HypoSUMOylation in embryonic stem cells generate head-trunk embryo-like structures [scRNAseq]

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. Transcriptomic profiling by MARS-seq (scRNA-seq) of cells at D1, D3, D8 D10, D18, cells from aggreWell gastruloids, µfluidic device-derived gastruloids and cells from embryo-like structures. Control ES cells (D1) were treated with ML-792 for 48h (D3) in serum+Lif medium. Drug was washed and cells were allowed to recover for 5 days (D8), before undergoing another ML-792 treatment for 48h (D10) in serum+Lif medium. Drug was washed and medium was changed to N2B27+Lif for 8 days (D18). ESC_N2B27Lif is a control condition that corresponds to untreated ES cells that underwent the medium switch. D18 cells were transferred to AggreWell plates and cultured for 3 days in N2B27+Lif (Gastruloid_AgW). ESC_AgW is a control condition that corresponds to untreated ES cells that underwent the medium switch and the plate transfer. D18 cells were transferred to a droplet-microfluidic device and cultured for 2 days (Gastruloid_F2) or 5 days (Gastruloid_F5) in N2B27+Lif. D18 cells were transferred to a droplet-microfluidic device and cultured for 4 days then extracted and embedded in 20% Matrigel for 3 additional days of culture in N2B27+Lif (ELS_M7).