Trans-omic Profiling Uncovers Molecular Controls of the Early Human Cerebral Organoid Formation

Defining molecular controls that orchestrate human brain development is essential for uncovering the complexity behind neurodevelopment and the pathogenesis of neurological disorders. Due to the difficulties in accessing embryonic and fetal brain tissues, the differentiation of human pluripotent stem cell (hPSC)-derived three-dimensional neural organoids has made it possible to recapitulate this developmental process in vitro and provide a unique opportunity to investigate human brain development and disease. To elucidate the molecular programs that drive this highly dynamic process, here, we generate a comprehensive trans-omic map of the phosphoproteome, proteome, and transcriptome of the initial stages of pluripotency and neural differentiation towards the formation of cerebral organoids. Our integrative analysis uncovers key phospho-signalling events underlying neural lineage differentiation, and their convergence on transcriptional (co-)factors and chromatin remodellers that govern downstream gene regulatory networks (GRNs). Comparative analysis with developing human and mouse embryos using these GRNs demonstrates the fidelity of our early cerebral organoids in modelling embryonic brain development. Finally, we demonstrate biochemical modulation of the AKT signalling as a key molecular switch for controlling human cerebral organoid formation. Our data provides a comprehensive resource to gain insight into the molecular controls in human embryonic brain development and also provide a guide for future development of protocols for human cerebral organoid differentiation. Overall design: To characterize early human organoid differentiation, we performed bulk and single-cell transcriptomic profiling of differentiating H9 hESC aggregates to the formation of the early neural organoid over a period of 8 days. Timepoints include 0h, 1h, 6h, day 1, day 2, day 4, day 6 and day 8 with 2 replicates per timepoint for bulk RNA-sequencing. Single-cell sequencing was performed on day 8 with one replicate.