NOTCH3 signalling controls human trophoblast stem cell expansion and differentiation.

Failures in growth and differentiation of the early human placenta are associated with severe pregnancy disorders such as pre-eclampsia and fetal growth restriction. However, regulatory mechanisms controlling development of placental epithelial cells, the trophoblasts, remain poorly elucidated. Using trophoblast stem cells (TSCs), trophoblast organoids (TB-ORGs) and primary cytotrophoblasts (CTBs) of early pregnancy, we herein show that autocrine NOTCH3 signalling controls human placental expansion and differentiation. The NOTCH3 receptor was specifically expressed in proliferative CTB progenitors and its active form, the nuclear NOTCH3 intracellular domain (NOTCH3-ICD), interacted with the transcriptional co-activator mastermind-like 1 (MAML1). Doxycycline-inducible expression of dominant-negative MAML1 in TSC lines provoked cell fusion and upregulation of genes specific for multinucleated syncytiotrophoblasts, which are the differentiated hormone-producing cells of the placenta. However, progenitor expansion and markers of trophoblast stemness and proliferation were suppressed. Accordingly, inhibition of NOTCH3 signalling diminished growth of TB-ORGs, whereas overexpression of NOTCH3-ICD in primary CTBs and TSCs showed opposite effects. In conclusion, the data suggest that canonical NOTCH3 signalling plays a key role in human placental development by promoting self-renewal of CTB progenitors.

The human placenta shapes the phenotype of decidual macrophages.

During human pregnancy, placenta-derived extravillous trophoblasts (EVTs) invade the decidua and communicate with maternal immune cells. The decidua distinguishes into basalis (decB) and parietalis (decP). The latter remains unaffected by EVT invasion. By defining a specific gating strategy, we report the accumulation of macrophages in decB. We describe a decidua basalis-associated macrophage (decBAM) population with a differential transcriptome and secretome compared with decidua parietalis-associated macrophages (decPAMs). decBAMs are CD11chi and efficient inducers of Tregs, proliferate in situ, and secrete high levels of CXCL1, CXCL5, M-CSF, and IL-10. In contrast, decPAMs exert a dendritic cell-like, motile phenotype characterized by induced expression of HLA class II molecules, enhanced phagocytosis, and the ability to activate T cells. Strikingly, EVT-conditioned media convert decPAMs into a decBAM phenotype. These findings assign distinct macrophage phenotypes to decidual areas depending on placentation and further highlight a critical role for EVTs in the induction of decB-associated macrophage polarization.

3D organoid formation and EVT differentiation of various trophoblastic cell lines.

3-dimensional trophoblast organoids (TB-ORG) represent a reliable model for studying extravillous trophoblast (EVT) lineage formation and differentiation. However, restricted access to first trimester placentae requires alternative cell sources for establishing placental organoids. Recently, we demonstrated EVT differentiation in JEG-3-derived organoids. Consequently, we herein tested whether other commonly used trophoblastic cell lines, ACH-3P, HTR-8/SVneo, and SWAN-71 were capable of self-organizing into organoids and subsequent EVT differentiation. Notably, only ACH-3P formed organoids under stemness conditions mimicking TB-ORG architectures, and induction of EVT differentiation provoked formation of HLA-Gpos areas. Hence ACH-3P-ORGs provide another organoid model for studying controlled EVT lineage formation and differentiation.

Transforming growth factor-ß signaling governs the differentiation program of extravillous trophoblasts in the developing human placenta.

Abnormal placentation has been noticed in a variety of pregnancy complications such as miscarriage, early-onset preeclampsia, and fetal growth restriction. Defects in the developmental program of extravillous trophoblasts (EVTs), migrating from placental anchoring villi into the maternal decidua and its vessels, is thought to be an underlying cause. Yet, key regulatory mechanisms controlling commitment and differentiation of the invasive trophoblast lineage remain largely elusive. Herein, comparative gene expression analyses of HLA-G-purified EVTs, isolated from donor-matched placenta, decidua, and trophoblast organoids (TB-ORGs), revealed biological processes and signaling pathways governing EVT development. In particular, bioinformatics analyses and manipulations in different versatile trophoblast cell models unraveled transforming growth factor-ß (TGF-ß) signaling as a crucial pathway driving differentiation of placental EVTs into decidual EVTs, the latter showing enrichment of a secretory gene signature. Removal of Wingless signaling and subsequent activation of the TGF-ß pathway were required for the formation of human leukocyte antigen-G+ (HLA-G+) EVTs in TB-ORGs that resemble in situ EVTs at the level of global gene expression. Accordingly, TGF-ß-treated EVTs secreted enzymes, such as DAO and PAPPA2, which were predominantly expressed by decidual EVTs. Their genes were controlled by EVT-specific induction and genomic binding of the TGF-ß downstream effector SMAD3. In summary, TGF-ß signaling plays a key role in human placental development governing the differentiation program of EVTs.

Ultra-High-Resolution 3D Optical Coherence Tomography Reveals Inner Structures of Human Placenta-Derived Trophoblast Organoids.

OBJECTIVE: 3D optical coherence tomography (OCT) is used for analyses of human placenta organoids in situ without sample preparation. METHODS: The trophoblast organoids analyzed were derived from primary human trophoblast. In this study a custom made ultra-high-resolution spectral domain OCT system with uniform spatial and axial resolution of 2.48 µm in organoid tissue was used. The obtained OCT results align to differentiation status tested via quantitative polymerase chain reaction, Western blot analyses, immunohistochemistry, and immunofluorescence of histological sections. RESULTS: 3D OCT enables a more detailed placenta organoid monitoring compared to brightfield microscopy. Inner architecture with light scattering "bridges" surrounding cavities were visualized and quantified in situ for the first time. The formation of these bridges and cavities is congruent to differentiated trophoblast organoids having developed syncytiotrophoblasts. CONCLUSION: Using 3D OCT in living placenta organoids is a fast tool to assess the differentiation status and resolve internal structures in situ, which is not possible with standard live cell imaging modality. SIGNIFICANCE: Only recently human placenta-derived organoids were established, allowing to have a highly reproducible and stable in vitro model to investigate not only developmental but also physiological and pathophysiological processes during early pregnancy. To our knowledge, this work is the first to analyze living human placenta organoids using 3D OCT. Thereby, the rapid and especially non-endpoint OCT qualitative analyses align to the differentiation stage of organoids, which will aid future advancement in this field.

Densities of decidual high endothelial venules correlate with T-cell influx in healthy pregnancies and idiopathic recurrent pregnancy losses.

STUDY QUESTION: Do high endothelial venules (HEVs) appear in the uterus of healthy and pathological pregnancies? SUMMARY ANSWER: Our study reveals that HEVs are present in the non-pregnant endometrium and decidua parietalis (decP) but decline upon placentation in decidua basalis (decB) and are less abundant in decidual tissues from idiopathic, recurrent pregnancy losses (RPLs). WHAT IS KNOWN ALREADY: RPL is associated with a compromised decidual vascular phenotype. STUDY DESIGN, SIZE, DURATION: Endometrial (n = 29) and first trimester decidual (n = 86, 6-12th week of gestation) tissue samples obtained from endometrial biopsies or elective pregnancy terminations were used to determine the number of HEVs and T cells. In addition, quantification of HEVs and immune cells was performed in a cohort of decidual tissues from RPL (n = 25). PARTICIPANTS/MATERIALS, SETTING, METHODS: Position and frequency of HEVs were determined in non-pregnant endometrial as well as decidual tissue sections using immunofluorescence (IF) staining with antibodies against E-selectin, intercellular adhesion molecule, von Willebrand factor, ephrin receptor B4, CD34 and a carbohydrate epitope specific to HEVs (MECA-79). Immune cell distribution and characterization was determined by antibodies recognizing CD45 and CD3 by IF staining- and flow cytometry-based analyses. Antibodies against c-c motif chemokine ligand 21 (CCL21) and lymphotoxin-beta were used in IF staining and Western blot analyses of decidual tissues. MAIN RESULTS AND THE ROLE OF CHANCE: Functional HEVs are found in high numbers in the secretory endometrium and decP but decline in numbers upon placentation in decB (P ≤ 0.001). Decidua parietalis tissues contain higher levels of the HEV-maintaining factor lymphotoxin beta and decP-associated HEVs also express CCL21 (P ≤ 0.05), a potent T-cell chemoattractant. Moreover, there is a positive correlation between the numbers of decidual HEVs and the abundance of CD3+ cells in decidual tissue sections (P ≤ 0.001). In-depth analysis of a RPL tissue collection revealed a decreased decB (P ≤ 0.01) and decP (P ≤ 0.01) HEV density as well as reduced numbers of T cells in decB (P ≤ 0.05) and decP (P ≤ .001) sections when compared with age-matched healthy control samples. Using receiver-operating characteristics analyses, we found significant predictive values for the ratios of CD3/CD45 (P < 0.001) and HEVs/total vessels (P < 0.001) for the occurrence of RPL. LIMITATIONS, REASONS FOR CAUTION: Analyses were performed in first trimester decidual tissues from elective terminations of pregnancy or non-pregnant endometrium samples from patients diagnosed with non-endometrial pathologies including cervical polyps, ovarian cysts and myomas. First trimester decidual tissues may include pregnancies which potentially would have developed placental disorders later in gestation. In addition, our cohort of non-pregnant endometrium may not reflect the endometrial vascular phenotype of healthy women. Finally, determination of immune cell distributions in the patient cohorts studied may be influenced by the different modes of tissue derivation. Pregnancy terminations were performed by surgical aspiration, endometrial tissues were obtained by biopsies and RPL tissues were collected after spontaneous loss of pregnancy. WIDER IMPLICATIONS OF THE FINDINGS: In this study, we propose an inherent mechanism by which the endometrium and in particular the decidua control T-cell recruitment. By demonstrating reduced HEV densities and numbers of T cells in decB and decP tissues of RPL samples we further support previous findings reporting an altered vascular phenotype in early pregnancy loss. Altogether, the findings provide important information to further decipher the etiologies of unexplained RPL. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Austrian Science Fund (P31470 B30 to M.K.) and by the Austrian National Bank (17613ONB to J.P.). There are no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

Pivotal role of the transcriptional co-activator YAP in trophoblast stemness of the developing human placenta.

Various pregnancy complications, such as severe forms of preeclampsia or intrauterine growth restriction, are thought to arise from failures in the differentiation of human placental trophoblasts. Progenitors of the latter either develop into invasive extravillous trophoblasts, remodeling the uterine vasculature, or fuse into multinuclear syncytiotrophoblasts transporting oxygen and nutrients to the growing fetus. However, key regulatory factors controlling trophoblast self-renewal and differentiation have been poorly elucidated. Using primary cells, three-dimensional organoids, and CRISPR-Cas9 genome-edited JEG-3 clones, we herein show that YAP, the transcriptional coactivator of the Hippo signaling pathway, promotes maintenance of cytotrophoblast progenitors by different genomic mechanisms. Genetic or chemical manipulation of YAP in these cellular models revealed that it stimulates proliferation and expression of cell cycle regulators and stemness-associated genes, but inhibits cell fusion and production of syncytiotrophoblast (STB)-specific proteins, such as hCG and GDF15. Genome-wide comparisons of primary villous cytotrophoblasts overexpressing constitutively active YAP-5SA with YAP KO cells and syncytializing trophoblasts revealed common target genes involved in trophoblast stemness and differentiation. ChIP-qPCR unraveled that YAP-5SA overexpression increased binding of YAP-TEAD4 complexes to promoters of proliferation-associated genes such as CCNA and CDK6 Moreover, repressive YAP-TEAD4 complexes containing the histone methyltransferase EZH2 were detected in the genomic regions of the STB-specific CGB5 and CGB7 genes. In summary, YAP plays a pivotal role in the maintenance of the human placental trophoblast epithelium. Besides activating stemness factors, it also directly represses genes promoting trophoblast cell fusion.

Metabolism of cholesterol and progesterone is differentially regulated in primary trophoblastic subtypes and might be disturbed in recurrent miscarriages.

During pregnancy, extravillous trophoblasts (EVTs) invade the maternal decidua and remodel the local vasculature to establish blood supply for the growing fetus. Compromised EVT function has been linked to aberrant pregnancy associated with maternal and fetal morbidity and mortality. However, metabolic features of this invasive trophoblast subtype are largely unknown. Using primary human trophoblasts isolated from first trimester placental tissues, we show that cellular cholesterol homeostasis is differentially regulated in EVTs compared with villous cytotrophoblasts. Utilizing RNA-sequencing, gene set-enrichment analysis, and functional validation, we provide evidence that EVTs display increased levels of free and esterified cholesterol. Accordingly, EVTs are characterized by increased expression of the HDL-receptor, scavenger receptor class B type I, and reduced expression of the LXR and its target genes. We further reveal that EVTs express elevated levels of hydroxy-delta-5-steroid dehydrogenase 3 beta- and steroid delta-isomerase 1 (HSD3B1) (a rate-limiting enzyme in progesterone synthesis) and are capable of secreting progesterone. Increasing cholesterol export by LXR activation reduced progesterone secretion in an ABCA1-dependent manner. Importantly, HSD3B1 expression was decreased in EVTs of idiopathic recurrent spontaneous abortions, pointing toward compromised progesterone metabolism in EVTs of early miscarriages. Here, we provide insights into the regulation of cholesterol and progesterone metabolism in trophoblastic subtypes and its putative relevance in human miscarriage.

Estrogen Signaling Drives Ciliogenesis in Human Endometrial Organoids.

The human endometrium is the inner lining of the uterus consisting of stromal and epithelial (secretory and ciliated) cells. It undergoes a hormonally regulated monthly cycle of growth, differentiation, and desquamation. However, how these cyclic changes control the balance between secretory and ciliated cells remains unclear. Here, we established endometrial organoids to investigate the estrogen (E2)-driven control of cell fate decisions in human endometrial epithelium. We demonstrate that they preserve the structure, expression patterns, secretory properties, and E2 responsiveness of their tissue of origin. Next, we show that the induction of ciliated cells is orchestrated by the coordinated action of E2 and NOTCH signaling. Although E2 is the primary driver, inhibition of NOTCH signaling provides a permissive environment. However, inhibition of NOTCH alone is not sufficient to trigger ciliogenesis. Overall, we provide insights into endometrial biology and propose endometrial organoids as a robust and powerful model for studying ciliogenesis in vitro.

Extravillous trophoblast invasion of venous as well as lymphatic vessels is altered in idiopathic, recurrent, spontaneous abortions.

STUDY QUESTION: Do extravillous trophoblasts (EVTs) invade non-arterial decidual vessels in healthy and pathological pregnancies? SUMMARY ANSWER: Our results reveal that trophoblast invasion of venous and lymphatic vessels is a frequent event during the first trimester of pregnancy and is compromised in  recurrent spontaneous abortion (RSA). In addition, the present data suggest that EVTs populate regional lymph nodes during pregnancy. WHAT IS ALREADY KNOWN: Human trophoblasts remodel and invade decidual spiral arteries. In addition, a recent report demonstrates that trophoblasts contact and invade decidual veins. STUDY DESIGN, SIZE, DURATION: Tissue samples of human first trimester deciduae basalis (n = 54, 6th-13th weeks of gestation) obtained from elective pregnancy terminations were used to study trophoblast invasion into veins and lymphatics, in comparison to arteries. Age-matched cases of idiopathic, recurrent spontaneous abortions tissue samples (n = 23) were assessed for cell numbers of EVTs in these decidual vessels. In addition, lymph nodes of four pregnant women were analysed for the presence of EVTs. PARTICIPANTS/MATERIALS, SETTING, METHODS: Localization, frequency and EVT-mediated targeting and invasion of arterial, venous as well as lymphatic vessels were determined in first trimester decidua basalis tissue sections using immunofluorescence staining with antibodies against CD31, CD34, ephrin B2 (EFNB2), ephrin receptor B4 (EPHB4), HLA-G, podoplanin, prospero-related homeobox 1 (Prox-1), alpha-smooth muscle actin 2 (ATCTA2), von willebrand factor (vWF) and proteoglycan 2 (PRG2). Arterial, venous and lymphatic-associated EVTs were further characterized according to their position in the vascular structure and classified as intramural (im) or intraluminal (il). MAIN RESULTS AND THE ROLE OF CHANCE: EVTs, specifically expressing PRG2, target and invade veins and lymphatics in first trimester decidua basalis since HLA-G+ trophoblast were detected in the vascular wall (intramural EVT, imEVTs) and in the lumen of these vessels (intraluminal EVT, ilEVTs). In total, 276 arteries, 793 veins and 113 lymphatics were analysed. While EVTs contact and invade arteries and veins to a similar extent we found that lymphatics are significantly less affected by EVTs (P = 0.001). Moreover, ilEVTs were detected in the lumen of venous and lymphatic vessels, whereas ilEVTs were only found occasionally in the lumen of arteries. Interestingly, RSA tissue sections contained significantly more arterial (P = 0.037), venous (P = 0.002) and lymphatic vessels (P < 0.001), compared to healthy controls. However, while RSA-associated arterial remodeling was unchanged (P = 0.39) the ratios of EVT-affected versus total number of veins (P = 0.039) and lymphatics (P < 0.001) were significantly lower in RSA compared to age-matched healthy decidual sections. Finally, HLA-G+/PRG2+/CD45-EVTs can be detected in regional lymph nodes of pregnant women diagnosed with cervical cancer. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: In this study, first trimester decidual tissues from elective terminations of pregnancies have been examined and used as a reference for healthy pregnancy. However, this collective may also include pregnancies which would have developed placental disorders later in gestation. Due to limitations in tissue availability our staining results for EVT-specific marker expression in regional lymph nodes of pregnant women are based on four cases only. WIDER IMPLICATIONS OF THE FINDINGS: In this study, we propose migration of HLA-G+ cells into regional lymph nodes during pregnancy suggesting that the human EVT is capable of infiltrating maternal tissues via the blood stream. Moreover, the description of compromised EVT invasion into the venous and lymphatic vasculature in RSA may help to better understand the pathological characteristics of idiopathic recurrent pregnancy loss. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Austrian Science Fund (grant P-25187-B13 to J.P. and grant P-28417-B30 to M.K.). There are no competing interests to declare.