Characterization of extracellular vesicles and miRNA released by cerebral organoids
Brian B. Silver, Rick D. Fannin, Kevin Gerrish, Erik J. Tokar
Abstract
• Extracellular vesicles (EVs) were isolated from cerebral organoids. • Cerebral-organoid-derived EVs were characterized via several methods, including Nanoparticle Tracking Analysis, Electron Microscopy, and Western Blot. • Cerebral-organoid-derived EVs contained RNA in sufficient quantities for robust analysis using droplet-digital PCR and NanoString. • NanoString was used to characterize miRNA content of the cerebral-organoid-derived EVs. • Cerebral organoids were found to release EVs containing miRNA targeting several pathways involved in neural development and neurodegenerative disease, highlighting the potential use of cerebral organoids for studying brain-related EVs. Environmental toxicants can contribute to the development of several neurodegenerative diseases. However, the mechanisms behind this pathology are still incompletely understood. Prompt diagnosis of impending neurodegeneration is crucial for early interventions to prevent cognitive decline. Towards this end, accurate biomarkers for early neurodegenerative processes and exposure risk are needed. Extracellular vesicles (EVs) are lipid particles released by cells which contain many bioactive molecules including miRNAs. EVs may serve both as a route of propagating neurotoxic phenotypes and as a source of biomarkers for neurological disease. However, the exact mechanisms though which EVs could spread the deleterious effects of toxicants and the full spectrum of their usage as biomarkers remain unclear. Organoid models have several advantages, including capabilities in high-throughput toxicant testing and applications in personalized medicine and disease models. However, few studies have examined EV release in brain organoids to determine if the EVs could contain useful biomarkers. We employed several technologies to characterize EVs released by human cerebral organoids and their associated miRNAs. We identified that cerebral organoids consistently release EV-associated miRNA in quantities sufficient for robust analysis with NanoString. Further, pathway analyses revealed that terms related to neurodegenerative disease and nervous system signaling are associated with the recovered miRNAs. Together, these data suggest that cerebral organoids have utility as a tool for the discovery of EV-associated miRNAs involved in neurodegenerative disease and neurotoxicity.