Deciphering gene regulatory networks modulating human trophoblast stem cell self-renewal and differentiation

Although critical for development, the placenta is one of the least understood organs in the body. Cells belonging to the trophoblast lineage mediate proper implantation and placentation as well as the hematopoietic, vascular, and immunological properties of the placenta. Defects in proper trophoblast differentiation cause early pregnancy failure and other pregnancy-related disorders, but the molecular mechanisms of human trophoblast differentiation remain poorly understood. So far, only a few transcription factors (TFs) are known to play important roles in trophoblast lineage specification, and their functions are primarily characterized in mice, not human. Furthermore, how these TFs form global gene regulatory networks with other regulators, or their target cis-regulatory elements is not well understood. The objective of the proposed research is to delineate transcriptional regulatory networks and global regulatory logics modulating trophoblast lineage differentiation by utilizing human trophoblast stem (TS) cells and their differentiation towards syncytiotrophoblast (ST) and extravillous cytotrophoblast (EVT) as model systems via systems and molecular biology approaches. We hypothesize that mapping trophoblast cell-specific enhancers will allow us to define novel key TFs that control the self-renewal and differentiation of human trophoblast lineages. Our preliminary studies in both mouse and human TS cells revealed that most previously known trophoblast lineage markers are located close to enhancer clusters (ECs) that we have mapped in each cell type, supporting our hypothesis. Our objectives are to 1) comprehensively define human TS cell, ST, and EVT- specific enhancers and ECs, and subsequently identify EC-associated putative key regulatory TFs, 2) functionally validate putative key TFs in self-renewal and differentiation of TS cells to ST and EVT in vitro and in vivo, and 3) reconstruct the core transcriptional regulatory networks modulating human TS cells, ST, and EVT by mapping both native protein interacting partners and chromosomal targets of key TFs. Our proposed studies will provide critical new data in this field, enable a systems-level understanding of early trophoblast differentiation, and create an important resource to gain further insights into the molecular regulatory mechanisms of how extra-embryonic cells are specified, maintained, and lineage-restricted during human development. Our results will help guide future biomedical advances for detecting and treating pregnancy- related disorders.