Induced Pluripotent Stem Cell-Derived Neural Organoids Incorporating Microglia for Interrogation of Neural Inflammation and Toxicity

While animal models have value in central nervous system (CNS) drug discovery and toxicology applications, they also have limitations including translation to human biology, efficiency, efficacy, and safety. There is a critical need to develop advanced neural microphysiological systems (MPS) that could complement and reduce the use of animal models. Most current in vitro models, however, do not reflect the complexity of non-neural cell types and cell-cell interactions found within neural tissue. To address these limitations, Stem Pharm has leveraged its proprietary synthetic hydrogel platform to enable the formation of complex, reproducible induced pluripotent stem cell (iPSC)-derived neural organoids containing microglia and vascular cells. The neural organoids are formed in 96-well plates from iPSC-derived neural precursor cells, microglia, endothelial cells and mesenchymal stem cells and are ready for screening 21 days after initial plating. Single cell transcriptional analysis demonstrates that the resulting organoids are cell-type diverse, containing multiple neuron types, astrocytes, microglia, and endothelial cells. Bulk and single cell RNA-seq analysis demonstrates high intraclass correlation and low coefficients of variation between biological replicates. Microglia incorporated into the organoids are distributed throughout the organoids, display ramified morphology resembling their in vivo morphology, and demonstrate a gene signature that strongly correlates with in vivo microglia expression. Modulation of microglia within the organoids to pro- and anti-inflammatory phenotypes was validated through stimulation with lipopolysaccharides, interferon gamma, TGFβ & IL-10, or IL-4 & IL-13 and appropriate transcriptional signatures were observed based on these stimuli. To demonstrate application for neurotoxicity screening, organoids were subjected to a screen of known neurotoxins and transcriptional profiles between control and treatment groups were analyzed. Perturbations in expected pathways including neural development, axon guidance, mitochondrial function and innate immune responses were detected. These data demonstrate the promising application of Stem Pharm’s advanced neural organoids incorporating microglia for facilitating translation between pre-clinical and clinical discovery and development.