Multiomics approach to identify key functional regulatory genes in human endometrium and trophoblast

Abstract

The human endometrium is a dynamically renewing tissue that undergoes cyclic regeneration, which is tightly regulated by ovarian hormones and complex gene networks. Disruptions in this process can lead to serious health conditions such as infertility, endometriosis, endometrial hyperplasia and endometrial cancer. During pregnancy, trophoblast differentiation is a crucial process for successful embryo implantation and placental development. Despite the fundamental importance of these processes, the identification of key functional regulatory cells and genes implicated remains elusive and largely unexplored.

The first part of this thesis aimed to identify potential stem/progenitor stem-cell markers within the endometrium by the organoid system. In contrast to the conventional two-dimensional culture model, organoids can more comprehensively recapitulate the morphological and functional characteristics of the originating tissue. Single-cell analysis identified Intercellular Adhesion Molecule 1 (ICAM1) as a potential stem cell marker in the endometrial organoid. Combined with a known stem cell marker Fucosyltransferase 4 (FUT4), FUT4+ ICAM1+ cells and their corresponding stem cluster were identified in both in vivo and in vitro data as potential stem/progenitor stem-cell of the endometrial epithelia. These cells exhibited enhanced clonogenic ability and were involved in stem-cell related biological processes and pathways. Moreover, these cells demonstrated adaptability by altering their gene expression levels in response to environmental changes, such as hormonal treatments and glandular development.

The second part of the thesis focused on investigating the role of senescence and cell cycle in regulating trophoblast differentiation. Trophoblast stem cells (TSC) served as an in vitro model to mimic villous cytotrophoblasts (VCT) and to study the trophoblast differentiation into extravillous trophoblasts (EVT). Notably, cell cycle and cellular senescence-related genes exhibited distinct expression patterns between VCT/TSC and EVT. Moreover, the differentially expressed genes during the differentiation process from TSC to EVT also participated in the cell cycle and cellular senescence. Using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) screening, the differentiation-related genes were determined, with some of them specifically expressed in the trophoblast. Furthermore, Covalent Ligation of Endogenous Argonaute-Bound RNAs-Crosslinking and Immunoprecipitation (CLEAR-CLIP) revealed the possible miRNA-mRNA pair that contributed to the regulatory function during trophoblast differentiation.

In conclusion, through multiomics analysis and experimental verification, this study identified a potential stem/progenitor cell marker and revealed the association between TSC-to-EVT differentiation and cell cycle and cellular senescence regulation. These findings provide valuable insights into the complex mechanisms governing human endometrium regeneration and trophoblast differentiation to EVT, with potential implications for reproductive health and disease.