Fig 1.
Generation and characterization of brain organoids.
(A) Diagram illustrating the process of generating brain organoids from hiPSCs for up to Day 70 of suspension culture. The illustration was created with BioRender.com. (B) The process of generating brain organoids is shown at four stages, from hiPSC monolayer culture to spheroid formation, suspension culture of Day 3 organoids, and suspension culture of Day 70 organoids. The scale bar is 100 μm.
Fig 2.
Radiosensitivity of different brain cell types after irradiation with a 0.5 Gy or 2 Gy dose of 250 MeV protons.
Immunostaining of (A) NEUN+ neurons, (D) GFAP+ astrocytes, and (G) MBP+ oligodendrocytes in Day 70 WT83 C6 organoids 24 hours after irradiation with 0.5 Gy and 2 Gy doses as well as a control group. Kruskal-Wallis test (P < 0.05) with Dunn’s multiple comparison test was conducted for control versus 0.5 Gy versus 2 Gy collected 30 minutes (n = 4 per group for B and E; n = 3 per group for H) or 24 hours (n = 4 per group for C and F, n = 3 per group for I) after irradiation. Box-and-whisker plots show the range and median of cell counts across regions of interest. Scale bars = 10 μm.
Fig 3.
DNA damage in brain organoids 30 minutes post-irradiation.
Immunostaining of DNA damage marker γH2AX in Day 70 WT83 C6 organoids 30 minutes after proton irradiation: (A) Control, (B) 0.5 Gy, and (C) 2 Gy. (D) Kruskal-Wallis test (P < 0.05) with Dunn’s multiple comparison test showing significant difference for control versus 0.5 Gy (P < 0.0001) and control versus 2 Gy (P < 0.0001) at 30 minutes (N = 3 organoids per group, n > 40 DAPI+ cell nuclei analyzed per group). The box-and-whisker plot shows the range and median of foci counts across regions of interest. Scale bars = 10 μm.
Fig 4.
DNA damage in brain organoids 24 hours post-irradiation.
Immunostaining of DNA damage marker γH2AX in Day 70 WT83 C6 organoids 24 hours after proton irradiation: (A) Control, (B) 0.5 Gy, and (C) 2 Gy. (D) Kruskal-Wallis test (P < 0.05) with Dunn’s multiple comparison test showing no change for control versus 0.5 Gy and significant difference for control versus 2 Gy (P < 0.05) at 24 hours (N = 3 organoids per group, n > 40 cell nuclei analyzed per group). The box-and-whisker plot shows the range and median of foci counts across regions of interest. Scale bars = 10 μm.
Fig 5.
DNA damage in brain organoids 48 hours post-irradiation.
Immunostaining of DNA damage marker γH2AX in Day 70 XB C2 organoids 48 hours after irradiation: (A) Control (B) 2 Gy. (C) Quantification of foci counts per cell nucleus with two-tailed Mann Whitney test (P < 0.05) showing no significant difference between unirradiated controls and irradiated organoids (N = 3 organoids per group, n > 40 cell nuclei analyzed per group). Box-and-whisker plot shows the range and median of foci counts across regions of interest. Scale bars = 10 μm.
Fig 6.
Hematoxylin and Eosin (H&E) staining of brain organoids.
H&E staining of unirradiated control and 2 Gy irradiated Day 70 XB C2 organoids 48 hours after irradiation. The magnification used for microscopy is shown above each image (A-C, G-I). (A-C) Representative section of a control organoid shows outer regions with laminar cytoarchitecture and large ovoid cell nuclei surrounded by abundant small circular cell nuclei. (D-F) 60X magnified outer region of unirradiated control organoids with distributed nuclear and eosinophilic material separated using ImageJ Color Deconvolution. (G-I) Representative section of an irradiated organoid shows outer regions with large ovoid cell nuclei and few small circular nuclei. (J-L) 60X magnified outer region of 2 Gy irradiated organoids with distributed nuclear and eosinophilic material separated using ImageJ Color Deconvolution. (M) Illustration of an organoid showing the region of interest that was used to quantify small circular nuclei, within 300 μm of the outer surface. The illustration was created with BioRender.com. (N) Quantification of small circular nuclei with two-tailed Mann-Whitney U test (P < 0.05) showing a significant difference in counts between unirradiated controls and irradiated organoids (n = 5). Box-and-whisker plot shows the range and median of small circular nuclei proportions, relative to large ovoid nuclei, across regions of interest.
Fig 7.
Gene expression changes of brain organoids 48 hours post-irradiation.
(A) Principal Component Analysis (PCA) plot of the Control and 2 Gy irradiated XB C2 organoids. (B) Expression heat map showing sample-to-sample distances where darker blue represents greater similarity between samples. (C) MA plot showing the log (base 2) fold change (y-axis) versus the mean of normalized read counts (x-axis) using DESeq2. The alpha value for significance on the MA plot is set to 0.05, where each dot represents a single gene and significantly DEGs are colored blue.
Fig 8.
GSEA hallmark pathways and volcano plot.
(A) Heatmap of significantly (FDR < 0.05) DEGs. The samples are clustered on the x-axis of the dendrogram based on their gene expression patterns. Columns 1–3 are the irradiated samples and columns 4–6 are the control samples. The dendrogram on the y-axis shows the relative expression patterns of individual genes across the samples. (B) Volcano plot showing DEGs with FDR < 0.05 and log2FC > |0.58| colored in red (upregulated) and blue (downregulated). The top 10 most significant DEGs are labeled. (C) Unsupervised gene set enrichment analysis results using MSigDB 7.5 C2 Canonical pathways for annotated genes, pre-ranked by fold change. On the graph, GSEA pathways are arranged according to their Normalized Enrichment Scores.
Table 1.
Top 10 most significant DEGs.
Fig 9.
Top GO categories and KEGG pathways related to DEGs.
(A) Enrichment plots of relevant pathways, showing running enrichment scores and positions of rank-ordered genes. (B) Gene ontology of the significant (FDR < 0.05) DEGs with the top 20 categories of each BP, CC, MF shown on the y-axis and fold change on the x-axis. The number of DEGs we identified in this category are shown in the graph to the right. (C) KEGG pathway analysis of the significant pathways with the largest number of DEGs. (D) KEGG visualization of the Cell Cycle pathway (hsa04110) adapted to show the replication fork in eukaryotes and overlaid with DEGs identified from the dataset. Red indicates upregulation, green indicates downregulation, and white indicates no change. (E) Expression ratios of DEGs in GO categories for the Molecular Function “microtubule motor activity”, Biological Process “mitotic cell cycle process”, and Cell Component “chromosome”, are shown sorted by their log2(FC) from highest to lowest. Upregulated genes are colored in red and downregulated genes are colored in blue.
Fig 10.
Ingenuity pathway analysis of DEGs.
(A) A graphical summary of significant DEGs with p < 0.05 and log2(FC) > |0.58| was generated using Ingenuity pathway analysis (IPA) to identify the major biological themes in the dataset. (B) IPA representation of overlapping canonical pathways with lines connecting pathways that share common genes. (C) Graph of canonical pathways with -log(p-value) > 1.3 (p-value < 0.05) sorted from most to least significant.