Symmetry breaking and germ layer emergence through optogenetic Wnt activation in gastruloids

Human gastrulation is a dynamic, complex, and poorly understood process. Starting from a seemingly homogenous population, cell and tissue organization during this process establishes the 3D organismal body plan. However, due to technical and ethical limitations, mechanisms of human gastrulation are largely unexplored. For example, it is generally unclear whether a spatially ordered, external cue is necessary for patterning, or whether the epiblast harbors the innate ability to differentiate and self-organize in the absence of asymmetric cues. Human pluripotent stem cells (hPSCs) fortunately offer the potential to model gastrulation, most notably through generation of 3D “gastruloids”. Here we use optogenetic control of Wnt signaling to establish a 3D hPSC gastruloid model and demonstrate the spatially-ordered emergence of the three germ layer lineages in the absence of exogenous asymmetrical cues. Overall design: scRNA-seq was performed on the 10X Genomics Chromium platform using the V3.1 3' chemistry, sequencing was done on a Novaseq 6000 paired end reads with depth aimed for 50k reads per cell.

人类原肠胚形成是一个动态、复杂且尚缺乏充分认知的过程。该过程起始于看似均一的细胞群体，其间的细胞与组织构建确立了三维生物体的躯体蓝图。然而受限于技术与伦理层面的约束，人类原肠胚形成的相关机制迄今尚未得到充分探索。例如，学界目前仍普遍不清楚：空间有序的外部信号是否为模式构建所必需，抑或是上胚层（epiblast）在缺乏不对称信号的条件下，是否具备自发分化与自组织的固有能力。幸运的是，人类多能干细胞（human pluripotent stem cells, hPSCs）为原肠胚形成的建模提供了可能，其中最具代表性的方式便是构建三维‘类原肠胚（gastruloids）’。本研究通过光遗传调控Wnt信号通路，构建了三维人类多能干细胞类原肠胚模型，并证实了在缺乏外源性不对称信号的条件下，三胚层谱系可实现空间有序的出现。实验整体设计：本研究依托10X Genomics Chromium平台，采用V3.1版3'端化学试剂盒开展单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）；测序在NovaSeq 6000测序仪上完成，采用双端读段模式，设定每细胞目标测序深度为5万条读段。