(628h) Size-Dependent Polystyrene Microplastic Exposure to the Developing Human Induced Pluripotent Stem Cell Derived Cortical Spheroids: Effects and Implications
AIChE Annual Meeting
2021 Annual Meeting
Food, Pharmaceutical & Bioengineering Division
Understanding and Engineering Cellular Responses - Virtual
Thursday, November 18, 2021 - 5:24pm to 5:43pm
Background: Plastics have been part of our ecosystem for about a century and their degradation by different environmental factors produce secondary microplastics. These microplastics have a size <5mm and can enter in living organisms through inhalation and ingestion. They have been seen to accumulate in organs like brain, lung and liver of animal species and even have been observed in the fetus of pregnant women. They can cause respiratory problems like bronchiolitis, neurological problems like neurotransmitter imbalance and reactive oxidative specie (ROS) production in various organisms. The effects of microplastics on humans have not been well investigated. Therefore, to understand the possible effects of polystyrene-microplastics (PS-MPs), a common type of microplastic observed in the environment, on human brain, a 3D model of human forebrain cortical spheroid has been derived employing induced pluripotent stem cell technology, which mimics the early developmental stage of human cerebral cortex.
Methods: To derive the 3D model of human cortical spheroids, iPSK3 cells were initially treated with dual SMAD signaling inhibitors, later with fibroblast growth factors (FGF2) and sonic hedgehog inhibitor (cyclopamine). To analyze the effects of PS-MPs, the spheroids were exposed to 100, 50, and 5 Âµg/mL concentrations of 1 Âµm and 10 Âµm size PS-MPs during day 4-30. The expression of neuronal marker Î²-tubulin III, antioxidant enzyme marker superoxide dismutase (SOD) and cell proliferation marker Ki67 was determined by immunocytochemistry. Flow cytometry was utilized to quantify marker expression. Cell viability after 30 days was measured using live/dead assay. The relative gene expression of ROS cleanup markers (catalase and SOD2), cell death marker (CASP3), premature neuronal marker (CDKN1B), hindbrain marker (HOXB4), forebrain cortical layer marker (TBR1), mature neuronal marker (TUBB3) and Nestin, ventricular zone marker (TBR2), and Ki67, were analyzed using RT-PCR.
Results: The immunocytochemistry showed expression of neural development and patterning markers, showing successful differentiation of cortical spheroids. The results of live/dead assay quantified by flow cytometry showed that the numbers of dead cells were twice the numbers of live cells for both 100 Âµg/mL and 50 Âµg/mL concentrations of 1 Âµm size PS-MP treated spheroids. While the ratio of dead to live cells was around 1:1 for untreated spheroids. The percentages of SOD2 and TBR1 markers were higher in PS-MP treated conditions compare to untreated control. The relative gene expression of SOD2 also showed higher expression for treated cells compared to untreated control. The relative expression of cellular stress, DNA damage, ROS response, cell proliferation, development, and patterning genes indicate PS-MP size and concentration dependent effects. Î²-tubulin III and Nestin gene expression decreased in PS-MP treated conditions compare to untreated control, which may indicate that the long-term exposure of developing forebrain tissues to PS-MPs have negatively affected neural maturation.
Conclusion: The results of both quantitative and qualitative measurements suggest that the size and concentration dependent effect of PS-MPs on developing forebrain cerebral spheroids can cause higher oxidative stress and inversely affect neural maturation. This study has significance in assessing environmental factors in neurotoxicity and degeneration.