Transcriptional regulation of temporal dynamics in human neurogenesis and neuronal maturation [CUT&Tag]

Human pluripotent stem cells (hPSCs) provide a valuable platform for studies of human neuronal differentiation, but the mechanisms regulating the timing of this process remain poorly understood. This study compares two differentiation systems with distinct timing of differentiation, transcription factor (TF)-induced forward programming and stepwise cellular differentiation. Our analyses reveal that divergent cellular trajectories drive distinct neurogenesis timing. Multi-omic analysis identifies crucial gene regulatory networks (GRNs) that govern cell fate determination and timing control. Modulation of these GRNs modulates neurogenesis and timing of neuronal maturation. Specifically, OLIG family TFs, enriched in the TF-induced system, promoted cell cycle exit through NOTCH signaling regulation; conversely, ablation of these TFs delayed neurogenesis. In contrast, NEUROD2 overexpression accelerated neuronal maturation through precocious activation of maturation gene modules. Collectively, these findings illuminate characteristics of the cell-intrinsic mechanisms that govern timing of differentiation; the studies also offer a framework for investigation into, and the rational design of, timing-controlled in vitro differentiation strategies. Overall design: We performed scRNA-seq on cells over the time course of TF-induced forward programming (hPSCs female and male cell lines, with mRNA or dox inducbile NEUROG1, NEUROG2, NEUROD1 overexpression induced neurogenesis) and stepwise cellular differentiation (dual-SMAD inhibition).Taken together, these data indicate that TF-induced forward programming represents a distinct differentiation shortcut from hPSCs to neuronal fate. In order to further decipher the regulatory landscape underlying the drastic cell fate changes, several experiments were performed including high-resolution time-course RNA expression profiling, chromatin accessibility analysis, and TF binding profiling by CUT-TAG (NEUROG1, NEUROG2, and NEUROD1). We next aimed to infer GRNs that may play a crucial role in facilitating rapid neurogenesis (OLIG1, OLIG2, OLIG3) and subsequent neuronal maturation (NEUROD2, MSC). To gain mechanistic insights, scRNA seq of cells induced from OLIG KO cell line, TF binding profiling by ChIP (OLIG2) and CUT-TAG (NEUROD2, MSC) were performed.