Self-organization of pluripotent stem cells into reproducible early embryonic developmental patterns through epigenome editing

During embryonic development, different embryonic cell types co-develop together where each cell type expresses specific fate-inducing transcription factors through activation of developmental non-coding regulatory elements and interactions with neighboring cells. Here, we hypothesized that cell-intrinsic activation of a minimum number of such endogenous regulatory elements is sufficient to self-organize PSCs into early embryonic models. Our results show that CRISPR activation of only two regulatory elements in the genome of pluripotent stem cells is sufficient to generate embryonic patterns that show spatial and molecular resemblance to that of pre-gastrulation mouse embryonic development. Quantitative single-cell live fluorescent imaging showed that emergence of the spatially-ordered embryonic pattern happens through intrinsic induction of cell fate that leads to an orchestrated collective cellular motion. Based on these results, we propose a straightforward approach to efficiently form 3D embryo models through intrinsic CRISPRa-based epigenome editing and independent of external signaling cues. CRISPRa-induced embryo models show highly consistent composition of major embryonic cell types that are spatially organized, with nearly 80% of the structures forming an embryonic cavity. Single cell transcriptomics confirmed the presence of main embryonic cell types in CRISRPa-induced embryo models with transcriptional similarity to pre-gastrulation mouse embryos and revealed novel signaling communication links between different embryonic cell types. Our findings demonstrate that self-organization of embryonic patterns through minimum intrinsic epigenome editing presents a promising approach for efficient production of stem cell-based embryo models with high consistency and will pave the way for future straightforward systematic CRISPR-based screening of embryo models Single-cell RNA-seq analysis was performed on 3D organoids grown using the protocol displayed below. We compared the generation of organoids either assembled from individual cell lines inducing either a control, Cdx2 or Gata6 sgRNA, or when assembling the 3 cell lines together, to assess self-organizing properties of embryonic cell types