Induction and long-term maintenance of hindbrain-like neural stem cells in xeno- and basic fibroblast growth factor-free conditions

Neurons exhibit region-specific identities corresponding to functional distinctions across different brain areas. Region-restricted neural stem cells (NSCs) have previously been generated from pluripotent stem cells; however, maintaining their regional identity over extended passages remains challenging. Here, we report the generation of hindbrain-like induced NSCs (Hb-LiNSCs) with upregulated hindbrain-specific markers and downregulated forebrain, midbrain, and spinal cord markers under xeno- and basic fibroblast growth factor-free conditions using three chemicals—CHIR99021 (at a high concentration), a potent activator of the Wnt pathway, A-83-01, a potent inhibitor of the TGF-β/Activin/Nodal pathway, and LDN193189, a potent inhibitor of the bone morphogenetic protein pathway. Hb-LiNSCs maintained their chromosomal integrity, multipotency, and differentiation capacity even after long-term culture for more than 60 weeks. This innovative approach enhances our understanding of neurodevelopmental and neurodegenerative processes in the hindbrain region and paves the way for developing targeted cell-based therapy as well as disease modeling for drug discovery.