Deciphering signalling and transcriptional networks in human uterine organoids

Many uterine disorders including infertility, endometriosis and endometrial cancer have their origins in a dysfunctional endometrium - the inner layer lining the uterus. The endometrium is highly dynamic as it undergoes a hormone-induced monthly cycle of growth, differentiation and degeneration. These remarkable regenerative abilities are attributed to the existence of stem cells driven by WNT signalling in concert with the sex hormones estrogen and progesterone. However, due to the lack of reliable experimental systems, the molecular mechanisms behind these processes remain poorly understood. Recently, we and others have established uterine organoids as a novel in vitro model of human endometrial epithelium. Uterine organoids are 3D structures that once derived from human endometrium can be kept long-term in culture and subjected to chemical and genetic manipulations. Most importantly, they faithfully recapitulate the architecture, expression patterns, hormonal responsiveness and secretory abilities of the in vivo uterine epithelium. We propose to use this model to determine how the WNT/Estrogen/Progesterone signalling pathways control expression of specific genes and thereby coordinate the monthly cycle on the molecular level. In particular, we aim to use a comprehensive molecular approach integrating chemically defined signalling, gene expression analysis and functional tests 1) to dissect WNT signalling requirements during renewal and differentiation of the endometrium with the special focus on the identification and validation of transcription factors directly regulated by this pathway and 2) to interrogate the crosstalk between WNT, Estrogen and Progesterone signalling in the context of transcriptional regulation of their target genes. The expected results will illuminate the fine balance between endometrial self-renewal and differentiation and provide new insights into mechanisms that underlie endometrial disorders including endometriosis and endometrial cancer.

Despite advances in reproductive medicine, pregnancy-related disorders associated with impaired placental and uterine development and function, still pose a large risk to the mother and embryo. Molecular studies of the underlying causes have been hampered by the lack of reliable models. This funding enabled us to establish trophoblast and endometrial organoids as novel in vitro models of the human placenta and the inner uterine lining, respectively. The endometrium is the highly dynamic inner uterine layer undergoing a hormone-induced monthly cycle of growth, differentiation and degeneration. We derived endometrial organoids and showed that they preserve the structure, expression patterns, secretory properties, and Estrogen responsiveness of their tissue of origin. Next, we used them to illuminate how different signaling pathways such as Estrogen and Notch control secretory vs. ciliated cellular identities and thus determine cellular make-up of the endometrium during menstrual cycle. The placenta is a vital organ to sustain mammalian development in utero. As a by-product of endometrial organoid derivation, together with Prof. Knöfler laboratory, we established 3D human trophoblast organoid cultures from purified first-trimester cytotrophoblast. Detailed molecular analysis revealed that while the proliferative outer layer represents the cytotrophoblast, the inner core consisted of multinucleated syncytiotrophoblast that resulted from cell fusion. Thus, the trophoblast organoids preserved the architecture of human placental villi in the inside-out orientation. In addition, trophoblast organoids can be induced to form the highly migratory and invasive extravillous trophoblast, that during development invades maternal decidua and remodels spiral arteries. Thus, we established trophoblast organoids that recapitulate the three main trophoblast lineages in vitro and provided the first 3D model of the human placenta. The derivation of trophoblast organoids was a major breakthrough, offering a novel and reliable tool to study molecular mechanisms driving placental development and disease. This innovative in vitro model has been broadly used and facilitated major findings and advancements in the placental field as evidenced by recent publications. Similarly, the endometrial organoids are an excellent system to study human endometrium and to expand our understanding of the hormonally regulated endometrial cycle in health and disease, with a special focus on endometriosis and endometrial cancer.