TMOD-44. Modeling gliomagenesis via engineered chromosomal aneuploidy and foundational genetic events throughout neurodevelopment of cerebral organoids

Abstract Aneuploidy is a hallmark of cancer and particularly prevalent in glioblastoma (GBM), where it is frequently observed alongside early tumorigenic mutations. Inferred tumor lineage trajectories from patient derived samples suggest that aneuploidy may act as a genetic driver of gliomagenesis; however, its role in initiating GBM remains poorly understood and has never been experimentally validated. To address this, we developed a novel platform that integrates engineered chromosomal aneuploidy with foundational genetic alterations using human cerebral organoids. Leveraging karyotype CRISPR-engineered aneuploidy technology (KaryoCreate), we introduced chromosome 7 gains and chromosome 10 losses—hallmark aneuploidies specific to GBM—into human embryonic stem cells (hESCs) that were differentiated into cerebral organoids alongside karyotype normal hESCs. This resulted in tumor-like clonal expansion of engineered populations validated via fluorescent lineage tracing, inferred copy number variation (CNV) events from single-cell transcriptomics, and fluorescent in situ hybridization (FISH) of Chr 7 and Chr 10 centromeric probes. To investigate alternate cell-of-origin candidates, we introduced doxycycline-inducible aneuploidy machinery into a hESC safe harbor locus and later induced Chr 7 gains and Chr 10 losses throughout organoid neurodevelopment. We further incorporated C228T TERT promoter mutations and CDKN2A deletions to recapitulate genetic alterations commonly found in patients. Over time, immunofluorescent staining and integrated single cell transcriptomic analyses show these models span the diverse transcriptomic cell states characteristic of GBM. This modular, temporally controlled platform enables systematic dissection of how early genomic instability and driver mutations converge to shape cellular fitness, lineage dynamics, and tumor-initiation in a developing human brain context.