Neural organoids incorporating microglia to assess neuroinflammation and toxicities induced by known developmental neurotoxins

The use of iPSC-derived complex <i>in vitro</i> 3D cellular constructs is a promising avenue to more accurately predict human neural toxicities and reduce the use of animal models. We have generated a neural organoid model which incorporates iPSC-derived microglia and enables interrogation of neuroinflammation induced by pre-clinical drug candidates of varying modalities and chemical compounds in industrial use. Herein we describe the generation and characterization of this model system and its utility in assessing toxicity. We exposed the neuroimmune organoids to a variety of developmental neurotoxins and measured cellular damage by release of LDH, GFAP, and NF-L into the cell culture supernatants. Additionally, to determine whether the compounds led to activation of microglia-mediated inflammation, we measured IL-8 secretion and assessed microglia-specific gene transcriptional analysis using bulk RNA sequencing. Spearman correlation matrices using both differentially expressed genes in the RNA sequencing data and pathway analysis using Gene Ontology Enrichment revealed that microglia may play a role in the toxicity of these compounds which has been widely overlooked in standardized neurotoxicity tests. Treatment of the organoids with lead acetate demonstrates a dose-response curve of IL-8 secretion and alterations in the microglial morphology. Our findings suggest that both direct neurotoxicity and indirect neuroinflammatory mechanisms contribute to the potentially harmful effects of these compounds in the developing central nervous system.