调控转录因子GATA2/GATA3诱导胎盘滋养层细胞及类器官的研究

孕期胎盘的发育完整对于整个妊娠过程非常重要，胎盘发育异常可引起很多疾病。小鼠的研究中，转录因子Gata2/3对于胎盘的发育至关重要，由于物种差异巨大，GATA2/3在人类胎盘发育中的调控机制并不明确。申请人预实验发现，敲除扩展潜能干细胞（hEPSCs）中的GATA2/3，严重影响hEPSCs向滋养层细胞分化。因此，我们提出假说：GATA2/3通过调控滋养层细胞特异基因表达而影响扩展潜能干细胞向滋养层细胞分化，同时影响滋养层干细胞（hTSCs）在体外形成胎盘类器官。为了验证假说，课题组拟1）构建GATA2/3双敲EPSC细胞系，和Dox诱导敲除hTSCs细胞系；2）研究双敲细胞向滋养层谱系分化的影响；3）诱导敲除GATA2/3在胎盘类器官形成中的影响。通过EPSCs向滋养层细胞的命运转变的机制研究和三维类器官的形成研究，本项目将进一步明确转录因子GATA2/GATA3的作用。

A successful pregnancy is dependent on a healthy placenta, which in turn requires the adequate functioning of all trophoblasts subtypes in unison. Pregnancy-associated complications typically stem from anomalies in trophoblast development, which is typically studied in human trophoblast stem cells (hTSCs). Nevertheless, utilizing hTSCs as a model for studying early placental development has been hampered by the limited genetic diversity of existing hTSC lines, and ethical constraints on utilizing human fetal tissue or embryos. Meanwhile, mechanistic insights into early trophoblast development has been much more established in animal models. Previous studies on mouse embryos have revealed that Gata2/Gata3 directly regulate many common downstream genes to orchestrate stem versus differentiated trophoblast fate..Notwithstanding the progress in animal studies, the role of GATA2/3 in human trophoblast lineage differentiation and placental development has remained elusive. Our preliminary results have identified that double knock-out of GATA2/3 in human expanded potential stem cells (hEPSCs) impedes its differentiation along the trophobalst lineage. We thus hypothesized that GATA2/3 may directly regulate trophoblast-specific genes and govern the formation of trophoblast organoids in vitro. To this end, we have generated GATA2/3 double knock-out hEPSCs and Doxorubicin-inducible GATA2/3 knock-out hTSCs. We will differentiate these cells into various trophoblast lineage subtypes and organoids to identity the role of GATA2/3 on trophoblast fate entry and transition as well as the mechanisms of trophoblast cytoarchitecture formation and maturation. Our study will provide novel platforms for and shed important mechanistic insights into elucidating early human placental development in unprecedented resolutions.