SP6调控人扩展潜能干细胞向滋胚层干细胞分化的作用和机制研究

人类滋胚层干细胞（TSC）是从早期胚胎中的全能细胞分化而来，其持续分化产生的滋胚层终端细胞是构成胎盘的主要细胞群。人扩展潜能干细胞（EPSC）具有早期胚胎细胞的全能性特征，可高效分化至TSC，因此是研究TSC特化机制的理想模型。本项目前期通过人转录因子CRISPR高通量筛选发现：SP6在EPSC向TSC分化过程起调控作用。敲除SP6可使EPSC向TSC分化受阻，而过表达SP6则促进这一进程。进一步实验证明，SP6可以结合在KRT7等滋胚层发育相关基因的转录起始调控区。因此我们认为，SP6可以单独或者和其他滋胚层特化相关因子协同，调控滋胚层谱系相关基因的表达和相应细胞谱系的命运决定。为验证这一设想，本项目拟通过“蛋白-DNA”结合高通量测序、细胞转录组学、ATAC-seq等技术手段，结合转录因子功能研究方法，对SP6在TSC分化形成过程中的调控功能和分子作用机制进行揭示。

Human trophoblast stem cells (TSCs) are differentiated from totipotent cells in early human embryos, and terminal descendant trophoblast cells produced by continuous differentiation of TSCs are the main cell populations constituting the human placenta. As human expanded potential stem cells (EPSCs) have the totipotent characteristics of early embryonic cells and can efficiently differentiate into TSCs, they are an ideal model for studying the processes and mechanisms of TSC specialization. We have screened the human transcription factor group with CRISPR/CAS9 and found that SP6 plays an important role in the process of EPSC to TSC differentiation. Knocking-out of SP6 can block the differentiation of EPSC to TSC, while overexpression of SP6 promotes this differentiation process. Further experiment proves that SP6 can bind to the transcription initiation regulatory region of genes related to trophoderm development, such as KRT7. Therefore, we propose that SP6 regulates the expression of genes related to trophoblast development and thus the differentiation of corresponding cell lineages in synergy with other factors. This project intends to use "protein-DNA" analysis, transcriptomics, ATAC-seq and other technical methods, combined with transcription factor function analysis, to reveal molecular mechanisms of SP6 in regulating TSC formation. This study helps to clarify the specialization and differentiation of human TSCs, and provides the theoretical basis for the prevention and treatment of placental-related diseases.