TTNPB Promotes Pluripotent-to-Neural Stem Cell Transition through Modulating Chromatin Accessibility and Choline Metabolism [ATAC-seq]

Efficient derivation of neural stem cells (NSCs) from human pluripotent stem cells (PSCs) is crucial for developmental biology and regenerative medicine. Here, we established a chemically defined system using CHIR99021 and TTNPB to generate advanced NSCs (ANSCs). Integrated chromatin accessibility, transcriptomic, and metabolomic analyses revealed molecular mechanisms underlying neural fate commitment. TTNPB synergistically enhanced CHIR99021-mediated neural induction by globally increasing chromatin accessibility and activating neural-specific transcription factors. Compared to NSCs and PSCs, ANSCs exhibited robust upregulation of neural markers and enhanced chromatin remodeling. Metabolomic profiling identified significant metabolic reprogramming during the PSC-to-ANSC transition, with specific enrichment of S-adenosylhomocysteine (SAH), ADP, and glutathione in ANSCs correlating with elevated neural gene expression. Functionally, choline supplementation—regulated via the PEMT pathway—enhanced NESTIN expression and promoted neuroectodermal fate commitment. Our findings establish CHIR99021/TTNPB as a potent synergistic pair for efficient NSC induction and identify choline metabolism as a critical axis governing neuroectodermal lineage specification. Overall design: ATAC-seq of human pluripotent stem cells (W24 and H9) and their derivatives NSC and ANSC. The pluripotent stem cells were collected from passage 30. The NSC and ANSC both were collected from passage 20.