indeed, increasing evidence suggests that miRNAs are involved in the regulation of trophoblast cell proliferation, apoptosis, migration and invasion.
In rst trimester placental explants, knockdown of Dicer signicantly enhances cytotrophoblast cell proliferation and the
expression of two pro-mitogenic signaling molecules within cytotrophoblasts, ERK and SHP-2 (Forbes et al., 2012). There are
some studies that have identied individual miRNAs involved in regulating trophoblast proliferation. For instance, miRNA155 inhibits cell proliferation by down-regulating cyclin D1/p27 in HTR-8/SVneo cells (Dai et al., 2012). MiR-141 is highly
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expressed in rst trimester trophoblast cells as well as in HTR8/SVneo cells and JEG-3 cells (Morales-Prieto et al., 2012).
Downregulation of miR-141 decreases trophoblast proliferation, indicating the participation of this miRNA in modulating
trophoblast cell growth in early pregnancy (Morales-Prieto et al., 2011). Similarly, miR-378a-5p enhances trophoblast cell
proliferation and survival, in part by downregulating Nodal, in HTR8/SVneo cells (Luo et al., 2012). MiR-21 is abundantly expressed in rst trimester trophoblast cells (Morales-Prieto et al., 2012, 2011). This small RNA stimulates cell proliferation in
ovarian epithelial carcinomas by targeting PTEN protein (Lou et al., 2010), but it is unclear whether miR-21 can regulate trophoblast cell growth in a similar way.
The roles of miRNAs in modulating trophoblast cell differentiation towards an invasive pathway are also reported. The
microRNAs that inhibit trophoblast cell invasion include miR-34a which targets Notch1 and Jagged1 (Pang et al., 2010) or
PAI-I (Umemura et al., 2012), and miR-29b that targets Mcl-1, MMP2, VEGFA, and integrin b1 (Li et al., 2013). MiR-210 also
has inhibitory effects on trophoblast cell migration and invasion and the target genes that mediate these effects include Ephrin-A3 and Homeobox-A9 (Zhang et al., 2012). Several invasion-promoting miRNAs are also identied, such as miR-195 (Bai
et al., 2012), miR-376c (Fu et al., 2013), miR-378-5p (Luo et al., 2012) and miR-20b (Wang et al., 2012). Interestingly, miR195, miR-376c, and miR-378-5p target different components in Activin/Nodal signaling, such as ActRIIA, ALK7 and Nodal,
resulting in enhanced trophoblast cell invasion. Our unpublished data also reveals that miR-18a can promote EVT cell invasion by targeting Smad2 (Fig. 2). Activin A and Nodal share the same type II receptors (ActRIIA and ActRIIB) and signaling
mediators (Smad2/3), but they exhibit opposite roles in regulating trophoblast cell invasion. Based on these ndings, we propose that the level of these small RNAs may contribute to balancing Activin A signaling and Nodal signaling in trophoblasts.
Fig. 2. Regulation of Nodal/Activin A signaling pathway by miRNAs. Activin A and Nodal share the same receptors (ActRIIA and ALK4) and signaling
mediators (Smad2/3 and Smad4). Several miRNAs are experimentally demonstrated to target different molecules in this signaling pathway. These include
miR-378-5p that targets Nodal, miR-376c that targets ALK7, miR-195 that modulates ActRIIA, and miR-18a that targets Smad2. The imprinted H19 gene
encodes miR-675, which negatively regulates the expression of NOMO1, an antagonist of Nodal signaling. In severe preeclamptic placentas, the expression
levels of these miRNAs are evidently downregulated, and the expressions of their targets are upregulated. High concentration of Activin A that is observed in
preecplamtic placenta can stimulate Nodal expression. We propose that the proper levels of these small RNAs are critical to balance Activin A signaling and
Nodal signaling in trophoblast. Aberrant expression of these miRNAs may contribute to the dysregulation of trophoblast cell apoptosis and invasion in PE
placenta.
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The nding that these miRNAs are aberrantly expressed in preeclamptic placenta (discussed in Section 5) further supports
our hypothesis. MiR-20b was recently shown to regulate EPHB4 and ephrin-B2 expression in human trophoblasts and endothelial cells (Wang et al., 2012). It has been previously demonstrated that EPHephrin interactions can pattern cytotrophoblast invasion by generating repulsive signals to direct cytotrophoblast invasion toward the uterine wall and allowing
invasive cytotrophoblast migration only toward ephrin-B2 expressing arterioles but not EPHB4-expressing veins (Red-Horse
et al., 2005). It is therefore suggested that miR-20a may have an important role in vascular remodeling during human placental development.
Several miRNAs are also involved in other aspects of placental functions. For instance, miR-148a and miR-152 target and
down-regulate HLA-G expression and abolish the LILRB1-mediated inhibition of NK cell killing (Manaster et al., 2012). miR210 and miR-518c have been experimentally validated to target HSD17B1, a steroidogenic enzyme that catalyzes the conversion of estrone to 17b-estradiol (Ishibashi et al., 2012). A variety of miRNAs have been associated with different cell activities in other cell types, especially cancer cells, and various target genes pertaining to these cellular events have been
experimentally validated. It remains unclear whether these results can be applied to the modulation of placental cell functions. However, the existing evidence seems to suggest that miRNAs ne-tune homoeostasis in the placenta by targeting a
large number of genes and participating in various cellular events.
It is much efcient to understand the functional mechanisms of miRNAs by identifying and clustering the targets of different small RNAs into similar signaling pathways, no matter these small RNAs exist in the same family, gene cluster or are
independent. This strategy has been reected in many studies, including ours, in different tissues (Bai et al., 2012; Ishibashi
et al., 2012; Kim et al., 2012; Luo et al., 2012; Renthal et al., 2010). However, a single miRNA can target multiple genes, and
identifying which miRNA plays a role in different cell types largely depends on the microenvironment encountered by the
cells and the properties of the cell. It becomes useful to elucidate the real function of a unique miRNA by picturing its global prole of targeting genes in the context of specic cellular event using the combined methods of gene chip, proteomics,
bioinformatics and molecular biology techniques (Leivonen et al., 2011; Yang et al., 2009). Whats more, while the in vitro
studies discussed above provide initial evidence for the potential role of miRNAs in regulating trophoblast activities, their
precise physiological roles during placental development requires further investigation, particularly in vivo studies.
4.4.4. The imprinted H19 gene functions via encoding miR-675 in the placenta
Many long imprinted non-coding RNAs act as precursors of small regulatory RNAs, including small nucleolar RNAs, miRNAs and piRNAs, known as antisense trans-acting regulators of gene expression. H19 was the rst imprinted noncoding transcript to be identied, and the imprinting of the H19-Igf2 locus on chromosome 11p15.5 has been proven to be regulated via
a chromosomal looping mechanism, resulting in maternal H19 and paternal Igf2 monoallelic expression. The H19 gene does
not encode a protein but instead encodes a capped, spliced and polyadenylated 2.7 kb RNA (Brannan et al., 1990; Lustig et al.,
1994; Matouk et al., 2007). H19 is thought to regulate adjacent genes including Igf2 and insulin 2, in either an enhancercompetition model or a boundary model (Li et al., 1998).
H19 gene is abundantly expressed in placenta and fetal tissues while being postnatally down-regulated (Goshen et al.,
1993; Lustig et al., 1994). In the placenta, H19 is abundant in villous CTBs and column CTBs, but not in STBs (Adam et al.,
1996; Mutter et al., 1993), which indicates its potential to modulate trophoblast cell proliferation. Deletion of the H19 gene
and the H19/Igf2-imprinting control region (also known as H19 DMD) in mice leads to increased levels of Igf2 and fetal overgrowth as well as hyperplasia of all layers in the placenta. It has long been suggested that the increase in placental weight in
these knockout mice arises from the duplication of the Igf2 transcripts.
Recently, it is revealed that miR-675 is produced from the rst exon of the H19 gene, and that the pre-miR-675 region has
been highly conserved for 148 million years (Smits et al., 2008; Wilusz et al., 2009). In mice, exon 1 of H19 harbors a miRNAcontaining hairpin that serves as the template for two distinct miRNAs, miR-675-5p and miR-675-3p (Huntzinger and Izaurralde, 2011). These ndings suggest that H19 can also act via a post-transcriptional mechanism apart from imprinting regulation. A very recent study from Reik and colleagues (Keniry et al., 2012) clearly described the physiological function of H19
in placental development. Using several genetically manipulated mouse models, they show that miR-675 is expressed exclusively in the placenta during late gestation. This is a period when placental growth normally begins to cease, but the placentas that lack H19 continue to grow. Several targets of the miR-675, including the growth-promoting gene Igf1r, are
upregulated in the H19 null placentas. The inhibitory effect of miR-675 on proliferation is observed in a range of embryonic
and extra-embryonic cell lines. Moreover, the stress-response RNA-binding protein, HuR, is identied as a negative regulator
of miR-675 processing by specically binding to the H19 gene (Keniry et al., 2012). This elegant study is the rst to reveal
that the main physiological role of H19 is to act as a developmental reservoir of miR-675 and limit prenatal placental growth
by regulating miR-675 processing. Our recent study in the human placenta trophoblasts agree with the report by Keniry et al.
(2012). We demonstrate that H19 gene can encode miR-675 and inhibit human trophoblast cell proliferation. We also show
that Nodal Modulator 1 (NOMO1) is one of the target genes involved in the growth-suppressive effect of miR-675 (Gao et al.,
2012). Considering the highly conserved features of H19 gene throughout mammalian evolution (Smits et al., 2008), this
imprinted gene may be regulated and act as a reservoir of the growth-restrictive miR-675 during human placental
development. It may also be involved in the molecular pathology of pregnancy complications or cancer, but more investigation is required.
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5. Implications for the pathogenesis of preeclampsia (PE)

5.1. Overview of Preeclampsia
Preeclampsia (PE) is the leading cause of maternal morbidity, mortality and premature delivery affecting approximately 2
7% of pregnancies (Cartwright et al., 2010). These numbers are dramatically higher in developing countries, such as Columbia,
where 42% of all pregnancy-related deaths are attributed to this syndrome (Noris et al., 2005). This condition is typically diagnosed after 20 weeks of gestation by the presence of non-preexisting hypertension (P160/110 mmHg) and proteinuria (P5 g
in 24 h) in the mother and is accompanied by problems in multiple organs, such as pulmonary oedema, seizures, oliguria,
abnormal liver enzymes associated with persistent epigastric or right upper-quadrant pain, or persistent and severe CNS
symptoms (Gaiser, 2008; Sibai, 2003). Although preeclampsia is considered to be a late-pregnancy disorder based on clinical
data, the molecular events leading to its onset have been suggested to occur early in pregnancy. It is generally accepted that
the presence of a placenta, and more specically, the trophoblast cells, is a major cause of preeclampsia.
Two broad categories of preeclampsia have been suggested, placental PE and maternal PE (Redman and Sargent, 2005).
Maternal PE is considered to arise from the interaction between a normal placenta and an abnormal maternal response constituting of metabolic or microvascular disease such as long-term hypertension or diabetes. In this regard, the clinical manifestation is more likely to be inuenced by environmental or maternal nutrition factors, and usually occurs at relatively late
stage of pregnancy. In contrast, placental PE is considered to originate from a mal-functioning placenta under hypoxic and
oxidative stress. However, the classication of these two types of PE has not been well dened, and the combination of
maternal and placental contribution has been most commonly presented.
The pathological changes in the PE placenta have been reported in an increasing number of studies. Most notably, the
anchoring villi that give rise to the invasive trophoblast cells are the most severely affected (Gaiser, 2008; Noris et al.,
2005; Red-Horse et al., 2004; Silasi et al., 2010; Wang et al., 2009). Interstitial EVTs are signicantly reduced both in number
and density in the deciduas of these compromised pregnancies (Noris et al., 2005). Furthermore, the degree of their invasion
into the uterine parenchyma varies but is frequently much shallower than in non-compromised pregnancies (Red-Horse
et al., 2004). In the most severe cases that occur early in pregnancy, endovascular EVT migration and blood vessel invasion
is consistently incomplete, and it is extremely difcult to nd remodeled maternal vessels that contain enEVTs (Red-Horse
et al., 2004; Zhou et al., 1997a). Furthermore, EVT cells fail to induce the expression of stage-specic antigens that are normally upregulated as they penetrate the uterine blood vessels, indicating a defect in their differentiation towards the endovascular phenotype (Lala and Chakraborty, 2003; Red-Horse et al., 2004; Wang et al., 2009; Zhou et al., 1997a). An increase in
EVT cell apoptosis is also observed in PE placentas (Lala and Chakraborty, 2003). Poor EVT differentiation and an accumulation of intermediate cytotrophoblast cells have been observed and may contribute to the overall smaller PE placental size
(Cheng and Wang, 2009). Whats more, a decrease in the expression of the GCM-1 transcription factor and an increase in
syncytial shedding are observed due to insufcient spiral artery remodeling, which partially occurs because the villi are exposed to a higher velocity blood ow (Baczyk et al., 2009; Cartwright et al., 2010). In addition to the increased syncytial shedding, there is a switch from the normal apoptotic to the necrotic pathway, increasing the pro-inammatory characteristics of
the debris compared with normal pregnancies. These ndings are consistent with the observed increase in systemic inammation in the third trimester of PE pregnancies (Cartwright et al., 2010; Lockwood et al., 2011).
Currently, our understanding concerning this compromised pregnancy is improving. It is generally agreed that this syndrome is complex, that it is difcult to dene the causes, and that it is not equivalent to a disease with a dened pathogenesis. To date, a two-stage model of preeclampsia has been widely accepted. The rst stage is the poor development of the
early placenta and utero-placental blood supply that occurs in the rst half of pregnancy when there are no clinical features
of the disorder. The second stage is the manifestation of the maternal signs of the condition that arises from factors released
by the placenta as it is placed under an increasing amount of oxidative stress (Redman and Sargent, 2005). The model is further modied by Roberts and colleagues (Roberts and Hubel, 2009) based on current knowledge. Poor placentation has been
suggested to occur as early as the rst trimester of pregnancy before the blood vessel remodeling. It should also be considered that the maternal factors, including genetic, behavioral and environmental factors, may reciprocally interact with the
rst stage in multiple ways.
Currently, the only treatment available for PE is still premature delivery/termination of pregnancy. If left untreated, PE
can results in serious health consequences for both the mother and the fetus. The major challenge is to effectively diagnose
the condition early, and develop preventive and therapeutic strategies that will minimize the burden of PE, all of which will
largely depend on the in-depth study of its pathogenesis.
5.2. The injury from hypoxiareoxygenation

Hypoxiareoxygenation, or ischemiareperfusion, causes cell injury in different tissues (Grifths and Halestrap, 1993;
Hernandez et al., 1987; Hess and Manson, 1984; Levinson et al., 1986; Li and Jackson, 2002; McCord, 1985; Oliver et al.,
1990; Robin and Theodore, 1982). During hypoxia, the lack of oxygen leads to the excessive storage of electrons and superoxide formation. Hypoxia can also increase the production of oxygen free radicals by stimulating TNF-a expression (Conrad
et al., 1998; Wang and Walsh, 1996b). Reoxygenation leads to a burst of reactive oxygen specie (ROS) production, which
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further induces cell necrosis, apoptosis and many other pathological changes (Hung and Burton, 2006). Physiologically,
hypoxiareoxygenation occurs during placenta development, and certain antioxidant enzymes expressed in villous
tissue can protect the tissue from such oxidative stress (Hung and Burton, 2006; Myatt and Cui, 2004; Watson et al.,
1997, 1998).
Excessive lipid peroxidation (Wang and Walsh, 1998) and decreased activity of superoxide dehydrogenases (SODs) are
observed in the PE placenta (Wang and Walsh, 1996a, 1998, 2001), indicating an overproduction of ROS at the expense of
antioxidant defenses. Such oxidative stress is most likely the cause of the increase in placental cell apoptosis and further
necrosis (Hung et al., 2001; Shibata et al., 2001), which leads to the release of trophoblast cell debris into the maternal circulation. Based on this recognition, an encouraging clinical trial of antioxidant vitamins C and E was carried out to investigate the potential on reducing the risk for PE. The randomized trials showed a substantial alleviation of the maternal
symptoms, but did not reduce the risk of preeclampsia or intrauterine growth restriction (Raijmakers et al., 2004; Rumbold
et al., 2006). Larger multicenter trials are needed for a nal conclusion. However, hypoxiareoxygenation and the resulting
oxidative stress are still considered vital for the onset of preeclampsia.
5.3. Placental factors that contribute to preeclampsia
During pregnancy, the placenta is highly active to produce various hormones and factors. Some of the secreted factors can
be detected in the maternal circulation, even at the very early stages of gestation. A dysfunctional placenta from a PE patient
releases factors of different concentrations and isoforms into the maternal circulation compared with normal placentas.
Accordingly, identifying differential factors in the PE serum/plasma is a feasible way to nd predictive biomarker candidates
for the disease. Moreover, these candidates partially reect the defect in the placenta and may cause the maternal clinical
features. In this regard, they are extremely valuable for elucidating the pathogenesis of PE.
Many differential plasma/serum proteins in PE patients have been reported, and most of them are thought to be derived
from the placenta. These include VEGF, PlGF, sFlt-1, sEng, Activin A, Inhibin A, hCG, cystatin C, pregnancy-associated plasma
protein-A (PPAP-A), Placental protein 13 (PP13), fetal hemoglobin (HbF), a1-microglobulin (A1M), soluble c-Met (sMet), leptin, IGF-I, corticotropin-releasing factor (CRF) and CRF-binding protein (CRF-BP), ADAM12, P-Selectin, adrenomedullin, and
auto antibodies against the angiotensin II type 1 (AT1) receptor (AT1-AA), among others (Fig. 3). The number of PE biomarkers is increasing in conjunction with the advancement of techniques. In this article, we will only review some recent advances and focus in particular on the advances that are supported by in vivo experiments.
5.3.1. Angiogenic and anti-angiogenic factors
The most prominent PE biomarkers are circulating angiogenic factors such as VEGF, PlGF and one of their antagonizing
receptors, sFlt-1. Soluble Flt-1 is generated through alternative splicing and is capable of binding and neutralizing PlGF
and VEGF. The placenta is a rich source of these factors, and maternally circulating PlGF decreases while sFlt-1 consistently
increases in the third trimester. In PE patients, these changes can be detected earlier and are more pronounced, making it
possible to predict the onset of the disease with the ratio of sFlt/PlGF at approximately 5 weeks prior to the clinical manifestation. A decrease in the VEGF serum levels is also observed 5 weeks before the onset of the disease. Furthermore, low
PlGF levels during early pregnancy are correlated with a much greater risk for early-onset preeclampsia (Levine et al.,
2006, 2004). Studies from many other investigators yield similar results (Chaiworapongsa et al., 2005; Hertig et al., 2004;
Polliotti et al., 2003; Shibata et al., 2005). In a prospective nested case-control study by Thadhani et al., it was shown that
serum PlGF and sFlt-1 levels may predict preeclampsia earlier than weeks 12 of gestation (Thadhani et al., 2004). Reports
from Akolekar et al. further suggest that the combination of maternal serum PlGF level with maternal characteristics, obstetric history and uterine artery pulsatility index (PI) may effectively screen for PE as early as weeks 1113 (Akolekar et al.,
2008; Savvidou et al., 2009).
The more remarkable studies come from rodent models. When sFlt-1 is introduced into pregnant rats via tail vein injection, the animals exhibit hypertension, proteinuria and defects in the renal and placental vessels that highly mimic symptoms in PE (Maynard et al., 2003). More recently, the clinical features and placental defects of PE have been recapitulated
in a mouse model with placenta-specic lentiviral overexpression of sFlt-1 (Kumasawa et al., 2011). Human trophoblast cells
from PE placentas produce signicantly more PlGF, sFlt-1, and sEng compared with those from normal placentas (Gu et al.,
2008). Furthermore, prior immune-depletion of sFlt-1 prevents the conditioned medium of the PE placenta from impairing
vessel formation (Ahmad and Ahmed, 2004).
Endoglin is expressed in the vascular endothelium and placental STBs, and is a co-receptor for TGF-b1 and TGF-b3. It is
involved in angiogenesis, and its anti-angiogenic soluble form (sEng) may be produced through proteocleavage and exists in
the circulation of pregnant women. Interestingly, its concentrations are elevated in the sera of women with preeclampsia at
911 weeks before the clinical symptoms manifest. The increase in concentration is more prominent in PE complicated by
the HELLP symptom (Levine et al., 2006). Consistent with this observation, simultaneous adenoviral administration of sEng
and sFlt-1 in pregnant rats leads to clinical features of the HELLP syndrome, which is an extremely severe subtype of PE
(Venkatesha et al., 2006).
The molecular mechanisms regulating the excessive release of these angiogenic factors in the PE placenta remain to be
claried. Understanding the mechanisms may be extremely valuable to elucidate the cause of the placental defects in PE
and to identify potential targets for the treatment of PE. As has been discussed in Section 4, hypoxia plays an important role
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in stimulating sFlt-1 expression in the human placenta (Nevo et al., 2006). Heme oxygenase-1 (HO-1) catalyzes the process
that liberates carbon monoxide (CO) and Fe2+ and can protect the cells from ischemiareperfusion injury. In endothelial cells
and placental villi explants, HO-1 reduces sFlt-1 and sEng production, and antagonizes VEGF-induce sFlt-1 release, as well as
interferon-c and TNF-a-enhanced sEng release. COreleasing molecule-2 (CORM-2) or CO can also decrease sFlt-1 release.
Therefore, the HO-1/CO pathway may be a negative regulator of sFlt-1 and sEng release in the placenta (Cudmore et al.,
2007). HO-1 is hypothesized to be a target for the treatment of preeclampsia (Cudmore et al., 2007), but this has not yet been
proven. Very recently, a report from Kumasawa et al. indicates that low-dose statins, drugs generally used for hypercholesterolemia, may treat PE. This conclusion is based on the ability of pravastatin to enhance PlGF production and ameliorate the
PE symptom in an sFlt-1-induced rat PE model (Kumasawa et al., 2011). More investigation is needed to fully elucidate the
mechanisms of statins function in the placenta.
However, conicting data exist as to whether these angiogenic factors are specic biomarkers for PE. The results from
several groups reveals that the impaired expression patterns of PlGF or sFlt-1 are also linked to small-for-gestational age
(SGA) pregnancies, regardless of whether these pregnancies are affected by preeclampsia (Bersinger and Odegard, 2004;
Than et al., 2008). There are reports showing that pregnancies with IUGR without maternal symptoms of PE also present elevated levels of sEng, indicating that sEng may instead be a marker for placental pathology (Jeyabalan et al., 2008; Stepan
et al., 2007). The association of these factors with other pregnancy disorders, such as gestational diabetes mellitus (GDM)
and preterm birth, has not been well-characterized. On the other hand, the accuracy of using these factors to predict PE
is under review. A recent meta-analysis shows that they are poor predictors of preeclampsia in clinical practice (Kleinrouweler et al., 2012). This problem may be resolved by combining these factors in various patterns. Data from Erez et al. (2008)
shows that the ratios of PlGF/sEng and (sFlt-1 + sEng)/PlGF are more informative than any of the individual molecules as PE
predictors (Erez et al., 2008), and that sequential changes in the rst and second trimesters permit a sensitive and accurate
risk assessment. Large scale studies will be needed to clarify these important issues. Although these challenges exist, circulating sFlt, PlGF, sFlt/PlGF, and sEng are widely considered to be reliable biomarkers for severe PE. In fact, trials screening
sFlt-1 and PlGF levels have been launched by Roche in Europe as a test for preeclampsia in the second trimester.
5.3.2. Catechol-O-methyltransferase (COMT) and 2-methoxyestradiol (2-ME)
A natural metabolite of estradiol, 2-ME, is generated by COMT from hydroxyestradiol in the placenta and increases in concentration during the third trimester (Berg et al., 1983; Casey and MacDonald, 1983). In women with severe preeclampsia,
placental COMT activity is suppressed (Barnea et al., 1988) and plasma levels of COMT and 2-ME are signicantly lower than
in women without PE (Kanasaki et al., 2008). Furthermore, a functional Comt polymorphism that causes low enzyme activity
is associated with fetal growth restriction (Sata et al., 2006). An in vivo study reveals that COMT-decient pregnant mice have
a preeclampsia-like phenotype, as well as thrombotic vascular lesions, hypoxia, and elevated expression of HIF-1a and sFlt-1
in the placenta. Administration of 2-ME ameliorates all preeclampsia-like features and placental defects (Kanasaki et al.,
2008). It has been suggested that hypoxia and placental insufciency may lead to a deciency in placenta-derived estrogens
and hydroxyestradiols (Rosing and Carlstrm, 1984; Takanashi et al., 2000). It is likely that a feedback loop between 2-ME,
sFlt-1 and hypoxia plays an important role in the pathogenesis of PE. The utility of 2-ME as a plasma and urine diagnostic
marker for PE needs to be validated in a large scale investigation.
5.3.3. Corin
A recent in vivo study reveals the effect of impaired Corin expression in the uterus on the onset of preeclampsia (Cui et al.,
2012). Corin (also known as atrial natriuretic peptide-converting enzyme) is a transmembrane cardiac protease that activates atrial natriuretic peptide (ANP), a cardiac hormone that is important in regulating blood pressure (Chan et al., 2005;
Yan et al., 2000). In humans, Corin variants are associated with hypertension (Dries et al., 2005). During pregnancy, Corin
is expressed in the uterus and placenta (Cui et al., 2012). In PE patients, mutations in the corin gene are identied and uterine
Corin expression is decreased, which may reduce the ability of Corin to process pro-ANP. Most interestingly, corin- or ANPdecient mice mimic characteristics of preeclampsia by developing high blood pressure and proteinuria, and showing a
marked impairment in trophoblast invasion and the remodeling of the uterine spiral arteries. The pregnancy-induced hypertension cannot be rescued by cardiac Corin expression, indicating that such hypertension in pregnancy is not due to preexisting high blood pressure. Consistently, ANP is capable of promoting human trophoblast cell invasion in vitro. This study
strongly indicates that the defects in Corin and ANP can contribute to preeclampsia. However, other reports described a high
level of plasma Corin, pro-ANP and ANP in PE patients (Irons et al., 1997; Tihtonen et al., 2007), which contrasted with the
rodent data and the local changes of these proteins in the placenta/uterus. The role of Corin derived from sources such as the
heart during pregnancy need to be further elucidated.
5.3.4. Notch signaling
As stated in Section 4.2.4, Notch signaling regulates human EVT cell differentiation towards an endothelial-like phenotype (Hunkapiller et al., 2011). Compared with gestational-week-matched placentas from pre-term births in which iEVT
and enEVT differentiation is normal (Zhou et al., 1997a) and perivascular and endovascular EVTs are strongly positive for
Jag1, the Jag1-positive EVTs are signicantly reduced in PE placentas. The reduced Jag1 expression is in tight association with
failed vascular remodeling, and the downregulation of Jag1 is more predominant in HELLP cases (Hunkapiller et al., 2011).
Currently, it remains unclear whether altered Jag1 expression in PE is a cause or an effect of failed endovascular invasion.
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However, because placenta-specic deciency in Notch2 leads to reduced uterine vessel remodeling and placental perfusion
(Gasperowicz and Otto, 2008), it is suggested that Notch signaling may be an important part of the pathogenesis of PE.
5.3.5. Nodal, Activin A and Inhibin A
Nodal, Activin A and Inhibin A are predominantly secreted by the placenta during pregnancy, and the circulating levels of
Activin A and Inhibin A rise at the third trimester. Studies from several groups have shown a marked elevation of the serum
levels in PE patients in comparison with the controls in early to mid-gestation (Fraser et al., 1998; Muttukrishna et al., 1997,
2000; Salomon et al., 2003; Zwahlen et al., 2007). In one of our longitudinal study performed in a population of Han Chinese,
the elevation of serum Activin A levels from gestational weeks 25 to term is observed in patients with severe PE (Yu et al.,
2012). These data suggest the value of Activin A and Inhibin A as early predictive markers for PE. However, the test sensitivity
and accuracy were relatively low.
As expected, upregulation of Nodal, Activin A and two of their receptors, ActRIIA and ALK7, are observed in PE placentas
(Bai et al., 2012; Luo et al., 2012; Yu et al., 2012; Fu et al., 2013). It is not yet clear whether the upregulation of Nodal/ActivinA
signaling plays a role in the pathology of preeclampsia or it is just a manifestation of the disease. Meanwhile, the upregulation of Activin A in preeclamptic placentas conicts with its ability to promote invasion, considering the restricted invasive
properties of trophoblast cells in preeclamptic placentas. Recently, our in vitro study in HTR8/SVneo cells reveals that a pathological concentration of Activin A is not able to promote invasion, but is able to promote apoptosis of trophoblast cells by
enhancing Nodal expression and signaling through ALK7. Although further in vivo studies are needed, it is most likely that
the upregulated Nodal/Activin A signaling in PE placentas may contribute to the placental defect by inducing excessive apoptosis. Furthermore, we recently identied several miRNAs, namely, miR-378a-5p, miR-195, miR-376c and miR-18a, that target Nodal, ActRIIA, ALK7 and Smad2, respectively. Interestingly, the expressions of these miRNAs in PE placentas are
decreased compared with normal controls (Bai et al., 2012; Luo et al., 2012; Fu et al., 2013). Lastly, the imprinted H19 gene
encoded miR-675 is downregulated, while one of its targets, NOMO1, is increased in early-onset PE placenta (Gao et al.,
2012). Based on these results, we proposed that aberrant expression of these miRNAs may contribute to the augmentation
of Nodal/Activin A signaling in PE placentas, which results in trophoblast cell defects (Fig. 2).
5.3.6. HGF/c-Met
HGF/c-Met signaling is critical for placenta development, particularly in regulating trophoblast cell invasion and endothelial tubulogenesis. The Placental production of HGF is decreased in women who develop complications of preeclampsia or
intrauterine growth restriction (Furugori et al., 1997; Somerset et al., 1998; Yang et al., 2012). The change of HGF in the serum of PE patients has not yet been elucidated, but we recently nd an evident decrease in the soluble form of its c-Met
receptor, sMet, in PE plasma and describe its potential to predict PE at mid-gestation (Zeng et al., 2009). A large-scale validation of this work is currently being performed. In contrast, we also nd that the production of sMet in PE placentas is
higher than in normal controls, which may result from the ADAM activities induced by oxidative stress and excessive shedding of c-Met (Yang et al., 2012). We currently cannot explain the contradictions in the change of circulating and placental
sMet in PE patients. We hypothesize that a cleanup mechanism may exist to clear excessive amounts of circulating sMet and
are currently exploring this possibility.
5.3.7. hCG
As previously described, hCG is an important hormone in modulating placental development. Elevated serum hCG concentrations in PE patients was described very early on (Crosignani et al., 1974). Retrospective studies later demonstrate that
an elevation of mid-gestational hCG concentrations are associated with a risk for PE (Ashour et al., 1997; Audibert et al.,
2005; Muller and Bussieres, 1996). It is suggested that such a predictive value is more predominant in multiparous but
not nulliparaous women (Ashour et al., 1997). In a recent microarray study, b-hCG and LHB genes are expressed higher in
PE placentas compared with normal pregnant placentas, and maternal serum levels of b-hCG and LHB are accordingly higher
in PE patients (Lapaire et al., 2012; Sitras et al., 2009). The recent ndings regarding the potential roles of hyperglycosylatedhCG (H-hCG) on EVT invasion and the regulation loop between hCG and EG-VEGF help clarify the roles of hCG. More investigation is required to fully understand how defects in hCG function participate in the pathogenesis of PE.
5.3.8. miRNAs
Several studies have shown aberrant placental miRNA proles in preeclampsia by using microarray-based placental miRNA proling, Taqman PCR or qPCR techniques (Bai et al., 2012; Enquobahrie et al., 2011; Luo et al., 2012; Mayor-Lynn et al.,
2011; Pineles et al., 2007; Zhu et al., 2009). However, there is conicting data among the current studies. For example, miR210 and miR-377 are reported to be upregulated in PE placenta (Enquobahrie et al., 2011), whereas a reprot from MayorLynn et al. describes contrasting results (Mayor-Lynn et al., 2011). Such inconsistencies may be due to the differences in sample size, where the samples were taken from the placenta, or the use of different experimental settings and platforms. Our
recent study reveals signicant variations in the miRNA levels between placental tissues taken from the chorionic plate (fetal
side) or the basal plate (maternal side) (Xu et al., Unpublished results). Therefore, careful validation of differential miRNA
expression using appropriate specimens and a large sample size are crucial.
Many studies aim to identify how these differential miRNAs contribute to the onset of PE by focusing on their inuence on
the behaviors if trophoblast cells and the identication of the miRNA target genes. For instance, miR-210 is the most
1008
up-regulated miRNA in preeclampsia (Pineles et al., 2007) and inhibits trophoblast cell migration and invasion by targeting
Ephrin-A3 and Homeobox-A9 (Zhang et al., 2012). This miRNA also targets iron-sulfur cluster scaffold homologue (ISCU) (Lee
et al., 2011) and modulates mitochondrial respiration (Muralimanoharan et al., 2012). Additional targets of miR-210 include
HSD17B1, which is involved in steroid production (Ishibashi et al., 2012). These studies suggest that aberrant expression of
miR-210 may cause various defects in placental function. As we discuss in Section 4 and show in Fig. 2, several miRNAs target
Nodal/Activin A signaling, contributing to the deregulation of trophoblast cell apoptosis and invasion in PE placenta (Bai
et al., 2012; Gao et al., 2012; Luo et al., 2012). Preeclampsia has been associated with abnormal calcium metabolism (Thway
et al., 2004), and miR-1 has been shown to inuence calcium signaling by negatively regulating the calmodulin coding
mRNAs, Mef2a and Gata4 (Ikeda et al., 2009). A reduced level of miR-1 may therefore inuence the risk of preeclampsia
by affecting calcium signaling (Enquobahrie et al., 2011). MiR-584 is one of the down-regulated miRNAs in PE placenta
(Enquobahrie et al., 2011), and targets the lactoferrin receptor. The lactoferrin receptor has critical roles in mediating the
multiple functions of lactoferrin, including immune activation and platelet aggregation, two events that are closely associated with preeclampsia (Askie et al., 2007; Liao and Lonnerdal, 2010). The level of miR-34c-5p is low in preeclampsia
(Enquobahrie et al., 2011). Functional studies of miR-34b and miR-34c together with miR-34c-5p have shown that these
miRNAs decrease cell proliferation in human ovarian carcinoma cells in a p53 dependent manner (Corney et al., 2007). It
is hypothesized that miR-34c-5p possibly plays its role in the pathogenesis of preeclampsia by regulating the p53 pathway.
Further functional characterization of the differential miRNAs would not only reveal new mechanisms for the development
of preeclampsia, but also provide some potential therapeutic targets.
Interestingly, it has been demonstrated that the miRNA levels in plasma from pregnant women are much higher than
those from non-pregnant women and their levels correspond to the stage of pregnancy (Chim et al., 2008; Gilad et al.,
2008; Miura et al., 2010; Morales-Prieto et al., 2011). The circulating miRNAs are most likely coming from the villous trophoblast cells via the exosomes (Luo et al., 2009). It is therefore suggested that the plasma miRNAs may serve as unique
markers for monitoring pregnancy outcomes (Chim et al., 2008). A recent study has shown the increased miR-210 and decreased miR-152 levels in plasma of preeclamptic patients (Gunel et al., 2011) Fig. 3. The current data describing miRNAs in
the maternal plasma of PE patients are still lacking, but there is signicant potential for using plasma miRNAs as the noninvasive prenatal diagnosis of preeclampsia.
5.4. Genetic alterations in association with PE
It remains unclear whether preeclampsia has a genetic basis. Evidence from family studies, twin studies, and the effect of
genetic aberrations in the fetus are supporting the heritability of the disease (Laivuori, 2007). An exponential number of genetic association studies of preeclampsia have been carried out in the last decade, and most of them are performed by either
comparing genetic polymorphisms in candidate genes associated with placental function or preeclampsia or genome-wide
scanning. A comparison of the prevalence of genetic polymorphisms in candidate genes between PE and normal pregnancies
can provide direct evidence for the association of genetic polymorphisms in certain genes with the disease, and is relatively
easy and less expensive. However, the candidate genes are chosen based on the known genes that are relevant to preeclampsia. As a result, the information available is limited. The genome-wide scanning method globally identies the chromosomal
Fig. 3. The reciprocally complex interaction between the fetus and the mother contributes to preeclampsia pathogenesis. Poor development of the early
placenta and utero-placental blood supply that occurs in the rst half of pregnancy results in the release of many factors from the placenta to the maternal
circulation. Aberrant levels of these factors and the necrotic trophoblast debris in the maternal circulation cause the impaired functions in many maternal
organs including liver, kidney, blood vessels and brain as well as in maternal systemic inammation. The confounding maternal factors, including genetic,
epigenetic and environmental factors, may reciprocally interact with the poor placentation in multiple ways.
1009
regions that are transmitted with the disease within families. It is a powerful tool to uncover genes that are susceptible to
disease, but it is much more expensive and requires more professional statistical analysis.
The genetic studies of PE have been comprehensively are reviewed (Chappell and Morgan, 2006; Lachmeijer et al., 2002;
Laivuori, 2007; Wilson et al., 2003). In general, genes that are implicated in thrombophilia (e.g., factor V, prothrombin, methylenetetrahydrofolate reductase), angiogenesis (e.g., endothelial nitric oxidesynthase (NOS3), VEGF), invasion (e.g., MMP-9,
HLA-G, HB-EGF), hemodynamics (e.g., angiotensin converting enzyme (ACE), angiotensin II type 1 and type 2 receptors
(AGTR1, AGTR2), angiotensinogen (AGT)), hypertension (e.g., Corin, COMT), oxidative stress (e.g., 2-ME), and cytokines
(e.g., TNFa, IL-4) have been identied. Genome-wide studies also reveal signicant linkages with several loci for PE: 2p12,
2p25, and 9p13. A study of Dutch families strongly suggests that patients with STOX1 missense mutations are predisposed
to developing PE (van Dijk et al., 2005). Unfortunately, different groups have reported conicting results regarding the association of these genes or chromosome loci with PE. As a result, genetic basis for preeclampsia has not been denitely
determined.
The suggested guidelines for genetic association studies are as follows: a large number of patients; control subjects
matched for ethnic background, age and gender; adjustments for multiple comparisons; a polymorphism with a functional
change in physiology; replication of the results in an independent group of subjects; and the publication of the high-quality
association studies with negative results (Bird et al., 2001). Apart from all these criteria, the appropriate denition of subtypes is important to understand the complexity of preeclampsia, which most likely emerges from diverse patho-mechanisms. Large-scale genetic association studies that include several phenotypes of this syndrome will lead to the
introduction of greater heterogeneity and false-positive results. Furthermore, it is reasonable to propose that multiple genes
rather than a single major gene are associated with preeclampsia. Therefore, combining single nucleotide polymorphisms
(SNPs) and haplotype analysis will be powerful to detect or exclude associations.
6. Summary and perspectives
Exciting progress has been made in recent years, ranging from how trophoblast cell differentiation and placental development are regulated to the discovery of novel biomarkers and potential therapeutic targets of preeclampsia. It is well accepted that oxygen tension within the placenta plays an important role in trophoblast cell differentiation and placental
development. Recent studies provide further insights into how hypoxia exerts its effects by modulating signaling pathways
and miRNA expression. Our understanding of how hormone- and growth factor-regulated control of trophoblast cell activities is also expanding. However, the key factors and signaling pathways that determine human trophoblast cell fate remain
elusive, and the interactions among different signaling molecules in the control of human trophoblast cell differentiation requires further investigation. The microenvironment of trophoblast cell differentiation is very complicated and changes
dynamically throughout gestation, during which trophoblast cells interact with many other cell types. The regulatory network that maintains appropriate cellcell interactions is far from clear.
In recent years, emerging evidence points to an important role of miRNAs in regulating trophoblast cell functions. However, the role of these small RNAs in the differentiation of different trophoblast subtypes remains poorly understood. Aberrant expression of miRNAs in preeclamptic placentas suggests a link between the deregulation of placental miRNAs and
preeclampsia, but the precise mechanisms through which these miRNAs contribute to the pathogenesis of preeclampsia
have not yet been completely elucidated. Clarifying how the expression of the essential or predominant miRNAs are regulated by transcriptional factors and signaling molecules and identifying the gene networks regulated by those miRNAs will
be a novel step in understanding placental development during normal and compromised pregnancies. The detection of miRNAs in maternal plasma raises the exciting possibility of using them as novel biomarkers.
Our understanding of preeclampsia has improved signicantly during the past decades, and a variety of biomarkers have
been identied. However, most biomarkers or signaling pathways can only t certain aspects of preeclampsia and there are
still no ideal predictive and therapeutic strategies for this disorder. It has become clearer that there are several subtypes of PE
in which different pathways are affected, and different strategies should be developed to predict and treat each subtype. It is
most likely that placental PE, due to poor placentation and dysfunctional utero-placental perfusion, is an early-onset disease
in which markers of placental dysfunction (angiogenic and anti-angiogenic factors) are more predictive or diagnostic. The
most severe early-onset manifestation of PE is possibly due to a dual contribution of maternal and placental components.
Apart from the traditional features of PE, many other confounding factors should be taken into account in the investigation.
These factors may include obesity, infection, smoking, drinking, sex and weight of the fetus, history of assisted reproductive
technology, and environmental factors such as altitude, temperature, toxic endocrine-disrupting chemicals, and so on. The
heterogeneous clinical and biochemical properties of PE make it unlikely that a single specic diagnostic feature for PE will
be recognized. We are currently at the point of properly categorizing subtypes of the diseases, which is vital to clarify the
pathogenesis of the complications and to translate ndings of bench research into effective clinical treatments.
Acknowledgements
This work is supported by grants from the Chinese National Special Fund for Basic Research Project (2011CB944400), the
National Natural Sciences Foundation (81025004) and the Knowledge Innovation Program of the Chinese Academy of
1010
Sciences (No. KSCX2-EW-R-06) to Y.L.W., and by grants from the Canadian Institutes of Health Research (MOP-81370 and
CCI-92222) to C.P. J.B. was supported by an Ontario Graduate Scholarship. The authors appreciate Drs. Enkui Duan, Haibin
Wang and Qi Chen for their critical advices. We apologize for unintended omission of any relevant references.
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Molecular Aspects of Medicine

Placental trophoblast cell differentiation: Physiological

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Model systems for examining placental trophoblast cell functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 989

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Blaschitz et al. (2000)

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Pattillo and Gey (1968)

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4.4. MicroRNAs (miRNAs)

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5. Implications for the pathogenesis of preeclampsia (PE)

5.2. The injury from hypoxiareoxygenation

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