TTNPB Promotes Human Pluripotent Stem Cell‐to‐Neural Stem Cell Transition via Modulation of Chromatin Accessibility and the S‐(5′‐adenosyl)‐L‐homocysteine/Choline Metabolic Network

ABSTRACT Efficient derivation of neural stem cells (NSCs) from human pluripotent stem cells (PSCs) is crucial in regenerative medicine. Here, we report that the combined application of the retinoic acid receptor agonist TTNPB and the GSK3β inhibitor CHIR99021 in a chemically defined medium enabled the induction of a highly advanced NSCs (ANSCs) population from PSCs. ANSCs display robust neuroectodermal gene expression and a heightened capacity for neural lineage commitment. The combination of TTNPB and CHIR99021 markedly enhanced global chromatin accessibility, particularly at neuroectoderm‐specific regulatory elements such as PAX6 and SOX1 , in parallel with reduced accessibility at the loci of pluripotency factors. Notably, TTNPB alone also exerts a marked effect in enhancing chromatin accessibility. Untargeted metabolomic analysis identified a distinct neural‐ectoderm associated metabolic state in ANSCs, prominently characterized by elevated choline, alongside S‐(5′‐adenosyl)‐L‐homocysteine, adenosine 5′‐diphosphate, and glutathione. Exogenous addition of these metabolites was sufficient to induce neuroectodermal marker expression, highlighting the instructive role of the metabolic network in neural fate induction. Moreover, functional studies showed that ANSCs enabled engraftment into depressed rat hippocampi and restored depression‐like behavioral deficits. Our study presents a novel small‐molecule strategy that leverages TTNPB‐centered epigenetic remodeling and metabolic reprogramming as dual mechanisms driving neural differentiation.