Aberrant neurodevelopment in human iPS cell-derived neural organoid model of Alexander disease

3D-cultured unguided neural and cortical organoids derived from human iPS cells carrying a GFAP (R239C) mutation - an identified cause of Alexander disease (AxD) - and their isogenic controls were analyzed with scRNA-seq to investigate the effect of the GFAP mutation on brain development, cell type composition, and gene expression. Results of this analysis showed impaired astro- and neurogenesis in both types of organoids (unguided and cortical), including a lack of cells acquiring the astrocyte fate, and an increased abundance of cells differentiating into lineages other than neuroectodermal. The results also suggested dysregulation of extracellular matrix, membrane, and cytoskeleton components, which might have also affected their differentiation trajectories. Overall design: The AxD cell line with a GFAP (R239C) mutation originated in Alexander disease patient fibroblasts, and an isogenic control cell line was prepared by mutation correction with CRISPR/Cas9, as described previously (Battaglia et al. 2019). 3D organoids were prepared using either unguided neural protocol (resulting in more diverse cell composition) and more targeted cortical protocol (DUAL SMAD inhibition directing cells towards cortical lineage). Organoids were harvested after 165 days in culture, with a variety of cell types including astrocytes present at this stage. Fixed single-cell suspensions from four samples (unguided control and AxD, cortical control and AxD) were prepared with 10X Genomics pipeline and scRNA-seq was performed to investigate changes in cell type composition, gene expression, and cell-cell interaction between the AxD cell line and controls.