Fig. 1. 3D iPSC-derived neuron and astrocyte asteroid coculture rapidly recapitulates neuronal and astrocytic interactions and signatures.

a Experimental schematic diagraming 2D culture of hiPSC-derived neurons and astrocytes, 3D asteroid coculture, and immunolabeling and single-cell transcriptomic analysis at indicated timepoints. Created with BioRender.com. b Representative immunolabeling of 21 DIV3D asteroids with cell-type markers, including astrocytic precursor markers (VIM), astrocytic markers (EAAT1, GFAP), neuronal precursor markers (SOX2, NES), neuronal markers (MAP2, NEUN), and synaptic markers (DLG4/PSD95, SYP). Scale bar = 50 µm. c UMAP of 26,789 single-cell transcriptomic profiles by scRNA-seq after filtration and cell-type identification, including all cells from 7, 14, and 21 DIV3D (see “Methods”). Five main cell types are identified: excitatory neurons (EX_NEU, dark green), inhibitory neurons (IN_NEU, light green), neuron precursors (NEU_P, teal), astrocytes (ASC, dark red), and astrocyte precursors (ASC_P, light red). d Dot plot of key cell-type marker transcript expression including cycling (CYC), astrocytic (ASC), pan-neuronal (PAN-NEU), inhibitory neuronal (GABA), excitatory neuronal (GLU), and synaptic markers (SYNAPSE) in cell-type clusters. e UMAP projection colored by timepoint across the 7–21 days in 3D culture (DIV3D) time course (blue gradient) highlighting the model trajectory. f Percent composition of cell-type clusters across the 7–21 DIV3D time course, indicating a decrease in precursor cell proportions and a correlated increase in mature NEU and ASC proportions. g, h Quantification of select immunolabeled cell-type markers (g) and scRNA-seq transcript expression (h) across the 7–21 DIV3D time course (blue gradient), indicating a decrease in precursor markers (SOX2, VIM) and an increase in mature neuronal marker (MAP2). Data obtained from 5 independent asteroids. (ANOVA, ns: P > 0.05, *P < = 0.05, **P < = 0.01). i UMAP feature plot indicating gene expression of neuronal synaptic markers DLG4 (PSD95) and SYP. j UMAP integration of the asteroid scRNA-seq dataset (Asteroid, red) with a cerebral organoid scRNA-seq dataset (Cerebral Organoid, blue³⁸) highlighting overrepresentation of the asteroid cells in more mature cell clusters (see “Methods” and Supplementary Fig. 7).