Development of Robust and Reproducible Murine Brain Organoids Endowed With Networks of Functional Neurons and Specific Brain-Region Signature

Abstract

Brain organoids are in vitro three-dimensional (3D) self-organized neural structures, which promise to become relevant tools for disease modelling and drug screening. High-throughput drug screening assess a large amount of compounds using reliable and fast procedures. Organoids obtained from human induced pluripotent stem cells have high batch-to-batch variability, long differentiation time (10-20 weeks), and high cost required for production, thus limiting their use for standardized large-scale drug screening studies. Here, we developed, for the first time, a highly standardized, reproducible and fast (5 weeks) murine brain organoid model starting from multipotent neural stem cells (NSCs) isolated from embryonic subgranular zone (SGZ). Global transcriptome, protein expression, and cell metabolism data show that murine brain organoids progressively differentiate trough defined early, intermediate, and mature stages. During differentiation, brain organoids acquire a self-organized neural structure with a compact and interconnected neuronal layer at its edge. We measured neuronal activity on whole-brain organoids by combining electrophysiological techniques (patch-clamp and MEA recordings), transsynaptic tracing and calcium imaging analysis, and we found that, in less than 5 weeks of in vitro culture, these organoids own functional neurons organized in 3D networks with synaptic connections. Furthermore, we validated the potential of murine brain organoids to acquire a specific brain-region signature; by adding the morphogen WNT3a, we were able to generate brain organoids enriched in neurons with CA3 hippocampal region signature. Overall, our results showed the establishment of a novel fast, robust and reproducible murine 3D in vitro brain model that may represent a useful tool for high-throughput drug screening and disease modelling.