Aberrant neurodevelopment in human iPS cell-derived astrocyte-neuron co-culture model of Alexander disease
Co-cultures of neurons and astrocytes derived from human iPS cells carrying a GFAP (R239C) mutation - an identified cause of Alexander disease (AxD) - were analyzed with single-cell RNA sequencing to assess cell population composition, differential gene expression, and cell-cell interaction differences in AxD co-cultures compared to controls. Oxygen-glucose deprivation (OGD) challenge was applied to identify differences in stress response. Our results suggested differentiation impairment especially in AxD astrocytes, represented by an enriched population of less differentiated cells and downregulation of mature astrocyte genes in AxD astrocytes-containing co-cultures. Furthermore, AxD co-cultures showed increased stress response represented by upregulation of metallothioneins and increased susceptibility to the OGD challenge. Overall design: Neurons and astrocytes were derived from human iPS cells and cultivated in a 2D co-culture system. GFAP (R239C) cell line originated in Alexander disease patient fibroblasts and isogenic control cell line was prepared by mutation correction, as described previously (Battaglia et al. 2019). Co-cultures were challenged by OGD for four hours, followed by a 16 h recovery period, to assess the effect of mild stress on AxD and control co-cultures. Six samples in total (corrected neurons + corrected astrocytes, corrected neurons + AxD astrocytes, AxD neurons + AxD astrocytes, control and OGD set of samples) were analyzed with scRNA-seq, focusing on identification of cell populations, differences in their representation across samples, differential gene expression, and cell-cell interaction patterns.