We next compared the generated iNPCs with primary GTICs, isolated and characterized by Pollard et al.5, by performing genome-wide analyses. Genome-wide expression data demonstrated that transformed iNPCs closely resemble primary GTICs at the molecular level, though some differences were readily observed among the different groups. WTiNPCs were most similar to p53KDiNPCs, while Ras/EGFR/SrciNPCs and p53KD-Ras/EGFR/SrciNPCs were very similar to each other and intermediate between the WTiNPC/p53KDiNPC cluster and primary GTICs (Supplementary Data 1, analysis of the variance (ANOVA) q value <0.01 and variance filter=0.2 using Qlucore). The corresponding heatmap showed five large clusters of genes that were differentially expressed among WTiNPCs, p53KDiNPCs, Ras/EGFR/SrciNPCs, p53KD-Ras/EGFR/SrciNPCs and GTICs (Fig. 1e, clusters I–V and Supplementary Data 2). Importantly, genes in cluster V showed higher expression in the GTICs, Ras/EGFR/SrciNPCs and p53KD-Ras/EGFR/SrciNPCs compared with ESCs, WTiNPCs, and p53KDiNPCs (Fig. 1e, cluster V and Supplementary Data 2). Of the five clusters, cluster V showed the highest number of significant enrichments on pathways analysis, and was enriched for several cell growth-, proliferation- and cancer-associated pathways. Notably, DNA methylation analysis confirmed a more undifferentiated phenotype for Ras/EGFR/SrciNPCs and p53KD-Ras/EGFR/SrciNPCs (Supplementary Table 1).
