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

虽然胎盘对发育至关重要，但它是人体中最不被了解的器官之一。属于滋养层细胞系的细胞调节着床和胎盘的形成，以及胎盘的造血、血管和免疫学特性。正常滋养细胞分化缺陷会导致早期妊娠失败和其他妊娠相关疾病，但人类滋养细胞分化的分子机制仍然知之甚少。到目前为止，只有几个转录因子(TF)在滋养层细胞系中发挥重要作用，而且它们的功能主要是在小鼠身上，而不是在人类身上。此外，这些转录因子如何与其他调节者形成全球基因调控网络，或者它们的目标顺式调控元件还没有被很好地理解。本研究的目的是通过系统和分子生物学方法，以人滋养层干细胞(TS)及其向合体滋养细胞(ST)和绒毛外细胞滋养细胞(EVT)的分化为模型系统，描述调控滋养层细胞系分化的转录调控网络和全球调控逻辑。我们假设，绘制滋养层细胞特异性增强子的图谱将使我们能够定义控制人类滋养层细胞系自我更新和分化的新的关键因子。我们在小鼠和人类TS细胞上的初步研究显示，大多数先前已知的滋养层谱系标记位于我们在每种细胞类型中定位的增强子簇(ECs)附近，支持我们的假设。我们的目标是1)全面定义人类TS细胞、ST和EVT特异的增强子和ECs，并随后确定EC相关的假定关键调控因子，2)在体内外从功能上验证TS细胞自我更新和向ST和EVT分化的关键TF，以及3)通过定位天然蛋白质相互作用伙伴和关键TF的染色体靶标来重建调控人TS细胞、ST和EVT的核心转录调控网络。我们提出的研究将在这一领域提供关键的新数据，使我们能够在系统水平上了解早期滋养细胞的分化，并创造一个重要的资源，以进一步深入了解胚胎外细胞在人类发育过程中如何指定、维持和谱系限制的分子调控机制。我们的结果将有助于指导未来在检测和治疗妊娠相关疾病方面的生物医学进展。

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.