Fig 1: Schematic illustrations of experimental paradigm and the differentiation protocols.(A) hiPSC-derived from five healthy individuals are differentiated into astrocytes (in-house lines: C1, C2, and C3; commercial lines from ACS: C4 and C5) and from one individual into neurons. (B) Differentiation of cortical region–specific astrocytes was performed, modifying established protocols (78, 79). NPCs using an established protocol [Shi et al., 2012 (29)] were differentiated into either neurons or GPC (glial precursor cells) after 30 days from NPC and then further differentiated into mature astrocytes. (C) Immunocytochemistry images show that three lines of hiPSC-derived astrocytes express the astrocytic marker, GFAP. (D and E) There is a calcium response to ATP in iPSC-derived astrocytes. Representative traces of calcium (D) and the percentage of cells (E) in response to ATP. (F) hiPSC-derived astrocyte enables uptake of glutamate (Glutamate Assay Kit, ab83389/K629-100, Abcam). (G and H) Composition of hiPSC-derived neuron and astrocyte coculture assessed using MAP2 (neuronal marker) and GFAP (astrocytic marker) immunocytochemistry together with representative images of a neuronal, astrocyte, and coculture (G) and the quantification (H). (I) Uniform Manifold Approximation and Projection (UMAP) plot showing the clustering of the integrated dataset using the cells from all the samples (basal and αsyn-O–treated astrocytes, neurons, and coculture samples). (J) Dot plots showing the expression of the astrocyte and neuron marker genes in the clusters identified in the single-cell RNA-seq data. (K) UMAP overlaid with the correlation coefficients, showing the correlation of the two astrocyte clusters with the astrocytes from Leng et al. (27). (L) Gene Ontology (GO) terms associated with the genes up-regulated in astrocyte clusters 1 and 2. (A) and (B) were created with BioRender (https://biorender.com/)