Figure 3.

Subclone phenotypes present distinct developmental and genetic traits. (A) Neurosphere assay (Materials and Methods) testing the differentiation potential of GNV019 subclones in vitro. Secondary neurospheres derived from individual subclones were plated and analyzed 10–26 days after growth factor withdrawal. Spontaneous differentiation always yielded neuronal (βIII-tubulin⁺) and glial (GFAP⁺) progeny. Quantification of (giant) multinucleated cells (mnCells) based on counting 1,158±172 cells per condition, i.e. proliferative (Prol) vs. 4-day growth factor withdrawal (Diff) in vitro (n=3 independent experiments, each). (B) Correlation heatmaps of gene expression and genotype data. Left: To focus on transcriptional differences the 1,000 most variably expressed genes were identified from genome wide gene expression datasets of GNV019 parental cells and subclones / hESCdNPs / U87 and human fibroblasts (Materials and Methods). Expression values of these genes were correlated between all samples, and respective Pearson correlation coefficients from every single correlation analysis were illustrated as heatmap and clustered using Euclidean distance and average linkage analysis. Right: 550,316 B allele frequency values from the SNP array dataset (Materials and Methods) were correlated between all case GNV019 samples. Pearson correlation values from every single correlation analysis were then illustrated as heatmap and clustered using Euclidean distance and average linkage analysis. Note the high correlation values. Color keys indicate Pearson correlation values. Note the consistent alignment of tumor subclones. PA+3/5/10, GNV019 parental cells at the respective cell culture passage. FIBRO, human fibroblasts; U87, glioma cell line; hESCdNP, non-cancerous human ES cell-derived neural precursor cells (19). Scale bars: A, 20 µm. Significance levels: *p<0.05; **p<0.01. See also Supplementary Fig. S4.