Glial-enriched stem-cell 3D model resembling the cellular landscape of the human brain mimics the glial-immune neurodegenerative phenotypes of multiple sclerosis.

The role of central nervous system (CNS) glia in sustaining self-autonomous inflammation and driving clinical progression in multiple sclerosis is attracting increasing scientific interest. Here, we applied a single transcription factor (SOX10)-based protocol for accelerating oligodendrocyte differentiation from human induced pluripotent stem cell (hiPSC)-derived neural precursor cells to produce three dimensional, multilineage organoids integrating submillimetric self-organizing forebrain organoids (consisting of neurons, astrocytes, oligodendrocyte precursors cells, and myelinating oligodendrocytes). To achieve an immunocompetent organotypic model, hiPSC-derived microglia was also incorporated. Within an 8-week time frame, organoids reproducibly generated a rich diversity of mature cell types, with single-cell transcriptional profiles similar to the human adult brain. This cellular system is able to respond to complex inflammatory stimuli and to properly mimic macroglia-microglia neurodegenerative phenotypes and crosstalk, as seen in chronic active multiple sclerosis. The results obtained pave the way for the implementation of this novel 3D model in the identification of druggable targets for inflammatory neurodegeneration as drug screening platform. For each condition we pooled 30 organoids. We compared WT vs SOX10 induced clones and organoids exposed or not to inflammatory CSF. We performed droplet-based scRNA-seq, libraries were prepared using the Chromium Single Cell 3ʹ v3, according to the manufacturer's protocol (10x Genomics) without modifications, targeting 5,000 cells per sample. Using the Illumina Novaseq platform, all samples were sequenced at 50,000 reads per cells