Fig. 1: Generation and validation of glial-enriched brainstem organoids.
a, Schematic representation of timely morphogen stimulated patterning of hES cells and hiPS cells toward brainstem organoids and their subsequent application for DMG tumor, CAR T cell treatment and microglia-enriched tumor microenvironment modeling. b, Heat map of z score measuring relative brain region identity on the basis of VoxHunt similarity mapping for various supplemented concentrations of FGF2 or FGF4 (n = 3 experimental repeats, with n = 3 organoids pooled; total n = 9 organoids per condition). c, Immunofluorescence 3D images of a 200-µm-thick organoid slice on day 21 labeled for F-actin (white), SOX2 (yellow) and HOXB1 (red). Right, zoomed-in view of area in white inset. Scale bars, 250 µm (main image) and 25 µm (inset). A representative image of n = 2 slices from n = 2 organoids is shown. d, VoxHunt spatial correlation map of day 120 brainstem organoids with E18.5 mouse brain. The pons area is delineated in red (n = 9 organoids from three independent batches). e, Integrated UMAP representation of developing brainstem organoids from different time points, colored by cell annotation. f, Area plot following the relative distribution of cell types over time. Cell types are color-coded as in e. g, UMAP of the HNOCA34 colored for brainstem organoid presence score. A high score indicates a high likelihood that these HNOCA cells are present in the brainstem organoid dataset. Areas annotated by a dashed line indicate lineages as annotated in the HNOCA. Inset, UMAP colored by coarse regional annotation. h, Heat map showing the log2 fold compositional changes in the brainstem organoid dataset compared to the HNOCA. Positive values correspond to an increased abundance of cells from the indicated regional identity or glial lineage. NPC, neuronal precursor cell. i, Cell clusters in the HDBCA35 with gained coverage in brainstem organoids relative to the HNOCA. The horizontal line indicates the threshold used to define a cluster as gained or not. j, UMAP of the HDBCA showing, in shades of red, the HDBCA clusters gained in brainstem organoids, mostly related to oligos and glioblasts. Gray represents clusters below the threshold used to define gained. Inset, UMAP colored by coarse regional annotation. For e–j, n = 84 organoids in total with 5–24 organoids pooled per time point (details in Supplementary Table 1).
