Extended Data Fig. 8: Differentiation potential is impaired in H3K27M cells.

a-b, Characterization of the 19 PCW human brainstem astrocytes (n = 258 cells), a predominant best match to H3K27M HGG. a, By PCA, the first principal component separates the two populations. b, Heat map of expression of genes most strongly positively and negatively correlated with PC1. c, Western blot of K27M-mutant H3 protein and total H3 protein confirms presence of mutation and knock-out in each replicate of K27M and KO lines respectively. d-g, Analysis of bulk RNA-seq data for DIPG cell lines (n = 2 independent experiments per condition, biological replicates). d, PCA plot. SCM, stem cell media; DM, differentiation media. e, Volcano plots of differential expression analysis between cells in DM vs. SCM for K27M lines (top) and KO lines (bottom). Red color highlights differentially expressed genes present in the human brainstem astrocyte 2 gene signature (left), and any brainstem or pontine astrocyte gene signature (right). P-values (two-sided Wald test) were adjusted using the Benjamini-Hochberg correction. f, Boxplots of log2 fold change of expression for genes in selected developmental signatures, between cells in DM vs. SCM for K27M lines (red) and KO (blue). Statistical significance was assessed using a two-tailed Student’s t-test (p-values: Hindbrain astrocyte: 1.46x10^-13; Human astrocyte: 6.85x10^-5; OPC/Oligodendrocyte: 0.14; Excit. Neuron: 0.12; ns: not significant). Boxplots: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range. g, Volcano plot of differential expression analysis between K27M and K27M-KO cell lines in DM; differential expression analysis was performed as described above. h, Representative morphology of GFAP + cells among cell lines at 60X magnification. Experiment was repeated, and images are shown, for n = 2 biologically independent replicates per condition. i, Bubbleplot of projection of K27M-KO cell lines onto developmental atlas using ssGSEA, shown for the neuroectodermal cell types. The color of the bubbles indicates the change in ssGSEA score for each signature between cell lines in SCM and DM, while the size of the bubbles indicates the ssGSEA score in DM. Cell types are stratified into two rows based on direction of change of the score upon differentiation. No bubbles are shown for clusters with non-specific gene signatures.