Human placenta and trophoblast development: key molecular mechanisms and model systems

Abnormal placentation is considered as an underlying cause of various pregnancy complications such as miscarriage, preeclampsia 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 self-renewing human trophoblast stem cells and 3-dimensional 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.

section_content: Formation of the placenta, the unique exchange organ between mother and fetus, is essential for successful human pregnancy and fetal health. Derived from extraembryonic tissues, the placenta rapidly develops during the first weeks of gestation dynamically changing its structure and function [1, 2]. Throughout pregnancy the placenta fulfils a plethora of tasks ranging from physiological adaption of the mother to immunological acceptance, nourishment and support of the developing embryo. Placental villi, bathed in maternal blood, represent the transport units of the organ, delivering nutrients and oxygen to the developing fetus and clearing its waste products. During the 9 months of gestation these villi undergo dynamic morphological changes. Mesenchy mal villi of early pregnancy develop into highly vascular ized structures, efficiently extracting substances from the maternal circulation [3]. By term, the extensive branching morphogenesis of villi creates an overall epithelial surface of about 12-14 m 2 ensuring adequate nutritional supply, at a time when the fetus shows high growth rates. Besides ful filling the needs of the developing fetus, the placenta also profoundly changes the metabolism of the mother by secret ing numerous hormones into the maternal blood stream [4]. These hormones affect most maternal tissues and organs, and effectively modulate the maternal physiology to promote the maintenance of pregnancy, mobilization of nutrients, par turition and lactation. Moreover, some placental hormones are also released into the fetal circulation thereby regulating fetal development, growth and timing of delivery [5, 6]. 

Failures in placental formation, can compromise embry onic growth and development. Indeed, abnormal placenta tion is a feature of diverse pregnancy complications such as miscarriage, stillbirth, preterm labour, intrauterine growth restriction (IUGR) and preeclampsia [7] [8] [9] [10] [11] [12] [13]. Albeit these dis orders can have multiple causes, including fetal aberrations and maternal factors [14, 15], placental defects, inappropriate adaption and remodelling of the uterine vascular bed, and as a possible consequence malperfusion occurs in a consider able number of cases, particularly in severe IUGR and early onset preeclampsia [16] [17] [18] [19]. Accordingly, transcriptomic analyses of preeclamptic placentae revealed different sub classes of the disease with specific gene modules for placen tal dysfunction [20] [21] [22]. Disorders with underlying placental abnormalities not only increase morbidity and mortality of mother and fetus, but may also negatively affect longterm health [23, 24]. Mothers with preeclampsia and/or infants born with growth restriction have a higher risk for developing types 2 diabetes, hypertension or cardiovascular disease in later life [25] [26] [27]. In addition, developmental programming of the embryo, due to structural, developmental or functional defects of the placenta, may also predispose the fetus to a variety of other chronic adult diseases [28]. For example, abnormal fetal growth impairs neuronal development elevat ing the risk for psychiatric disorders in adulthood [29]. 

Considering the complex role of the placenta in fetal-maternal communication, it may not be surprising that its diverse functions at different stages of normal and abnor mal pregnancies remain poorly understood. Within the first weeks of pregnancy the human placenta generates epithelial trophoblast with diverse biological roles including attach ment of the conceptus to the uterine wall, establishment of early histiotrophic nutrition (nourishment of the fetus by decidual glandular secretions) and adaption of the maternal uterine vasculature [30, 31]. Different types of trophoblasts, including stem cells, progenitors and differentiated subtypes with multiple functions develop [32]. However, their spe cific roles, particularly in the context of pregnancy disor ders, remain largely elusive. Our ability to understand how inadequate placentation contributes to pregnancy disorders is confounded by the fact that the pathogenesis of these dis orders develops during the first trimester of pregnancy, when availability of placental tissue for in vitro studies is ethically more limited, and crucially, when we cannot accurately pre dict which placentae would have gone on to develop pathol ogy later in gestation. Furthermore, culture conditions that allow for selfrenewal and longterm propagation of primary trophoblasts, a prerequisite for detailed molecular investiga tions, have only been recently established [33, 34]. 

As a result, our knowledge of the functional aspects of placental development is largely based on investigations performed in mice. In diverse knockout studies key regu latory genes have been unravelled, some of which are also expressed in the human placenta [35] [36] [37] [38]. Although spe cific trophoblast lineages considerably differ between mice and men, both species show haemochorial placentation, resulting in direct contact of maternal blood with fetal derived trophoblasts. Mouse studies have not only identi fied specific regulators of major biological processes in the placenta, such as placental vascularization, labyrinth and junctional zone formation and function, but also delineated the importance of the decidualized maternal endometrium (decidua) in governing placental development, trophoblast differentiation and fetal growth [39]. Notably, correct spec ification and functionality of distinct placental trophoblast subtypes at early stages of development could be crucial for subsequent organ development in the fetus itself. Studies in mice, carrying mutations that provoke lethality around mid gestation suggest that the preceding placental defects could be causative for fetal demise [40]. In particular, failures in heart, neuronal and vascular development were found to be associated with placental abnormalities. Notably, restoring gene function in the placenta compensated for the embry onic defects in these mutants. An equivalent role of the human placenta in subsequent organogenesis seems likely. For example, failures in adapted perfusion of the placenta could alter haemodynamics in the fetoplacental circulation and thereby impair cardiac development [41]. 

However, despite some similarities, considerable dif ferences haven been noticed between murine and human placental development and structure. Besides deviations in gross morphology and specific trophoblast cell types, blas tocysts implant differently in mice, trophoblast invasion is very shallow and remodelling of uterine arterial ves sels largely depends on maternal factors [42]. Moreover, key regulators of placental development differ between mouse and men [37], as also outlined below, making the mouse an imperfect model of human placentation. Hence, establishment and further improvement of appropriate human model systems are highly warranted. Considering the crucial role of the placenta in pregnancy complica tions and longterm health, better insights into molecular mechanisms of human implantation and early placental development should advance options for therapeutic treat ment of pregnancy pathologies. However, the critical steps of normal and pathological placentation have hardly been elucidated. Herein, we summarize our current knowl edge of human placental development and its underlying mechanisms. Structural changes of the human placenta throughout pregnancy and the specific roles of trophoblast subtypes will be discussed. Further, we will focus on dif ferent stem and progenitor cells present in chorionic villi and elucidate key regulatory pathways controlling placen tation, trophoblast development and differentiation.
section_name: Introduction

section_content: Our knowledge about the first weeks of human placental development (Fig. 1 ) is largely based on the interpretation of anatomical structures of early implantation sites present 1 3 in hysterectomy specimens of the Boyd (Centre of Tropho blast Research, Cambridge) and Carnegie (Human Devel opmental Anatomy Center, Washington DC) collections [1, 43]. In addition, preimplantation and implantation studies in mice [44, 45], as well as histological analyses of species with comparative placental development, such as the great apes [46, 47], contributed to our understanding of early placentation events in humans. The precursor of all trophoblast cells is the trophectoderm (TE) constituting the outer layer of the human blastocyst. The TE develops approximately 4-5 days after fertilization. Its formation represents the first lineage decision during development, segregating the TE from the inner cell mass (ICM), the lat ter giving rise to the embryo proper (Fig. 1a ). Interaction of the socalled polar TE, adjacent to the ICM, with the uterine luminal epithelium results in implantation around day 6-7 postconception at which time the first steps of placental development commence. After implantation, stem cells of the TE (TESC) generate the first trophoblast lineages, early mononuclear cytotropho blasts (CTBs) and the multinuclear primitive syncytium (PS) at day 8 postconception [32, 48, 49]. The PS represents the first invasive placental cell type which further expands into the maternal decidua (Fig. 1b ). At this time the ICM simul taneously develops into a bilaminar epithelial structure con sisting of epiblast (Ep) and hypoblast (Hy; also termed prim itive endoderm), giving rise to the embryo and the primitive yolk sac (pYO), respectively. Lineage tracing studies in pri mates show that the Hy also gives rise to the extraembryonic mesoderm (ExM), which in turn forms the mesenchymal compartment of chorionic villi and the umbilical cord [50]. However, the Ep may also contribute to the ExM, as ExM cell express markers traditionally associated with this line age [51]. Around day 15 postconception the Ep forms the three embryonic germ layers and the amnion. Approximately at day 9 vacuoles appear in the PS, which upon fusion form a network of lacunar spaces eventually breaching the maternal uterine capillaries (UC) around day 12-13 thereby forming discontinuous maternal blood sinusoids (MS) [1]. Around day 10 postconception the development and morphogenesis of placental villi commences. At the time of PS expansion, rows of proliferative CTBs break through the expanding syn cytial mass thereby forming primary villi (PV) (Fig. 1c ). The PV extend into the underlying maternal decidua and, like the early multinuclear structures, erode uterine blood vessels and glands (UG). During the following days PV are trans formed into secondary villi, achieved by migration of ExM cells into the primary structures. Concurrently, the epithelial surface branches and expands tremendously by continuous proliferation and cell fusion of developing villous cyto trophoblasts (vCTB). The latter process generates the outer multinuclear syncytiotrophoblast (STB) layer, providing the interface between mother and fetus for nutrient transport and gas exchange in floating villi. The STB is thought to arise from asymmetrical cell division, differentiation and fusion of villous cytotrophoblasts (vCTBs) with the preexisting syncytium and secrete critical pregnancy hormones into the maternal circulation, such as human chorionic gonadotro phin (hCG) and placental lactogen [52, 53]. 

Around day 17 postconception secondary villi develop into tertiary villi (TV) that contain placental vessels, at a time when the fetal allantois extends and fuses with the chorionic plate at later stage (Fig. 1d ). These vessels begin as haemangiogenic foci which differentiate from the ExM. These haemangiogenic foci develop into primitive endothe lial tubes. The recruitment of pericytes stabilizes these tubes allowing further expansion of the placental vascular network via increases in capillary length and diameter finally con necting placental vessels with the vasculature of the fetus after the fourth week of pregnancy [3]. Interestingly, the pla centa leads the way in vascular development in the embryo, with the first blood vessels evident when the embryo proper still exists as three germ layers [54]. Consequently, all of the cell lineages involved in early placental haemangiogen esis and vasculogenesis are thought to arise in the placenta de novo via differentiation directly from the ExM, as the umbilical circulation does not connect the fetal and placental systems until 32 days postconception. The placental vascu lature continues to undergo extensive expansion the latefirst and second trimester as a result of branching angiogenesis. Towards the end of pregnancy the placental capillaries elon gate and form loops that are pushed up against the STB layer of terminal villi, decreasing the exchange distance between the maternal and fetal circulations and thereby maximizing oxygen and nutrient transport to the fetus [55]. 

Besides developing chorionic villi, proliferating CTBs at distal sites also expand laterally around day 15 postcon ception to form the trophoblastic shell, which represents the outermost site of the placenta encircling the embryo (Fig. 2a ). The shell lacks maternal cell types and is thought to be critical for anchorage of the placenta to the decidua and protection of the embryo from oxidative stress [56]. Dur ing the early phases of placentation the trophoblastic shell gives rise to the second differentiated trophoblast cell type, the invasive extravillous trophoblasts (EVTs). However, once mature placental villi have formed, EVTs originate from the differentiation of CTBs in the tips of anchoring villi (Fig. 2b ). In these villi, rows of proliferative proximal cell column trophoblasts (pCCTs) develop, representing the progenitor cell pool of differentiated EVTs. Upon forma tion of the distal cell column, cells cease mitosis, but do not exit the cell cycle to reach a quiescent state. Instead, dCCTs enter endoreduplicative cycles and undergo polyploidiza tion and senescence upon differentiation into EVTs [57]. By 15-16 days postconception two distinct EVT popula tions can already be identified; interstitial cytotrophoblasts (iCTBs) invade the decidual stroma, whereas endovascular cytotrophoblasts (eCTB) colonize the maternal spiral arter ies [58, 59]. Invasion of uterine stroma by iCTBs provokes numerous effects during early pregnancy. These cells inter act with decidual stromal cells, macrophages and uterine natural killer (uNK) cells (Fig. 2b ) in order to regulate immunological acceptance of the placental/fetal allograft and control EVT function [60, 61]. Besides migration into the spiral arteries, iCTBs also invade decidual glands, lym phatics and veins (Fig. 1d ) [62] [63] [64]. Whereas breaching of glandular structures could be required for early histiotrophic nutrition of the embryo, invasion of lymphatics and veins might be necessary for fluid drainage, adaptation of immune cell trafficking and hormonal adaption to pregnancy. Indeed, EVTspecific proteins, such as diamine oxidase (DAO), are detectable in the serum of pregnant mothers prior to the onset of the maternal-placental circulation [65]. As iCTBs reach the underlying myometrium, they undergo a final differentiation step into multinucleated trophoblast giant cells losing their invasive capacity. 

Migration of EVTs into the maternal spiral arteries rep resents another key step of human placentation (Fig. 2b ). In early pregnancy these vessels are extensively remodelled within the decidua and as far as the first third of the myo metrium. To achieve this, iCTBs are recruited to the spiral arteries by uNK cells and macrophages, which surround these vessels from early pregnancy and initiate the remod elling process [66, 67]. iCTBs then breach the spiral arteries, and differentiate into eCTBs that migrate along their lumen and adopt a vascular adhesion phenotype that allows them to interdigitate into the endothelial layer, whereby they induce endothelial cell apoptosis and completely replace the mater nal endothelial cells within these vessels [68]. Concurrently, iCTBs induce apoptosis or dedifferentiation of the smooth muscle layer and basal lamina of the spiral arteries, thereby contributing to vessel remodelling [67, 69, 70]. This results in a dramatic change in the spiral arteries during which nar row vessels with relatively high resistance are transformed into highly dilated, lowresistance conduits (Fig. 2b ). These remodelled vessels change the nature of blood flow entering the intervillous space later in pregnancy to ensure that the increase in volumetric blood flow to the uterus during preg nancy is delivered at an appropriately low speed to ensure maximal perfusion and prevent damage to the villi. 

As well as remodelling the spiral arteries, eCTBs also form trophoblast plugs during the first weeks of pregnancy that occlude the spiral arteries in the decidua basalis under lying the implantation site (Fig. 2a ). These plugs completely prevent blood flow until 6-7 weeks of gestation, after which narrow channels in the plugs begin to form, which may allow a limited flow into the intervillous space that can be detected by Doppler ultrasound [71]. Trophoblast plugs disintegrate completely near the end of the first trimester, and this is associated with significant onset of flow of oxy genated maternal blood into the intervillous space around 12-13 weeks of gestation [71] [72] [73]. This significant increase in flow is also thought to coincide with the completion of trophoblastindependent remodelling of the upstream radial arteries, which may act as the ratelimiting vessels regulating the volumetric flow of maternal blood into the intervillous space [71, [74] [75] [76]. As a result of trophoblast plugging, the placenta exists in a low oxygen environment for the majority of the first trimester, and this is thought to be key to promote placental development, vasculogenesis and angiogenesis. Indeed, a premature rise of oxygen levels could provoke production of reactive oxygen species and, as a consequence, oxidative damage of the fetal-placental unit, as incomplete plugging of the maternal arteries and disorganized early onset blood flow has been noticed in mis carried pregnancies [9, 11]. At the margin of placenta, where high oxygen flow is first initiated, the trophoblast layer may degenerate (Fig. 2a ), providing a potential mechanism for the regression of villi and formation of the mature discoidal shape of the placenta [30]. The establishment of a vascular connection between the mother and fetus by the end of the first trimester marks the transition from histiotrophic to hae motrophic nutrition. In humans, the placenta is accordingly defined as haemochorial since placental villi are in direct contact with maternal blood filling the intervillous space.
section_name: Development and functional properties of the early chorionic villus and its different cell types

section_content: Throughout the past few decades, research with primary trophoblasts has been dependent on the availability of pla cental tissues from different stages of pregnancy. Samples are most easily obtained after delivery of normal term preg nancies and of pregnancy disorders at late stages. However, at the end of gestation it is impossible to determine if pla cental pathology is the cause or the consequence of a given pregnancy complication. Moreover, distinct trophoblast functions, for example invasiveness and motility of EVTs, are significantly reduced at term [77]. Hence, among the different developmental processes of trophoblasts, only STB formation in vitro can be effectively studied using human term placenta. CTBs from term placentae isolated by trypsin digestion and Percoll gradient centrifugation spontaneously fuse into multinuclear structures when seeded on plastic or extracellular matrix (ECM)coated dishes and upregu late markers of STBidentity such as hCG [78, 79]. Using this model numerous soluble factors, such as epidermal growth factor (EGF), as well as key regulatory genes pro moting trophoblast syncytialization have been characterized [80] [81] [82] [83]. Several trophoblastsecreted proteins increase cell fusion and transcription of hormone genes through eleva tion of cAMP levels, the latter activating crucial regulators in STB, such as cAMPresponsive element binding pro tein (CREB) and glial cells missing (GCM1) [82, 84, 85]. Notably, the villous trophoblast epithelium also expresses the fusogenic proteins syncytin1 and 2, encoded by the human endogenous retroviruses (HERV) HERVW and HERVFRD, interacting with their respective receptors, the sodiumdependent neutral amino acid transporters (ASCT1 and ASCT2) and major facilitator superfamily domain con taining 2a (MFSD2a), respectively [86]. Syncytin expres sion is controlled by a placentaspecific enhancerbinding GATAbinding proteins and GCM1 [87, 88]. The latter is critical for branching morphogenesis and syncytialization in mouse placenta, and was also shown to increase cell fusion of human CTBs [89, 90]. Additionally, other transcriptional regulators, such as activating enhancerbinding protein 2α (AP2α), distalless homeobox 3 (DLX3) and peroxisome proliferator activated receptor gamma (PPARγ), regulating hormone expression and syncytialization of term tropho blasts, have been identified [38, 91]. However, to what extent molecular mechanisms of cell fusion may differ between first and third trimester remains largely unknown. DNA microar ray and RNAseq data of early and late STBs, generated by in vitro cell fusion or analysed by singlecell sequencing of whole placental tissues, have been published [34, [92] [93] [94] [95] ]. Yet, functional fusion studies with first trimester primary vCTBs are rarely performed due to the restrictions on using this material in many laboratories, and the limited amount of placental tissue obtained. Likewise, regulation of early PS formation remains enigmatic. Possibly STB, generated by treatment of human embryonic stem cells (hESC) with bone morphogenetic protein 4 (BMP4), could be representative of the early PS, since gene expression in that model is different to that of term STB [96]. 

Signalling pathways and key mechanisms controlling EVT migration, invasion and function have been widely investigated [97, 98]. Due to the limited availability of first trimester placental tissues different trophoblast cell lines have been utilized as cellular model systems [99]. However, most of these cell lines differ considerably from primary EVTs with respect to gene expression patterns and human leukocyte antigen (HLA) profiles [100, 101], question ing their origin and suitability as EVT models. In contrast invasive trophoblasts, isolated from first trimester placenta, express the correct HLA genes and cellspecific markers of the in situ EVT. In vitro, EVTs can be generated from puri fied CTBs or villous explant cultures, the latter recapitulat ing cell column proliferation and differentiation [102, 103]. After attachment to matrix, purified CTB cultures sponta neously induce markers of the migratory trophoblast, for example HLAG, proteoglycan 2 (PRG2), erythroblastic oncogene B2 (ErbB2) and the EVTspecific proteins integrin α1 (ITGA1) and α5 (ITGA5) [64, [104] [105] [106], and upregulate matrixmetalloproteinases and other proteolytic enzymes for invasion into the decidua [107, 108]. Several transcription factors, including GCM1, AP2α, signal transducer and acti vator of transcription 3 (STAT3), and FOS like 1 (FOSL1), were shown to control trophoblast invasion and EVTspe cific gene expression in different trophoblast cell models [90, [109] [110] [111]. Furthermore, within the early placenta hypoxia inducible factor 1 (HIF1) is only expressed by EVTs, and low oxygen levels promote in vitro EVT formation [112] and elevation of the EVT progenitor marker Notch1 [113]. Another critical pathway regulating EVT function is canoni cal Wnt signalling [114]. Activity of nuclear Wntdependent T cell factor 4 (TCF4)/βcatenin complexes is induced dur ing EVT formation and silencing of TCF4 impaired motility and EVTmarker expression [115, 116]. 

Although many trophoblastspecific transcription factors have been tested in the context of CTB proliferation and motility [38, 97], their specific roles in commitment and differentiation of the invasive trophoblast lineage remain elusive. This is explained by the fact that, until recently, selfrenewing cell culture models were lacking and CTB preparations (containing EVT progenitors) rapidly differen tiate when seeded in 2dimensional (2D) layers on ECMs. In 2018, however, culture conditions for induction of the EVT lineage have been established in longterm expanding trophoblast models [33, 34]. 

EVT formation could be largely driven by an autocrine differentiation program operating independently of the surrounding environment. Besides spontaneous 2D differ entiation in vitro, ectopic trophoblast of tubal pregnancies and anchoring villi, implanted into the kidney capsule of SCID mice, induce EVT markers and switch their integ rin expression like EVTs invading the decidua basalis [117, 118]. Notably, CTBs derived from preeclamptic placental tissues show defects in EVT differentiation, suggesting that failures in this process could contribute to abnormal placen tation in this disease [119]. On the other hand, EVTs isolated from preeclamptic tissues were shown to revert their gene expression pattern back to normal [120], suggesting that the decidua could play a role in shaping EVT function. These distinct results could possibly be explained by the different molecular subtypes of preeclampsia which may or may not have abnormal placentation as an underlying cause [20, 22, 121]. Despite the limited lifespan of 2D primary cultures, in vitro EVT formation and differentiation also occurs in dis crete steps as it has been described in vivo. Whereas many EVTmarkers, such as HLAG, ITGA5 or TCF4 are induced in the distal, nonmitotic part of the cell column, ITGA1 and DAO, for example, are expressed in deeper regions of the decidua where EVTs have detached from anchoring villi [65, 104]. Notably DAO, induced in pure CTB cultures in an autocrine fashion, appears at later stages of in vitro differen tiation compared to HLAG [65]. These data suggest that the different steps of EVT differentiation can be recapitulated in 2D CTB cultures. 

DAO expression in situ is mostly detectable in a subset of EVTs approaching veins and arteries [65] suggesting that the DAOsecreting cells could represent another specific EVT subtype. Along these lines, singlecell RNAseq of first tri mester placental/decidual tissues revealed between one and three different EVTs signatures, depending on the respective analyses [93-95, 122, 123]. Likewise, varying results with respect to the numbers of vCTB (1-3), placental macrophage (1-2) and stromal cells populations (2-3) were obtained in the RNAseq analyses [93-95, 122, 123]. The diverging data could be explained by differences in gestational age of placental samples, variations in the methodology (direct RNAseq of single cells after enzymatic digestion of tissues vs. RNAseq of HLAG purified cells), different cell cycle phases of the populations, as well as vast differences in the numbers of sequenced placental single cells (ranging from dozens up to several thousands). Whereas these analyses could be helpful to identify novel (surface) markers of specific placental subtypes, their origin and developmen tal regulation remain elusive warranting further functional investigations. For example, whether putative EVT subtypes are specified by an intrinsic developmental program of the anchoring villus, or merely represent phenotypic differences induced by the different components within the decidua remains unknown.
section_name: In vitro formation, identity and molecular control of differentiated placental trophoblast subtypes

section_content: CTB preparations from early pregnancy placentae undergo both cell fusion and EVT formation, and as a result CTBs were thought to be a homogenous bipotential 'stem cell like' population [124]. However, others found that EVT and STB develop from different subpopulations of vCTB, and that traditional CTB isolates contain both vCTBs and CCTs. In vivo, STBs are formed from the vCTBs that reside in a monolayer around the majority of the villus, and pure vCTBs can only form STBs in culture [113]. In contrast, precursors of the EVT lineage (CCTs) reside in proliferative multilay ered clusters within villus tips and proximal cell columns [102, 125]. The notion that CTBs were not a homogenous bipotential population was first raised by findings that sequential trypsinization led to the isolation of trophoblasts with different properties [126], and this was supported by subsequent findings that multilayered clusters of cells in the tips of first trimester villus explants were exclusively able to produce EVT outgrowth, but not regenerate the STB [127]. This EVT progenitor population was subsequently isolated and, unlike standard CTB preparations, proliferated slowly in culture, with around 20% of cells differentiating into HLAG positive EVTs, and no evidence of STB differ entiation [125]. The subsequent development of alternative methods to isolate highly purified CCTs, has allowed much higher rates of EVT differentiation (> 90%) to be achieved, and has enabled a greater understanding of the signalling pathways that distinguish EVT and STB progenitor popula tions [113]. 

The presence of distinct progenitors for EVT and STB means that vCTBs should not be considered a 'stem cell' population, and rather that a less differentiated 'true' tropho blast stem cell (TSC) population, that acts as the precursor to both progenitor populations, must reside within placental villi. Such a TSC population would overcome the limita tions of many traditional primary trophoblast models such 1 3 as heterogeneity between different cell isolations, and the limited lifespan of isolates in culture, and thus has been an intense focus of the field over the past decade. TSC popula tions have previously been isolated from preimplantation blastocysts in a number of animal models (porcine, bovine, rhesus monkey, and murine), of which murine TSCs are the best characterized [128] [129] [130]. Murine TSCs can be main tained in an undifferentiated state in the presence of fibro blast growth factor 4 (FGF4) and heparin, whilst removal of FGF4 from these cultures induces their differentiation into cells of the extraembryonic ectoderm, ectoplacental cone, and trophoblast giant cells [131]. 

Despite our knowledge of TSCs from animal models, the isolation of human TSCs proved considerably more chal lenging, and as a result was not achieved until recently. This difficulty in deriving human TSC may have in part arisen from the distinct anatomical differences between the human placenta and that of many animal models, as neither markers critical to murine TSC selfrenewal, or culture conditions used to propagate these cells, proved to be transferable to the human TSC derivation [132]. Indeed, key differences in the expression of lineageassociated factors have been observed between species [133, 134] as discussed below. 

The recent establishment of a human TSC model will allow us to begin to truly unpick these speciesspecific dif ferences to understand the unique nature of human tropho blast lineage differentiation in the mammalian context, and will play an important role in translating data between spe cies. The first human TSC population was isolated by enzy matically digesting first trimester placental villi, purifying α6 integrinpositive vCTBs, and then culturing these cells in a novel medium formulation that maintained these vCTB in culture far longer than had previously been possible [33]. After several passages, these cultures were taken over by the proliferative subset of TSCs, allowing the establishment of TSC lines that could be maintained in an undifferenti ated state for up to 5 months [33]. Similar populations were also established from the TE outgrowths of human blas tocysts [33]. However, the significant breakthrough in this paper arose from the authors' ability to identify conditions in which the human TSC within vCTB preparations could be expanded longterm (discussed below). Transcriptomic analysis revealed that human TSCs express critical mark ers of trophoblast identity and selfrenewal [33], such as cytokeratin7, GATA3, TEAdomain transcription factor 4 (TEAD4) and tumour protein p63 (TP63) [135, 136]. How ever, markers associated with murine TSCs were either absent or weakly expressed by human TSCs (or any other primary human trophoblast isolates examined) [33], suggest ing that different transcriptional networks could be impor tant for human trophoblast development. 

To date, TSCs have not been able to be isolated from third trimester placentae [33, 34]. However, the ability to do this would provide a significant advance by enabling researchers to link TSC function to pregnancy pathologies in order to understand how these pathologies may have developed. Fur thermore, this model of TSC derivation requires prolonged culture, which may lead to adaptation to in vitro conditions that may mask important differences between normal and pathological tissues. Therefore, whilst this has provided a quantum leap forward in our ability to investigate human TSCs, work remains to be done to identify unique cell sur face markers of this TSC population, or ways to isolate these cells directly from fresh placental tissue of normal and path ological pregnancies. 

Isolation of human TSCs was attempted for many years. Indeed, a number of candidate TSC populations were in existence prior to the breakthrough publication by Okae et al., and it will be of interest in the future to determine how these populations respond to the TSCspecific culture conditions defined by these researchers. The first significant potential candidate human TSC population in the literature was isolated after trypsin digestion of first trimester chorion from which the villi had been removed [137]. When cultured on gelatin, this 'trophoblast progenitor' (TBPC) population expressed POU class 5 homeobox 1/OCT4, cytokeratin7 and GATA4, but lacked expression of more differentiated trophoblast markers including GCM1 and hCG [137]. Cul ture on Matrigel provoked differentiation, resulting in down regulation of OCT4, and induction of STB and EVT markers [137]. However, more recent work demonstrates that these cells predominantly differentiate into EVTs, suggesting that they are more akin to an EVT progenitor population than a true human TSC [138]. 

Several groups have attempted to isolate human TSC pop ulations by exploiting a characteristic of many adult stem cell populations; the ability to rapidly efflux Hoechst 33342, resulting in a characteristic 'streak' of low intensity staining (termed a sidepopulation) when analysed by flow cytometry [139, 140]. As the trophectoderm expresses 90fold more of the primary Hoechst efflux pump ABCG2 than ICM derived hESCs, the sidepopulation technique is particularly promis ing for human TSC isolation [141]. Takao et al. identified a sidepopulation within both primary first trimester CTBs (constituting 0. 12% of cells) and the HTR8/SVneo cell line (constituting 0. 53% of cells) that uniquely coexpress inter leukin 1 receptor type 2 (IL1R2) and interleukin 7 receptor (IL7R) [142]. More recently, the sidepopulation technique was combined with a novel trophoblast isolation protocol to isolate a candidate human TSC population from first tri mester villous tissue that is distinct from both TBPC and the sidepopulation isolated by Takao et al. [143]. These sidepopulation trophoblasts form a distinct population more closely related to vCTB than EVT at both the transcriptomic and methalomic level [143, 144]. Furthermore, these cells express markers that maintain the stem cell state including WNT5A, Kruppellike factor 4 (KLF4) and OCT6, as well as markers involved with both murine and human tropho blast lineage differentiation, including E74like ETS tran scription factor 5 (ELF5), TEAD4, BMP4, and fibroblast growth factor 2 (FGF2) [143]. However, further work is required to confirm the stem cell status of sidepopulation derived candidate human TSC populations via their differ entiation into the mature trophoblast lineages.
section_name: Origin, localization and identity of trophoblast stem and progenitor cells

section_content: Whilst TSC populations are a key focus in the field, it is important to remember that trophoblasts in vivo do not exist or differentiate in isolation. Placental development is also crucially dependent on stem cell lineages within the villus core that play critical roles in influencing the morphogenesis of the branching architecture of the placenta, and in driv ing placental vascular development. Cells in the core of the placental villi arise from the ExM, which itself most likely originates from the Hy [145, 146]. In addition, progenitors from the yolk sac may migrate to the placenta, although these cells are more likely to colonize the placenta only after the umbilical circulation has opened up around 32 days post conception [49, 147]. During very early placental develop ment it is thus likely that the nontrophoblast lineages of the placenta arise de novo from sequential differentiation of the ExM. However, the exact lineage differentiation pathways, and the factors that regulate them, are only beginning to be understood. 

The earliest progenitor of vascular lineages in the placenta that has been isolated to date is a population of CD43, CD31 and CD144positive cells akin to a mesenchymoangioblast population [148]. Mesenchymoangioblasts isolated from other tissues have the potential to differentiate into all major vascular lineages including endothelial cells, smooth muscle cells, pericytes and mesenchymal stem cells [149], and in a similar manner placental mesenchymoangioblasts have been shown to form colonies containing both mesenchymal and endothelial cells [148]. This provides a likely candidate from which cells of the placental blood vessels originate. However, the question remains as to where the haematopoietic lineages (Hofbauer cells and red blood cells) arise from, as they are evident in the placenta from 18 days postconception, prior to the onset of the uterine circulation [54, 150]. It has been hypothesized that an even earlier precursor that could give rise to both mesenchymoangioblasts and haemangioblasts (from which blood lineages could derive) may exist in the early human placenta, although the relationship of this cell to the ExM that first invades the placental villi is unknown [49, 148]. Such a precursor could give rise to the early haemangiogenic foci, which may function in a similar way to haemangiogenic endothelium seen in other embryonic systems whereby red blood cells arise directly from the endothelial layer. Indeed, haematopoietic stem cells expressing RUNX1 (required for an endothelialtohaematopoietic transition) have been identified in murine placentae, suggesting a similar population of cells may exist in human placentae [151, 152]. Despite their hae matopoietic associations, Hofbauer cells are spatially isolated from haemangiogenic foci in early placentae [151]. Indeed, it seems that this population may arise from a precursor popula tion identified within the placental stroma that exhibits a fibro blastic morphology, but expresses the macrophage/monocytic markers CD115 and CD14 [153]. 

Finally, mesenchymal stem cells (MSCs) themselves, which reside in a perivascular niche in the placenta through out gestation, could directly contribute to the development of the placental vasculature via differentiation into endothe lial cells and smooth muscle cells. Placental MSCs can be differentiated in vitro into cells that express a number of endothelial markers including von Willebrand factor (vWF), CD31, VEGFR2 and CD144, and their propensity to differ entiate down this pathway is greater than MSC populations derived from bonemarrow, aligning with the concept that this is their biological function in vivo [154, 155]. How ever, whilst MSCs from other tissues are able to differentiate into smooth muscle cells and pericytes, to date the ability of placental MSC to differentiate into these cell types has not been demonstrated [49, 156]. The above suggests that while we are beginning to reveal distinct populations that may contribute to the development of cells within the vil lous core and vasculature, we still have little understanding of how the different progenitor populations within the core of the early placental villi relate to each other, nor of the different contributions these cells may make to the ongoing growth and development of the placenta across gestation. Future work to identify and propagate a progenitor cell at the apex of the placental core lineages may help to shed light on the relationships between these cell populations at both the functional and molecular level.
section_name: Origin, localization and identity of stem and progenitor cells of the villous core

section_content: The absence of a human TSC for most of the past decade has led researchers to use alternative selfrenewing model systems to mimic early placental development, the most popular of which has been human embryonic stem cells (hESCs). In vivo, cells of the ICM (from which most hESC lines are derived) form the embryo proper and not the pla centa [157]. However, in vitro, BMP4 treatment can be used to induce hESC to differentiate into trophoblastlike cells [158, 159]. In such models, morphological differentiation into trophoblastlike cells is first observed at the periphery of cell colonies that display a flattened phenotype, but over time cell differentiation spreads inwards towards the colony centre [157, 160, 161]. Whilst the wide variation in exact differentiation protocols used (i. e. concentration, time, start ing hESC line) can subtly vary the gene expression pattern [32], these models generally exhibit a downregulation in the expression of pluripotency factors (NANOG and OCT4) and induction of caudalrelated homeobox transcription fac tor 2 (CDX2), the master regulator of murine trophoblast development [162], provoking trophectoderm lineage dif ferentiation [163] [164] [165] [166] [167]. However, this model is not without limitations, as it can produce mixed cultures that express trophoblast, mesodermal and vascular endothelial cell mark ers, suggesting that it does not specifically induce differen tiation towards the trophoblast lineage [168] [169] [170]. Indeed, the generation of mixed mesodermal cultures from BMP4 treated hESCs has caused some authors to question whether BMP4 drives hESC differentiation into trophoblastlike or mesodermallike cells [169] [170] [171]. As a result, hESC mod els of trophoblast differentiation have been more recently refined to prevent mixed phenotype cultures by inhibiting Activin/Nodal and FGF2 signalling pathways, resulting in cultures that are 80-100% trophectoderm/trophoblastlike with undetectable levels of the mesodermal marker Brachy ury [167, 170]. TSClike trophoblasts, coexpressing CDX2 and p63, have also been isolated from this model by using low doses of BMP4, which could be differentiated into STBs and EVTs in vitro [172]. 

Further, putative TSCs have also been established from early stages of embryonic development. The manipulation of mixed potency morula blastomeres has allowed the deriva tion of a human TSC line (USFB6) from a single blastomere of an eightcell human embryo [173]. USFB6 cells maintain the expression of CTB markers when cultured in FGF2 with Activin/Nodal pathway inhibition and form EVTs and STB with an absence of mesodermal markers. However, USFB6 cells do have a more mesenchymal morphology than the human TSC population isolated by Okae et al., although it is unclear whether this is a consequence of the differing culture conditions the cells are maintained in [33, 173]. 

Human TSCs, selfrenewing on collagen IV in 2D, have been established from blastocysts and first trimester CTB preparations [33]. In contrast to mouse TSCs, requiring FGF4 and transforming growth factorβ (TGFβ) signal ling for maintaining stemness [131], treatment with EGF, and inhibition of histone deacetylase (valproic acid), Rho associated protein kinase (Y27632), TGFβ signalling (A8301) and glycogen synthase kinase3 (CHIR99021) were sufficient for continuous proliferation of human TSCs [33]. CHIR99021 inhibits βcatenin degradation thereby activating canonical Wnt signalling, which besides its role in EVT differentiation [115, 116], seems to be critical for trophoblast selfrenewal. Similar culture conditions were applied to develop longterm expanding 3dimensional (3D) trophoblast organoids (TBORGs) from first trimester pla cental tissues. In the first paper published on the derivation of TBORGs Haider et al. used EGF, the TGFβ signalling inhibitor A8301, the BMP signalling inhibitor Noggin, and the activators of Wnt signalling, Rspondin, CHIR99021 and prostaglandin E2, substances which have been utilized to successfully establish epithelial organoids from other human tissues [34, 174]. In a recent paper, confirming establish ment of TBORGs, EGF, FGF2, A8301, CHIR99021 and Rspondin were used [175]. In summary, these studies sug gest that activation of Wnt and EGF signalling and inhibition of the TGFβ pathway could be sufficient for the deriva tion and longterm expansion of human TSCs and placental organoids. 

TBORGs, growing in growth factorreduced Matrigel, mimic the in vivo structure of human placenta, express markers of trophoblast identity and stemness and actively secrete pregnancy hormones [34, 175]. TBORGs exclu sively contain trophoblast cells allowing researchers to study discrete steps of placental development in a simplified fash ion. STBs and EVTs can be generated in both 2D TCSs and 3D TBORGs suggesting that the respective progenitors are maintained in vitro. Yet, conditions for STB formation need considerable improvement, to further identify key tran scription factors and signalling molecules for STB commit ment and differentiation. Syncytialization of 2D TCSs was shown to require elevation of cAMP levels by forskolin [33], a rather unspecific treatment which has been used for dec ades to fuse primary CTBs. In contrast, growing TBORGs undergo spontaneous fusion towards the centre [34, 175], rendering the current conditions unsuitable for controllable induction of the SBT lineage. 

The EVT lineage could be induced in 2D TSCs by tran siently adding neuregulin and soluble Matrigel and by ele vating A8301 in the absence of valproic acid and EGF [33], conditions that were later also applied to generate invasive trophoblasts in TBORGs [175]. However, utilization of these culture conditions remains unclear, since EVT pro genitors lack neuregulin receptors consisting of heterodi mers of ErbB2 and ErbB3 [105]. The latter are exclusively expressed on EVTs promoting their survival [105], and therefore cannot be required for commitment of the EVT lineage. Also, EVTs originating from 2D TSCs, display an unusual spindleshaped morphology and lack critical markers such as insulin like growth factor binding protein 3 (IGFBP3), pappalysin 1 (PAPPA), DAO or PRG2 [33]. In contrast, others demonstrated that removal of the Wnt acti vator CHIR99021 was sufficient for induction of the EVT lineage in TBORGs [34]. Absence of the GSK3 inhibitor produced Notch1positive CCTs, the prime marker of EVT progenitors [113], which further differentiated into EVTs expressing all commonly accepted markers for these cells. 

Differentiation also occurred in a spatial correct orientation suggesting that these culture conditions closely recapitulate in vivo cell column formation and EVT differentiation [34].
section_name: Self-renewing model systems recapitulating placental development and differentiation

section_content: Our present view regarding key regulators of human tropho blast development is based on their expression patterns in the placenta and a few functional studies in isolated pri mary trophoblast, villous explant cultures and trophoblast cell lines [38]. Transcription factors, playing a pivotal role in mouse trophoblast development and differentiation, have been unravelled [35, 176]. Therefore, speculation on the putative functions of their analogous genes in the human placenta can be elaborated. For these comparisons one must assume that proliferative spongiotrophoblasts and CCTs, and giant cells and EVTs, respectively, are equivalent cell types in the two species. Indeed, several key transcription factors, for example AP2γ, encoded by the TFAP2C gene, or inhibitor of DNA binding 2 (ID2), are expressed in pro liferative CTBs and CCTs which, like their murine counter parts, could promote trophoblast development and prolifera tion [177, 178]. Others, such as achaete-scute family basic helix-loop-helix (bHLH) transcription factor 2 (ASCL2) and heart and neural crest derivatives expressed 1 (HAND1) are also expressed during human trophoblast development. However, their placental subtypespecific expression pat terns differ between mouse and men. Whereas murine Mash2/Ascl2 is a spongiotrophoblastspecific gene that con trols maintenance and proliferation [179], HASH2/ASCL2 is expressed in both cell columns and EVTs, suggesting that function of the human gene diverges [112, 180, 181]. Hand1 is critical for murine giant cell formation, while it is absent from first trimester human placenta [182, 183]. However, the particular bHLH protein is expressed in the outer layer of human blastocysts and BMP4treated hESC, indicating a possible role in early TE development [184, 185]. 

Furthermore, analyses of blastocysts and early gesta tional tissues revealed common expression patterns between murine and human TE and placenta [133, 186]. Some key factors, such as GATA3 controlling trophoblast selfrenewal and differentiation [187], are present in both TE and placen tal trophoblast of mouse and men [188]. However, timing and expression of several critical regulatory genes differ between the two species [189]. The prime markers of murine TE and ICM specification, Cdx2 and Oct4, respectively, are restricted to the respective tissues in murine preimplanta tion embryos and inhibit each other's expression [162, 190]. However, these genes have been reported to be coexpressed in the TE of cultured human blastocysts at 5 days post fertilization [188, 189, 191]. Compared to freshly flushed mouse blastocysts, colocalization of these key regulators in the human TE could either represent a functional dif ference between mice and men or be a consequence of the in vitro cultivation delaying downregulation of OCT4 in the TE compartment. Several critical genes of murine TE speci fication and selfrenewal, such as eomesodermin (Eomes) and Elf5 [192, 193], are absent from human TE [133], which could be key for a faster differentiation process in humans. However, ELF5 protein and/or mRNA were found to be expressed in vCTBs of the early human placenta and in self renewing CTBs of TBORGs [34, 186, 194]. Additionally, its presence in pCCTs was suggested [194]. However, ELF5 protein expression could not be confirmed recently [186] and Notch1 + EVT progenitors, generated in TBORGs, consider ably downregulated ELF5 mRNA [34], questioning its role in cell column proliferation. Hence, human ELF5 could be mainly required for vCTB expansion after implantation and during morphogenesis of placental villi. In conclusion, a dis tinct set of regulatory genes might control human TE speci fication and maintenance, despite some overlap with mouse. Along those lines, AP2γ, AP2α, GATA2 and GATA3, expressed in human TE, were sufficient for the induction of CTB/TEspecific genes and repression of OCT4 in the BMP4hESC model [195]. 

CDX2, the critical transcriptional regulator of murine TE specification, is also expressed by the human TE. How ever, discordant results with respect to its localization were published. Similar to mice, Kunath et al. observed nuclear expression in the TE of cultivated human blastocysts, whereas the factor was absent from the ICM [132]. In con trast, others found that CDX2 mostly localizes to the TE cytoplasm with variable levels between blastocysts [188, 189]. Therefore, further investigations are needed to clarify its localization and potential role in human TE development. In first trimester placenta CDX2 is present in the nuclei of vCTBs close to the chorionic plate, whereas no expression is seen in proliferative cell columns of the basal plate [113, 186]. Hence in the human placenta, similar to ELF5, CDX2 could have its main role after implantation by promoting TSC/CTB maintenance and proliferation. At subsequent developmental stages, and/or in regions distal from the cho rionic plate, selfrenewal of TSCs may not require CDX2, since only a few cells within TBORGs and 2DTSCs weakly express the gene, despite the fact that these cultures give rise to STBs and EVTs. Another key transcription factor of mouse TE development, TEAD4, could fulfil this role. Controlled by coactivators of the Hippo pathway Tead4 was shown to activate Cdx2 expression and TE development in mice [196, 197]. However, its precise role in human CTBs remains to be elucidated. Human TEAD4 localizes to the nuclei of proliferative CTBs of 2DTSCs and TBORGs, and of placental vCTBs in vivo, while its expression is downreg ulated in cell columns of anchoring villi [33, 34, 113, 186]. 

The latter might be achieved by CCTspecific induction of Notch1 in villi of the placental basal plate. Notch1 is present in proliferative pCCTs generating the transcriptional coacti vator Notch1 intracellular domain (N1ICD) by γsecretase mediated cleavage from the cytosolic region of the receptor [113]. N1ICD promoted trophoblast survival, and repressed markers of vCTB stemness, i. e. TEAD4 and p63, the latter by promoting its degradation via upregulation of interferon regulatory factor 6 (IRF6), and induced the EVT progenitor specific genes, MYC and VEcadherin [113]. 

So far, Notch1 represents the only functionally tested regulator for initiation of the EVT lineage (Fig. 3 ). It is expressed in a subset of cyclin Apositive pCCTs suggesting that the EVT progenitor cell pool could give rise to Notch1 negative transientamplifying cells, which further differen tiate into EVTs [113]. Noteworthy, pCCTs lack expression of TCF genes, suggesting that canonical Wnt signalling might not be critical for formation and/or maintenance of proliferative cell columns. Indeed, removal of Wnt activators promoted development of TCFnegative EVTprogenitors in TBORGs [34]. However, as these cells undergo differentia tion, they induce Wntdependent transcription factors and nuclear βcatenin, likely as part of an epithelial to mesenchy mal (EMT)like program operating during EVT formation [115, 198, 199]. Therefore, canonical Wnt signalling could play a dual role in human placental development (Fig. 3 ). In analogy to its role in other stem cell niches [200], Wnt activated TCF1, which is expressed in a subset of TBORGs and vCTBs [34], could be necessary for selfrenewal of TSC. In contrast, signalling through TCF4-βcatenin complexes could promote EVT differentiation and function. A switch in Notch receptor expression across the anchoring villus might also be critical in this process, as Notch2 is induced in dCCTs and EVTs, controlling motility of the latter [201, 202]. 

So far, little is known about altered functions/mutations of trophoblastspecific transcription factors in pregnancy disorders. However, a specific genotype of the winged helix protein STOX1, controlling the balance between cell column proliferation and EVT invasion, was found to be associated with a familial form of severe preeclampsia [203, 204].
section_name: Key regulatory transcription factors of human trophoblast development

section_content: Our current knowledge about human TSCs, their derivatives and specific key transcription factors predominantly relies on comparative expression patterns between mouse and human trophoblast. Based on these data and some functional stud ies, we herein speculate about putative markers discrimi nating the different human trophoblast subtypes (Fig. 4 ). Coexpression of CDX2, and TEAD4, among others such as GATA3 and AP2γ, is characteristic for trophectodermal stem cells (TESC). However, critical key factors for the development of postimplantation TSCs/early selfrenew ing CTBs are unknown, since in situ expression data and appropriate model systems are lacking. Accordingly, puta tive differences between expanding CTBs of primary villi/ trophoblastic shell and early proliferative vCTBs remain unknown. Possibly, induction of ELF5 and concomitant expression of CDX2 could be hallmarks of postimplanta tion TSCs. However, CDX2 is largely absent from vCTBs of distal villi of early placentae and its expression could not be maintained in selfrenewing TBORGs at higher pas sage numbers [34, 186]. Hence, CDX2positive cells of the first trimester human placenta could also represent residual TESC which might be distinct from selfrenewing TSCs and require different culture conditions for longterm mainte nance. Moreover, TCF1 could also mark TSCs due to its restricted expression in placental villi and its known role in other stem cell niches. Further, we postulate that precursors within the trophoblast epithelium, prone to fusion, could express a different set of pivotal regulators, as compared to selfrenewing TSCs. Whereas ELF5, TEAD4 and p63 are Formation of these precursors is also associated with the loss of TCF1 expression, whereas βcatenin-TCF4 complexes arise during EVT formation. CCT cell column trophoblast, IRF6 interferon regula tory factor 6, TCF Tcell factor, Wnt wingless present in all first trimester vCTBs, ovolike transcriptional repressor 1 (OVOL1) and GCM1 are restricted to a subset of progenitors in which STB formation could be initiated. 

Accordingly, both GCM1 and OVOL1 were shown to pro mote STB formation, the latter by repressing stemness and proliferationassociated genes [205]. N1ICD has been identified as a master regulator of EVT lineage induction inhibiting vCTB stemness genes. Hypoxia and contact to the decidual matrix could be critical triggers of EVT line age commitment and differentiation, respectively. Numerous transcription factors, controlling migration and invasion, are induced in EVTs in vivo and during 2D differentiation of primary CTBs. However, their specific role in developing EVT progenitors awaits further studies in the recently estab lished, selfrenewing trophoblast models. 

In summary, the previous absence of expandable systems hampered deciphering key steps of human trophoblast devel opment. However, establishment of selfrenewing cultures should allow delineating pivotal regulators of human pla centation in the near future. Moreover, in vitro modelling of pregnancy complications, for which failures of tropho blast growth and differentiation could be underlying causes, should be advanced by establishing 2DTSCs and TBORGs from abnormal placental tissues.
section_name: Conclusions

section_content: Acknowledgements Open access funding provided by Austrian Sci ence Fund (FWF). The present work was supported by the Austrian Science Fund (Grant P31470B30 ) to M. K. and a Health Research Council Sir Charles Hercus Research Fellowship to J. J. 

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