Effects of Aβ42 oligomer exposure on forebrain organoids.

(A) Schematic of the experimental design. Forebrain organoids were differentiated and exposed to Aβ42 oligomers for 8 days starting at day 90. (B–C) Immunofluorescence staining of cortical progenitor and neuronal layer markers in control (CTR) and Aβ42-treated organoids: TBR2, CTIP2, and SOX2 (B); SATB2, CTIP2, and BRN2 (C). Scale bars: 500 µm (overview), 100 µm (insets). (D) Immunofluorescence staining for FOXG1, MAP2, SOX2, and PAX6 in CTR and Aβ42-treated organoids. Scale bars: 500 µm. (E) Immunostaining for GFAP and S100B in CTR and Aβ42-treated organoids. Scale bars: 50 µm. (F) Aβ detected with the 6E10 antibody in CTR and Aβ42-treated organoids. Right, quantification of 6E10 signal intensity normalized to DAPI (violin plot; unpaired t test, P < 0.0001; n = 4–6 organoids per group). Scale bars: 100 µm. (G) UMAP visualization of integrated single-cell RNA-seq data from day 60 CTR and Aβ42-treated organoids, split by condition and colored by annotated cell type. (H) Relative cell-type composition of CTR and Aβ42-treated organoids, shown as stacked bars for each sequencing library (n = 2 per condition); statistics in Supplementary Table 1. (I) Volcano plot of differentially expressed genes from pseudobulk analysis comparing Aβ42-treated organoids with CTR; selected genes are labeled. (J) Gene set enrichment analysis (GSEA) of Gene Ontology Biological Process (GO:BP) terms using differentially expressed genes from the pseudobulk comparison in (I), ordered by normalized enrichment score. (K) Pseudotime trajectories inferred with Monocle3 and projected onto UMAP embeddings for CTR (left) and Aβ42-treated (right) organoids. (L) Distribution of pseudotime values in CTR and Aβ42-treated organoids (Wilcoxon rank-sum test).

Development and characterization of a 2D–3D hybrid organoid model for analyzing Aβ42-induced pathology.

(A) Heatmap of the top 30 most significantly differentially expressed genes from bulk RNA-seq of day 90 3D Aβ42-treated and CTR organoids. Expression values are shown as scaled normalized expression across individual samples, with hierarchical clustering applied to both genes and samples. (B) Experimental workflow. Day 90 cortical organoids were either maintained as intact 3D organoids for bulk RNA-seq or sectioned into 300-µm slices and cultured on Matrigel-coated plates to generate a 2D–3D hybrid organoid system for synaptic and functional analyses. Hybrid cultures were treated with 5 µM Aβ42 oligomers for 6 days prior to downstream assays. (C) Representative immunofluorescence images of synaptophysin and MAP2 co-stained with VGLUT or GABA, used to determine synapse ratios. Scale bar: 10 µm. (D–G) Quantification of synaptic marker density in CTR and Aβ42-treated 2D–3D hybrid organoids: excitatory synapses (VGLUT/synaptophysin) and inhibitory synapses (GABA/synaptophysin). (H) Representative calcium imaging traces from CTR and Aβ42-treated 2D–3D hybrid organoids, acquired at 7.67 s per frame. (I) Quantification of spike frequency per cell from calcium imaging in CTR and Aβ42-treated hybrid organoids. (J) GABA response expressed as the post-/pre-treatment spike count ratio. Data are mean ± SEM. For immunofluorescence and functional analyses, n = 4–6 organoids per group. Statistical significance was assessed using unpaired t tests.

A microglia-integrated forebrain organoid model of Aβ42 exposure.

(A) Experimental scheme for the microglia-integrated forebrain organoid model. Forebrain organoids were treated with vehicle (0.1% DMSO; CTR) or Aβ42 oligomers (5 µM) for 6 days, followed by 48 h of co-culture with iPSC-derived microglia (iMGLs) to generate CTR+iMGL and Aβ42+iMGL conditions. The scheme also depicts iPSC differentiation through hematopoietic progenitor, immature microglia, and mature microglia stages. (B) Characterization of iMGLs. Left, flow cytometry of CD11b and CD45 (∼96% CD11b+; ∼50% CD45^low). Right, representative immunofluorescence images of iMGLs stained for IBA1 (green) with Aβ42 detected by 6E10 (red). Scale bars: 100 µm (overview), 50 µm (high magnification). (C) Aβ42 concentration in culture medium measured by ELISA 48 h after iMGL integration, in Aβ42-treated organoids with and without iMGLs (unpaired t test, P < 0.0001; n = 3 samples per group). Data are mean ± SEM. (D) UMAP visualization of integrated single-cell RNA-seq data from Aβ42-treated forebrain organoids (Aβ42) and Aβ42-treated organoids co-cultured with microglia (Aβ42+iMGL), split by condition and colored by annotated cell type; the microglial cluster is shown in yellow. (E) Relative cell-type composition of Aβ42-treated organoids with and without iMGL co-culture, shown as stacked bars for each sequencing library (n = 2 per condition); statistics in Supplementary Table 1. (F) Pseudotime trajectory inferred with Monocle3 and projected onto the UMAP embedding for the Aβ42+iMGL condition. Color scale indicates pseudotime from early (purple/blue) to late (yellow/red). (G) Distribution of pseudotime values in Aβ42-treated organoids with and without iMGL co-culture (Wilcoxon rank-sum test, P = 2.17 × 10⁻¹). (H) Predicted cell–cell interaction network between cell types in Aβ42+iMGL organoids. Node size and edge thickness scale with interaction strength; colors denote cell populations. (I, J) Outgoing (I) and incoming (J) microglial signaling across pathways, relative to other cell types in the organoid. (K) Experimental design for functional analyses in the 2D–3D hybrid forebrain organoid system. Organoid slices were treated with Aβ42 (5 µM) for 6 days, followed by 48 h of co-culture with iMGLs prior to calcium imaging. (L) Representative calcium imaging field showing neuronal activity labeled with Fluo-4 (green) and iMGLs labeled with CellTracker CMTPX (red) in Aβ42-treated hybrid organoids. Scale bar: 100 µm. (M) Neuronal calcium activity (spike counts per cell) in CTR+iMGL and Aβ42+iMGL conditions (unpaired t test; n = 3–4 organoids per group). Individual dots represent single cells; horizontal lines indicate mean ± SEM. (N) Microglial calcium activity expressed as area under the curve (AUC) of ΔF/F₀ traces in CTR+iMGL and Aβ42+iMGL conditions (unpaired t test, P = 0.0346; n = 3–4 organoids per group). Individual dots represent single microglia; horizontal lines indicate mean ± SEM. 3K is created in BioRender.

Cytokine secretion, CD8+ T cell migration, and microglia–T cell calcium activity in microglia-integrated Aβ42-treated forebrain organoids.

(A–E) Relative secretion of CCL4 (A), CCL5 (B), IL-1β (C), TGF-β1 (D), and CXCL10 (E) measured in conditioned media from CTR+iMGL and Aβ42+iMGL forebrain organoids. Data are mean ± SEM; unpaired t tests. (F) Experimental schematic of the 2D–3D hybrid forebrain organoid model used to assess CD8+ T cell migration. Organoids were treated with Aβ42 for 6 days and co-cultured with iPSC-derived microglia (iMGLs), followed by transwell migration assays using CD8+ T cells with or without chemokine receptor inhibitors targeting CCR5, CXCR3, and CCR1. (G) Quantification of CD8+ T cell migration toward CTR and Aβ42-treated organoids in the absence (−MG) or presence (+MG) of iMGL co-culture. (H) CD8+ T cell migration toward Aβ42+iMGL organoids following blockade of CCR5, CXCR3, or CCR1 on CD8+ T cells, compared with the untreated Aβ42+iMGL condition. (I) Experimental design for calcium imaging of microglia–T cell interactions. iMGLs were integrated into Aβ42-treated organoids on day 6, CD8+ T cells were added on day 7, and calcium imaging was performed on day 8. (J) Representative multi-channel image showing CD8+ T cells (CellTrace Violet, blue), intracellular calcium signal (Fluo-4 AM, green), iMGLs (CellTracker CMTPX, magenta), and Aβ42 (647 nm). Scale bar: 50 µm. (K) Time-lapse calcium imaging of iMGLs in the presence of CD8+ T cells over a 13-min window. Insets show individual iMGLs in direct contact with T cells or associated with Aβ42. Scale bar: 50 µm. (L) Microglial calcium activity in Aβ42+iMGL and Aβ42+iMGL+T conditions, quantified as spike count per cell, area under the curve (AUC), and spike amplitude. Individual dots represent single iMGLs. (M) Microglial calcium activity in CTR+iMGL+T and Aβ42+iMGL+T conditions, using the same metrics as in (L). (N) CD8+ T cell calcium activity in CTR+iMGL+T and Aβ42+iMGL+T conditions, quantified as spike count per cell, AUC, and spike amplitude. Calcium imaging data were acquired at 3.8-s intervals. (O) Schematic of the integrated human forebrain organoid platform, in which Aβ42-treated organoids are converted to a 2D–3D hybrid system permitting stepwise integration of iPSC-derived microglia and CD8+ T cells, with readouts comprising single-cell transcriptional profiling, real-time calcium imaging, and assays of immune–neural cellular interaction. Data are mean ± SEM; statistical significance was assessed using unpaired t tests. For cytokine analyses, n = 3 samples per group. For migration and calcium imaging analyses, n = 3–4 organoids per group, with individual dots representing single cells where indicated. 4O is created in BioRender.