Stem cell research & therapy · 2026
Dual roles of GDNF in enteric glial cell plasticity: direct transdifferentiation via the CaMKII/NeuroD1 pathway and cooperative regulation in a neural stem cell-inducing medium.
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Jia Wanying, Yan Hanlei, Huang Jingjing, Liu Wei, Fu Zihao, Tian Donghao, Xu Wenyao, Chen Xinlin, Gao Ya, Yu Hui
Аннотация
BACKGROUND: Hirschsprung disease (HSCR) is a congenital condition featuring aganglionosis in the distal colon, causing functional obstruction. While EGF and bFGF are well-characterized neurogenic factors, the precise mechanistic role of GDNF in modulating enteric glial cell plasticity remains incompletely understood.
METHODS: EGCs were identified via proteomic profiling and immunofluorescence in Ednrb⁻/⁻ mice modeling HSCR. EGC/PK060399egfr and primary EGCs were induced with neural stem cell-inducing medium (NSC-Med). Morphological changes, EdU assay, immunofluorescence, RT‒qPCR, and Western blotting were employed to assess the expression of stemness- and neuron-associated markers. Metabolomic and transcriptomic analyses were performed to evaluate metabolic remodeling and signaling pathways.
RESULTS: Following treatment with NSC-Med, immunofluorescence analysis revealed that neurospheres expressed high proportions of Nestin-positive (97.09%), Sox2-positive (50.11%), and p75NTR-positive (77.87%) cells. Metabolomic profiling revealed a significant enhancement of the Warburg effect in the NSC-Med group. Western blot analysis further revealed elevated expression of PKM2, along with significant increases in both extracellular and intracellular lactate levels following NSC-Med treatment. NSC-Med treatment significantly enhanced proliferation, as demonstrated by a 2.3-fold increase in EdU incorporation (P < 0.05). Transcriptomic analysis revealed the activation of the calcium signaling pathway in the GDNF group. Western blotting revealed a significant increase in CaMKII phosphorylation, and treatment with the calcium chelator BAPTA-AM attenuated GDNF-induced NeuroD1 upregulation.
CONCLUSION: NSC-Med promotes stem cell-associated features and gene expression in enteric glial cells. GDNF-a key component of NSC-Med-activates a neurogenic cascade via the calcium signaling pathway (CaMKII-NeuroD1 axis), which offers a potential targeted molecular strategy for HSCR therapy.
METHODS: EGCs were identified via proteomic profiling and immunofluorescence in Ednrb⁻/⁻ mice modeling HSCR. EGC/PK060399egfr and primary EGCs were induced with neural stem cell-inducing medium (NSC-Med). Morphological changes, EdU assay, immunofluorescence, RT‒qPCR, and Western blotting were employed to assess the expression of stemness- and neuron-associated markers. Metabolomic and transcriptomic analyses were performed to evaluate metabolic remodeling and signaling pathways.
RESULTS: Following treatment with NSC-Med, immunofluorescence analysis revealed that neurospheres expressed high proportions of Nestin-positive (97.09%), Sox2-positive (50.11%), and p75NTR-positive (77.87%) cells. Metabolomic profiling revealed a significant enhancement of the Warburg effect in the NSC-Med group. Western blot analysis further revealed elevated expression of PKM2, along with significant increases in both extracellular and intracellular lactate levels following NSC-Med treatment. NSC-Med treatment significantly enhanced proliferation, as demonstrated by a 2.3-fold increase in EdU incorporation (P < 0.05). Transcriptomic analysis revealed the activation of the calcium signaling pathway in the GDNF group. Western blotting revealed a significant increase in CaMKII phosphorylation, and treatment with the calcium chelator BAPTA-AM attenuated GDNF-induced NeuroD1 upregulation.
CONCLUSION: NSC-Med promotes stem cell-associated features and gene expression in enteric glial cells. GDNF-a key component of NSC-Med-activates a neurogenic cascade via the calcium signaling pathway (CaMKII-NeuroD1 axis), which offers a potential targeted molecular strategy for HSCR therapy.