2. Academic Validation
  3. Wnt activation and dual SMAD inhibition for induction and maintenance of hindbrain-like neural stem cell from hiPSCs

Wnt activation and dual SMAD inhibition for induction and maintenance of hindbrain-like neural stem cell from hiPSCs

  • Cell Rep Methods. 2026 Apr 20;6(4):101372. doi: 10.1016/j.crmeth.2026.101372.
Ziadoon Al-Akashi 1,  Denise Zujur 1,  Nicholas Boyd-Gibbins 1,  Nathalie Eileen Wiguna 1,  Masato Nakagawa 2,  Tetsuhiro Kikuchi 1,  Asuka Morizane 3,  Jun Takahashi 1,  Makoto Ikeya 4
Affiliations

Affiliations

  • 1 Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
  • 2 Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
  • 3 Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan; Department of Regenerative Medicine, Center for Clinical Research and Innovation, Kobe City Medical Center General Hospital, Hyogo, Japan.
  • 4 Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Electronic address: [email protected].
PMID: 41916305 DOI: 10.1016/j.crmeth.2026.101372
Abstract

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-free and basic Fibroblast Growth Factor (bFGF)-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 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.

Keywords

CP: neuroscience; CP: stem cell; hindbrain; neural stem cell; neuron; pluripotent stem cell.

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