(759e) Effects of Nanoscale Magnetite on Human Forebrain-like Tissue Development in Stem Cell-Derived Cortical Spheroids

Introduction: Iron is one of the most abundant transition metals found in the brain, and it is required for some essential neurological processes, including myelination, neurotransmitter synthesis, nerve impulse transduction, and energy metabolism. Disrupting iron homeostasis in the brain could lead to diseases such as long-term neurodevelopmental and neurodegenerative abnormalities. The potential long-term impact of nanoscale iron oxides on cellular stress and neuro-inflammation remains unknown. This study utilized the in vitro 3-D human brain organoid model to test the effect of 8 nm and 15 nm magnetite iron oxides (Fe₃O₄). Cell viability, proliferation, and oxidative enzyme expression were characterized. Cellular stress, inflammation, cell apoptosis, DNA damage, DNA repair, and reactive oxygen species (ROS) response were also investigated. The importance of this study suggested that there could be a connection between environmental factors and neurotoxicity, and neurodegeneration.

Methods: Human brain cortical organoids were derived by sequentially treating human iPSK3 cells with dual SMAD inhibitors to promote neural progenitors, followed by the combination of basic fibroblast growth factor 2 and sonic hedgehog inhibitor cyclopamine. The iron oxide nanoparticles (NP) of 8 nm and 15 nm were added to the culture from day 4-30 at concentrations of 0.00 µg/mL, 0.023 µg/mL, 2.3 µg/mL, and 23 µg/mL. The spheroids were characterized with iron staining assay, as well as RT-PCR, immunocytochemistry, and flow cytometry for various markers of cellular stress and neurons.

Results: Regardless of the size, the higher magnetite concentration treatment showed higher accumulation in the cells. Cellular death was not significantly different when compared with the control. The 15 nm NP treatment only increased the SOD2 and β-tubulin III expression for the 2.34 µg/mL. In general, gene expression for the 8 nm NP treated spheroids showed an increase in the markers of cellular stress and ROS response with a minor increase in DNA damage but not the apoptosis markers. On the other hand, the 15 nm NP-treated spheroids did not show significant changes for cellular stress markers and the DNA repair marker. Tp53 was similar across all the treatment and slightly higher for the 2.3 µg/mL group. The cellular stress and ROS response were comparable or lower to the untreated group.

Conclusion: Although the cell viability was not significantly different across the tested conditions, the neuronal spheroids exposed to 8 nm iron oxide showed negative effects (i.e., increased inflammation and ROS response genes), whereas those exposed to 15 nm iron oxide treatment demonstrate a slightly positive effect (i.e., decreased inflammation, apoptosis, and ROS response, and cleaned up genes). It is postulated that the cells treated with 8 nm may have higher number of iron oxide particles, and these excessive iron oxide particles may disturb the cellular iron metabolism and cause adverse effects. This hypothesis remains to be investigated in the future.