Abstract
Our goal was to investigate mitochondrial damage in a three-dimensional (3D) neural stem cell (NSC) organoid model using oxidative stress-induced NSCs as primary research method. To create an in vitro organoid model, we utilized NSCs that were exposed to oxidative stress by treating them with hydrogen peroxide (H2O2) at a concentration of 75 µM, leading to mitochondrial damage. Markers for oxidative stress, differentiation, and neurodegenerative diseases were analyzed to characterize organoid models by assessing gene expression and protein levels via histology, immunofluorescence staining, spectrophotometry, and Real-Time PCR. To determine extent of mitochondrial damage in organoid models, we compared mitochondrial membrane potential and total mitochondrial ratio. We independently evaluated mitochondrial damage in both spontaneously self-organized organoid model and oxidative stress organoid models. The 3D NSC organoid model was established through histological and immunofluorescent analyses, which revealed a well-organized cellular structure. Due to intentionally induced oxidative stress, cell distribution varied. We found that H2O2 reduced cell viability and stimulated proliferation at specific concentrations. The cells in oxidative stress model showed strong expression of neural markers MAP2 and TUBB3 compared to controls, as well as positive expression of Alzheimer's marker TAU on 28th day. The model also displayed mitochondrial membrane changes and increased mitophagy during culture process. Overall, we successfully developed an organoid model using multipotent NSCs, which demonstrated H2O2's crucial role in directing cell differentiation and behavior. The model exhibited expected matrix rearrangement, resembling typical organoids, suggesting its potential as an Alzheimer's model and utility in related research studies.