Background: Basic science research on stroke is lagging. Results from both animal and animal-derived neural cell models are, more often than not, less valid for human stroke. To address this lack of models for human stroke mechanisms, we developed induced pluripotent stem cell (iPSC)-derived human neurons and subjected them to excitotoxicity and oxygen-glucose deprivation (OGD) conditions. These conditions produced a disturbed nitric oxide (NO) metabolome similar to human stroke. A therapeutic intervention, the NO-based neuroprotective agent S-nitrosoglutathione (GSNO), was investigated for neuroprotection against excitotoxicity as well as OGD-induced neuronal nitric oxide synthase (nNOS) activation and the deleterious nNOS/peroxynitrite/calpain neurodegenerative system.
Methods: The iPSC were generated using skin fibroblasts from normal adult human subjects. Neural precursor cells (NPC) were prepared using embryonic body and NPC were differentiated into neurons.
Results: The differentiated iPSC neurons were positive for NeuN and β-tubulin (neuronal markers), and they responded to glutamate/NMDA-induced Ca2+ influx. The majority of iPSC neurons (~90%) were nNOS-positive, supporting them as a suitable model for NO metabolome studies. To examine this model’s nNOS/peroxynitrite/calpain system under stroke conditions, nNOS activation (pnNOS)-dependent toxicity in the iPSC-derived neurons was observed. High expression levels were found for pnNOS Ser1412, 3-NT, and p25 fragment in glutamate/NMDA- and OGD-treated cells. GSNO treatment reversibly blocked glutamate/NMDA- and OGD-induced pnNOS and 3-NT expression levels. Calpain activity was also blocked.
Conclusions: This study of iPSC-derived human neurons is the first of its kind to investigate excitotoxicity or OGD-mediated neurodegeneration mechanisms and therapeutic interventions under stroke conditions. The data provide evidence that the deleterious nNOS/peroxynitrite/calpain system in iPSC neurons is similar to human stroke and that GSNO treatment down regulates this deleterious system. These observations support that iPSC human neurons are a suitable model for studies on stroke-associated neurodegenerative mechanisms as well as therapeutic interventions.