Abnormal placentation is considered as an underlying cause of various pregnancy complications such as miscarriage, pre eclampsia and intrauterine growth restriction, the latter increasing the risk for the development of severe disorders in later life such as cardiovascular disease and type 2 diabetes. Despite their importance, the molecular mechanisms governing human placental formation and trophoblast cell lineage specification and differentiation have been poorly unravelled, mostly due to the lack of appropriate cellular model systems. However, over the past few years major progress has been made by establishing selfrenewing human trophoblast stem cells and 3dimensional organoids from human blastocysts and early placental tissues opening the path for detailed molecular investigations. Herein, we summarize the present knowledge about human placental development, its stem cells, progenitors and differentiated cell types in the trophoblast epithelium and the villous core. Anatomy of the early placenta, current model systems, and critical key regulatory factors and signalling cascades governing placentation will be elucidated. In this context, we will discuss the role of the developmental pathways Wingless and Notch, controlling trophoblast stemness/differentiation and formation of invasive trophoblast progenitors, respectively.

SummaryDefective placentation is the underlying cause of various pregnancy complications, such as severe intrauterine growth restriction and preeclampsia. However, studies on human placental development are hampered by the lack of a self-renewing in vitro model that would recapitulate formation of trophoblast progenitors and differentiated subtypes, syncytiotrophoblast (STB) and invasive extravillous trophoblast (EVT), in a 3D orientation. Hence, we established long-term expanding organoid cultures from purified first-trimester cytotrophoblasts (CTBs). Molecular analyses revealed that the CTB organoid cultures (CTB-ORGs) express markers of trophoblast stemness and proliferation and are highly similar to primary CTBs at the level of global gene expression. Whereas CTB-ORGs spontaneously generated STBs, withdrawal of factors for self-renewal induced trophoblast outgrowth, expressing the EVT progenitor marker NOTCH1, and provoked formation of adjacent, distally located HLA-G+ EVTs. In summary, we established human CTB-ORGs that grow and differentiate under defined culture conditions, allowing future human placental disease modeling.

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During placentation invasive extravillous trophoblasts (EVTs) migrate into the maternal uterus and modify its vessels. In particular, remodeling of the spiral arteries by EVTs is critical for adapting blood flow and nutrient transport to the developing fetus. Failures in this process have been noticed in different pregnancy complications such as preeclampsia, intrauterine growth restriction, stillbirth, or recurrent abortion. Upon invasion into the decidua, the endometrium of pregnancy, EVTs encounter different maternal cell types such as decidual macrophages, uterine NK (uNK) cells and stromal cells expressing a plethora of growth factors and cytokines. Here, we will summarize development of the EVT lineage, a process occurring independently of the uterine environment, and formation of its different subtypes. Further, we will discuss interactions of EVTs with arteries, veins and lymphatics and illustrate how the decidua and its different immune cells regulate EVT differentiation, invasion and survival. The present literature suggests that the decidual environment and its soluble factors critically modulate EVT function and reproductive success.

Wingless ligands, a family of secreted proteins, are critically involved in organ development and tissue homeostasis by ensuring balanced rates of stem cell proliferation, cell death and differentiation. Wnt signaling components also play crucial roles in murine placental development controlling trophoblast lineage determination, chorioallantoic fusion and placental branching morphogenesis. However, the role of the pathway in human placentation, trophoblast development and differentiation is only partly understood. Here, we summarize our present knowledge about Wnt signaling in the human placenta and discuss its potential role in physiological and aberrant trophoblast invasion, gestational diseases and choriocarcinoma formation. Differentiation of proliferative first trimester cytotrophoblasts into invasive extravillous trophoblasts is associated with nuclear recruitment of β -catenin and induction of Wnt-dependent T-cell factor 4 suggesting that canonical Wnt signaling could be important for the formation and function of extravillous trophoblasts. Indeed, activation of the pathway was shown to promote trophoblast invasion in different in vitro trophoblast model systems as well as trophoblast cell fusion. Methylation-mediated silencing of inhibitors of Wnt signaling provided evidence for epigenetic activation of the pathway in placental tissues and choriocarcinoma cells. Similarly, abundant nuclear expression of β -catenin in invasive trophoblasts of complete hydatidiform moles suggested a role for hyper-activated Wnt signaling. In contrast, upregulation of Wnt inhibitors was noticed in placentae of women with preeclampsia, a disease characterized by shallow trophoblast invasion and incomplete spiral artery remodeling. Moreover, changes in Wnt signaling have been observed upon cytomegalovirus infection and in recurrent abortions. In summary, the current literature suggests a critical role of Wnt signaling in physiological and abnormal trophoblast function.

The molecular mechanisms governing invasive differentiation of human trophoblasts remain largely elusive. Here, we investigated the role of Wnt-␤-catenin-T-cell factor (TCF) signaling in this process. Reverse transcriptase-polymerase chain reaction and Western blot analyses demonstrated expression of Wnt ligands, frizzled receptors, LRP-6, and TCF-3/4 transcription factors in total placenta and different trophoblast cell models. Immunohistochemistry of placental tissues and differentiating villous explant cultures showed that expression of TCF-3/4 strongly increased in invading trophoblasts. Some of these cells also accumulated dephosphorylated ␤-catenin in the nucleus. Wnt3A treatment of primary cytotrophoblasts and SGHPL-5 cells induced activity of TCF-luciferase reporters. Accordingly, the ligand provoked interaction of TCF-3/4 with ␤-catenin as assessed in electrophoretic mobility shift assays (EMSAs) and upregulation of Wnt/TCF target genes as observed by Western blot analyses. Wnt3A stimulated trophoblast migration and invasion through Matrigel, which could be blocked by addition of Dickkopf-1, mediating inhibition of canonical Wnt signaling. Dickkopf-1 also reduced basal migration, invasion, and proliferation of cytotrophoblasts, suggesting expression of endogenous Wnt ligand(s). Immunohistochemistry revealed that the percentage of extravillous trophoblasts containing nuclear ␤-catenin was significantly higher in placentas of complete hydatidiform mole pregnancies as compared to normal placentas. Thus, canonical Wnt signaling may promote invasive trophoblast differentiation, and exaggerated activation of the pathway could contribute to trophoblastic hyperplasia and local invasion.

We have tested the hypothesis that elevated concentrations of TNF alpha could impair trophoblast invasion. Using first-trimester placental explant cultures, we have demonstrated that the cytokine inhibits in vitro migration of extravillous trophoblasts (EVT) on collagen I, and invasion through Matrigel. To elucidate the underlying mechanism, proliferation and differentiation of EVT in vitro were analyzed by immunohistochemistry of serial sections, Western blotting, zymography, ELISA, and RT-PCR from RNA pools of mechanically separated cell populations. At 24 h of cultivation in the presence or absence of TNF alpha, anchorage and proliferation of trophoblasts had occurred to generate cell columns containing viable, post-mitotic, differentiated EVT [positive for integrins alpha 1 and alpha 5, matrix metalloproteinase (MMP)-2, and human leukocyte antigen-G1; negative for proliferating cellular nuclear antigen, cytokeratin 18 neoepitope, and in 5-Bromo-2-deoxy-uridine labeling]. At 72 h, control cells had broken away from the column to migrate through the extracellular matrix; whereas, in contrast, TNF alpha-treated EVT remained as contiguous cell columns, despite increased MMP-9 expression. Thus, in vitro MMP9 activity appears not to be essential for trophoblast migration. Expression of plasminogen activator inhibitor (PAI)-1 was elevated in TNF alpha-treated EVT, and adding antibodies that inhibit PAI-1 activity restored migration, whereas tissue-inhibitor-of-metalloproteinases-1-blocking antibodies were ineffective. Induction of PAI-1 by TNF alpha could be related to restricted trophoblast invasion in preeclampsia.