Mutant GFAP alters lineage commitment of neural organoids [AxDorganoids_bulk_experiment2]

Glial fibrillary acidic protein (GFAP) is a type-3 intermediate filament protein mainly expressed in astrocytes in the central nervous system. Mutations in GFAP cause Alexander Disease (AxD), a rare and fatal neurological disorder. How exactly mutant GFAP eventually leads to white and grey matter deterioration in AxD remains unknown. GFAP is also expressed in radial glial cells, which are neural stem cells in the developing brain. Here, we used AxD patient-derived induced pluripotent stem cells (iPSCs) to explore the impact of mutant GFAP during neurodifferentiation. Our results show that GFAP is already expressed in iPSCs. Moreover, we have found that mutations in GFAP can severely affect neural organoid development through altering lineage commitment in embryoid bodies. Together, these results support a key role for GFAP as an early modulator of neurodevelopment. We generated unguided neural organoids from an Alexander Disease patient-derived iPSC line (R239C-AxD-1), as well as from an isogenic control line derived from this patient line (R239R-Ctrl). In experiment 2, we used the AggrewellTM800 plate as well as 96-well round bottom ultra low attachment plates to generate embryoid bodies. We performed RNAseq at day 3,5,7 and 9 of differentiaton to investigate how the two different growth protocols influenced organoid development.