Development of placental trophoblast progenitors

Development of the human placenta and its specific epithelial cells, the trophoblasts, is critical for a successful pregnancy and fetal wellbeing. During placentation mononuclear cytotrophoblasts (CTBs) undergo two distinct differentiation pathways, by either fusing into multinuclear syncytiotrophoblasts or by forming invasive extravillous trophoblasts (EVTs). The latter migrate into the spiral arteries of the maternal uterus and provoke remodelling of these vessels thereby adapting blood flow and transport of nutrients and oxygen to the developing foetus. Abnormal changes in EVT differentiation, invasion and remodelling have been noticed in severe pregnancy disorders, such as early-onset preeclampsia or intrauterine growth restriction. However, the underlying pathological mechanisms have not been elucidated, since availability of first trimester placental tissues is limited and self-renewing trophoblast models have not been established. Recently, we identified the receptor Notch1 as a critical regulator of EVT development. Notch1 converts trophoblast precursors into EVT progenitors and maintains their proliferation and survival. We hypothesize that the particular receptor plays a pivotal role in EVT progenitor formation by inducing cell-specific transcriptional activators. However, the full set of Notch1 downstream effectors and upstream regulators has not been unravelled. Herein, we aim to further elucidate the role of Notch1 in EVT formation by analysing genome-wide Notch1 DNA-binding sequences and target genes. Novel high throughput techniques such as RNA sequencing of expressed mRNAs and nascent transcripts as well as chromatin immunoprecipitation-DNA sequencing will be utilised. Also, EVT progenitors will be isolated from early placental tissues using laser-captured microdissection and compared to primary cell preparations. Moreover, we recently established self-expanding human trophoblast organoids growing in 3D under defined culture conditions. Proliferative CTBs in this systems express markers of stemness and differentiate into EVT progenitors and HLA-G-expressing EVTs in the correct spatial orientation upon changes of the growth medium. We aim identifying upstream regulators of Notch1 in this system using the above mentioned RNA sequencing techniques. Expression pattern and function of selected Notch1-dependent transcriptional regulators as well as potential Notch1 inducers will be studied in primary CTBs and organoids. The present project will identify novel markers of EVT progenitors and give new insights into the developmental program of the EVT lineage. Further, characterisation of the CTB organoid model represents the prerequisite for reliable in vitro studies of human placental development and the establishment of self-renewing cultures from abnormal placentae of pregnancy disorders.

Correct development of the human placenta is crucial for wellbeing of the developing baby and the expectant mother. Failures in growth and differentiation of its most important cell type, the epithelial trophoblast, has been identified as an underlying cause of gestational diseases. However, our knowledge about early developmental processes of the placenta during normal gestation and in different pregnancy disorders is still scarce. Herein we focused on key regulatory signalling pathways that control stem cell niches, cell expansion and differentiation in different organs of the body, i.e. HIPPO, NOTCH and TGF- signalling, in the context of human placenta and trophoblast development. The idea of this project was to unravel the mechanistic effects of these pathways under physiological conditions to set the basis for further studies in tissues affected by pregnancy disorders such as preeclampsia, a high blood pressure disease of the mother or fetal growth restriction. The latter may program the fetus for the development of different diseases as an adult such as cardiovascular defects or metabolic syndrome. Using state-of-the-art cellular models such as self-renewing trophoblast organoids, that we developed some years ago, and human trophoblast stem cells, we could show that the nuclear protein, Yes-associated protein (YAP), a crucial transcriptional co-activator of HIPPO signalling, has a key role supporting the growth of trophoblast progenitors. While YAP activates the expression of genes promoting proliferation and stemness of the placental epithelium, it also represses genes that foster differentiation into the syncytiotrophoblasts, one of the differentiated cell types of the human placenta producing pregnancy hormones such as human chorionic gonadotrophin. The latter is well known to the public for its application in commercially available pregnancy tests. Similar to the HIPPO co-activator YAP, NOTCH3, one of the receptors of NOTCH signalling, also has a pivotal role in the expansion of the early human placenta by promoting growth of placental trophoblast, but inhibiting their differentiation. In addition, data of this project demonstrate that TGF- signalling is an important pathway to mature the other differentiated cell type of the human placenta, namely extravillous trophoblasts. These cells detach from the placenta and invade the maternal uterus during pregnancy thereby remodelling the uterine spiral arteries of the mother. This process is required to ensure adapted blood flow to the placenta and hence adequate nourishment and oxygen delivery to the developing baby. Failures in this processes were detected in preeclampsia and fetal growth restriction. TGF- accounts for many aspects of invading extravillous trophoblasts such as secretion of specific enzymes that control uterine communication and physiological adaption of the expectant mother. In summary, we delineated key signalling pathways of human placental development providing the basis for future investigations in different pregnancy disorders.