Fig. 1: Engineered NSCs form tumors in mice that recapitulate GBM.

A Workflow for generating four serial mutant Proneural eNSC genotypes: Wildtype (WT), TERT promoter (TERTp) C228T mutant, TERTp mutant + TP53 G743A (R248Q) mutant, TERTp mutant + TP53 mutant + PDGFRA D842V mutant (triple mutant = PRO). B Sanger sequencing validation of CRISPR/Cas9-mediated TERTp and TP53 exon 6 point mutations in 2 independent genetic clones of wildtype, single, and double mutant hESCs. C Immunofluorescence for stem cell markers OCT4 and PAX6 (488 nm), as well as DAPI, in WT, TERTp, TERTp + TP53 hESC clone set 1 and their differentiation-matched NSCs (scale bar = 50 μM). Shown is a representative image from 3 replicates independently plated for analysis. D Immunoblot analyses for mutant PDGFRA expression and activity in one set of four serial mutant eNSC clones. Representative of three replicates. E Extreme limiting dilution assay using clone set 1 of the four serial mutant eNSC genotypes. Data represent mean ± SEM, independently plated for analysis (WT and PRO: n = 4, TERTp and TERTp + TP53: n = 3, ANOVA, *P < 0.05, ****P < 0.0001). F Representative soft agar colony formation assay of the four serial mutant eNSC genotypes (clone set 1) and G quantification of 3 independent replicates. Data represent mean ± SEM (n = 3, ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). H Kaplan-Meier survival analysis of four serial mutant eNSCs (two genetic clones, C1 and C2, pooled per genotype) following orthotopic implantation in mice (PRO: n = 12 animals, other: n = 10 animals, log-rank, ****P < 0.0001). I T2-weighted MRI images of mice 100 days post-xenograft with four serial mutant eNSC lines (clone set 1). Shown are representative images from the experimental cohorts. Red outlines indicate pathological lesions in the coronal plane (scale bar = 1 mm; L left, P posterior, R right). J Immunofluorescence imaging of representative histological sections generated from PRO eNSC brain tumors (clone 1) stained for Human Nuclear Antigen (488 nm) and DAPI (scale bar = 0.5 mm). Inset images display magnified fields of view and merged images (scale bar = 50 μm). Shown is a representative subject from the experimental animal cohort. K Hematoxylin & Eosin (H&E) staining of a representative PRO eNSC brain tumor (clone 1) demonstrating characteristic histopathology of high-grade gliomas. Left: A pink necrotic center (n) is surrounded by pseudopalisading tumor cells (arrowheads) and vascular proliferation (v), (left, scale bar = 200 μm). Right: A magnified view of tumor hypercellularity and mitotic atypia (right, scale bar = 50 μm). Shown are representative images from a subject within the experimental animal cohort. Source data are provided as a Source Data file, including exact statistical analyses and p values.