高内涵筛选揭示BMP4对小鼠iPSC合成胚胎形成的促进作用及其机制研究

Understanding the key events and mechanisms of mammalian early embryonic patterning is a fundamental problem in Developmental Biology, providing guidance for the treatment of early developmental failure caused diseases such as infertility and congenital malformation. Yet the early embryo development during peri-implantation stage has remained mysterious because of the experimental hurdles that beset the study of this phase. Synthetic embryos formed by cultured embryonic and extraembryonic lineage stem cells emerge as a novel and convenient system to study mammalian early postimplantation embryo development in vitro. However, embryonic stem cell (ESC) based synthetic embryo has a limited role in personalized disease modeling. The mechanism of cell sorting and morphogenesis during synthetic embryo assembly remains elusive. Whether different cell lines have distinct in vitro developmental potential and the detailed transcriptome/epigenome profiling of synthetic embryo are still desirably needed. Here, we assembled the mouse gastrula embryo-like structure using induced pluripotent stem cells (iPSCs). We showed that compared to mouse ESCs, when co-cultured with extra-embryonic trophoblast stem cells (TSCs) and extraembryonic endoderm stem cells (XEN cells), iPSCs have reduced ability to form correct gastrula embryo-like structures, namely ITS and ITX embryos. Despite that, some ITS and ITX embryos underwent lumenogenesis, developed basement membrane, and initiated anterior-posterior axis as natural postimplantation embryos. We performed a high-content screen of small molecules and cytokines and identified that BMP4 best promoted the correct morphogenesis of ITS embryos. Further effort will be using RNA-Seq, ChIP-Seq and ATAC-Seq to identify the novel mechanism and signaling pathways regulating BMP4 triggered synthetic embryo formation, as well as the systematical comparison between the natural embryo and synthetic embryo. Meanwhile, biochemical experiments will be conducted to verify the mechanism revealed by bioinformatics study. Our study established a new strategy to increase the efficiency to generate synthetic postimplantation embryos and uncovered multifaceted roles of BMP4 during the formation of gastrula like embryos. Compared to in utero development, the in vitro assembly platform is more amenable to experimental manipulation and analysis to assess, and may be used to personalized new drug development, gene therapy, and regenerative medicine. Our study further pushes the profound understanding of the field of synthetic embryo, providing a unique and stable platform for basic and clinical research.

哺乳动物围着床期胚胎发育相关研究对揭示早期胚胎发育过程中的重要事件、指导早期发育缺陷及妊娠失败等疾病的治疗有重要的意义。近年，胚胎体外培养及合成胚胎为围着床期胚胎发育相关研究提供了新的模型。现有基于胚胎干细胞的合成胚胎在个性化疾病模型模拟方面存在劣势，不同多能干细胞系的体外发育潜能区别仍缺少研究，此外，现有合成胚胎体外培养成功率较低，自组装及形态发生所涉及的调控因素未知，合成胚胎的表观修饰与真实胚胎间的相似度亦待比较。因此，我们首次将小鼠诱导多能干细胞与胚外干细胞系3D共培养获得了模拟小鼠围着床期的合成胚胎。利用高内涵筛选系统，我们优化了培养条件并初步发现BMP4对合成胚胎发生具促进作用。我们将利用多组学及生化实验结合的手段，揭示BMP4促进合成胚胎发生的作用机制并比较其与真实胚胎的相似度，以更加全面深刻的理解合成胚胎模型，推其发展并使之高效稳定的应用于基础及临床相关研究。

研究哺乳动物的胚胎发育过程对理解发育生物学过程中的重要事件、指导不孕不育等相关疾病的治疗有着重要意义，而由于材料及伦理限制，人类胚胎早期发育过程中的具体事件及调控机制尚未被揭示。通过胚胎及胚外干细胞体外共培养建立的类胚胎模型是模拟植入后早期发育的新平台，而类胚胎发生的调控通路尚不清晰，效率尚低。本研究发现，小鼠诱导多能干细胞 (iPSC) 与胚外干细胞共培养可以形成 ITS（iPSC 和滋养外胚层干细胞）和 ITX（iPSC、滋养外胚层干细胞和 XEN 细胞）胚胎，类似于体内发育的早期原肠胚。为了促进基于干细胞的胚胎模型的高效、公正的分析，我们建立了基于机器学习的分析方法，对类胚胎进行自动化、高通量的多维特征提取，并使用从高内涵筛选系统收集的 3D 图像对 ITS 胚胎进行量化。利用此机器学习的高内涵筛选系统，我们发现不同的多能干细胞系形成胚胎样结构的能力不同，进一步地，通过对小分子和细胞因子的高内涵筛选，我们发现 BMP4 最能促进 ITS 胚胎的形态发生，转录组和表观组测序的结果表明，BMP4通过调控原肠运动及极化相关基因表达促进类胚胎发生，涉及MAPK及TGFb信号通路。综上，我们的研究建立了基于机器学习的高通量创新策略来分析类胚胎模型，并揭示了 BMP4 在类胚胎发生过程中的新作用。