HypoSUMOylation in embryonic stem cells generate head-trunk embryo-like structures [ChIP-seq]. HypoSUMOylation in embryonic stem cells generate head-trunk embryo-like structures [ChIP-seq]

Numerous models of synthetic embryos have recently been established to simulate mammalian development. Two main strategies have been developed to build mouse or human embryo-like structures (ELS): by assembling embryonic and extraembryonic stem cells or by challenging embryonic stem cells (ESCs) with a pulse of WNT agonist. However, both models did not fully recapitulate early organogenesis, particularly the emergence of brain derivatives. SUMOylation was recently identified as a general barrier to cell fate transitions. Here, we show that mouse ESCs exposed to a small-molecule inhibitor of SUMOylation alone generate adherent spheroids which, once in suspension, self-organize in gastrulating structures containing cell types spatially and functionally-related to embryonic and extraembryonic compartments. Alternatively, spheroids cultured in an optimized droplet-microfluidic device form elongated ELS characterized by a multi-axial organization of the body plan reminiscent of natural embryo morphogenesis. Single-cell transcriptomics further revealed various cell types including anterior neuronal cell types, Schwann cell precursors and somites. Mechanistically, transient SUMOylation repression gradually increases the global level of DNA methylation, which in turn represses transcription of Nanog and other pluripotency-associated genes, enhancing cellular plasticity of ESCs. Our morphogen-free protocol constitutes a new facet to study regulative mechanisms of early development by targeting reprogramming roadblocks to shape multicellular architecture. Overall design: Examination of SUMO-2_3, Kap1 and Zfp57 landscapes in control ES cells (D1) and in ES cells after SUMO recovery (D8), in duplicate, using Illumina Illumina Hiseq 4000.

近年来，诸多合成胚胎模型已被构建，用以模拟哺乳动物的发育进程。目前已开发出两种主要策略，用于构建小鼠或人类类胚胎结构（Embryo-like Structures, ELS）：一是通过胚胎干细胞与胚外干细胞的组装，二是通过脉冲式WNT激动剂处理胚胎干细胞（Embryonic Stem Cells, ESCs）。然而，这两种模型均无法完全复现早期器官发生过程，尤其是脑源性细胞的产生。
SUMO化修饰（SUMOylation）近期被证实为细胞命运转变的普遍障碍。本研究证实，仅经小分子SUMO化修饰抑制剂处理的小鼠胚胎干细胞，可形成贴附样球体；将该球体置于悬浮培养环境后，其会自组织形成原肠胚结构，其中包含与胚胎及胚外区域在空间和功能上相关的细胞类群。此外，在优化后的液滴微流控装置中培养的球体，可形成拉长的类胚胎结构，其体轴呈现多轴排布特征，与天然胚胎的形态发生过程相似。
单细胞转录组测序进一步揭示了多种细胞类群，包括前脑神经细胞类群、施万细胞前体及体节。从机制上来看，瞬时抑制SUMO化修饰会逐步提升全基因组DNA甲基化水平，进而抑制Nanog及其他多能性相关基因的转录，增强胚胎干细胞的细胞可塑性。本研究建立的无形态发生因子方案，为通过靶向重编程障碍以构建多细胞结构，进而探究早期发育的调控机制提供了新的研究视角。
实验整体设计：采用Illumina Hiseq 4000测序平台，对对照组胚胎干细胞（第1天，D1）以及经SUMO化恢复后的胚胎干细胞（第8天，D8）中的SUMO-2_3、Kap1及Zfp57结合图谱进行重复检测，每组设置2个生物学重复。