Temporal Transcriptional Regulation of Human Neuronal Differentiation via Forward Programming

Single‐cell profiling of TF‐induced forward programming versus stepwise dual‐SMAD differentiation reveals that divergent trajectories set the pace of neurogenesis. OLIG TFs advance cell‐cycle exit via NOTCH modulation, while NEUROD2 later accelerates maturation. The study elucidates transcriptional mechanisms governing differentiation timing, providing a reference for rationally designing timing‐controlled in vitro differentiation strategies. Abstract Human pluripotent stem cells (hPSCs) serve as a powerful model for studying human neuronal differentiation, yet the temporal control of this process remains poorly understood. This study compares two differentiation systems with distinct timing of differentiation: transcription factor (TF)‐induced forward programming and stepwise cellular differentiation by dual‐SMAD (DS) inhibition. The 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. Perturbation of these GRNs modulates the timing of neurogenesis and neuronal maturation. Specifically, OLIG family TFs, enriched in the TF‐induced system, promoted cell cycle exit via NOTCH signaling regulation; their ablation delays neurogenesis in this system. Additionally, NEUROD2 overexpression after neurogenesis accelerated in vitro neuronal maturation in both TF‐ and DS‐induced differentiating cells by enhanced activation of maturation gene modules. These findings elucidate transcriptional mechanisms governing differentiation timing and provide a framework for rationally designing timing‐controlled in vitro differentiation strategies. Single-cell profiling of TF-induced forward programming versus stepwise dual-SMAD differentiation reveals that divergent trajectories set the pace of neurogenesis. OLIG TFs advance cell-cycle exit via NOTCH modulation, while NEUROD2 later accelerates maturation. The study elucidates transcriptional mechanisms governing differentiation timing, providing a reference for rationally designing timing-controlled in vitro differentiation strategies. Abstract Human pluripotent stem cells (hPSCs) serve as a powerful model for studying human neuronal differentiation, yet the temporal control of this process remains poorly understood. This study compares two differentiation systems with distinct timing of differentiation: transcription factor (TF)-induced forward programming and stepwise cellular differentiation by dual-SMAD (DS) inhibition. The 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. Perturbation of these GRNs modulates the timing of neurogenesis and neuronal maturation. Specifically, OLIG family TFs, enriched in the TF-induced system, promoted cell cycle exit via NOTCH signaling regulation; their ablation delays neurogenesis in this system. Additionally, NEUROD2 overexpression after neurogenesis accelerated in vitro neuronal maturation in both TF- and DS-induced differentiating cells by enhanced activation of maturation gene modules. These findings elucidate transcriptional mechanisms governing differentiation timing and provide a framework for rationally designing timing-controlled in vitro differentiation strategies. Advanced Science, EarlyView.