ID #1055 Modelling Medulloblastoma Using iPSC-Derived Cerebellar Organoids and Inducible Oncogene Systems for Drug Screening

Abstract

Medulloblastoma is the most common malignant brain tumour in children and arises from disrupted cerebellar development combined with aberrant oncogenic signalling. Current medulloblastoma drug discovery largely relies on 2D monolayer cultures, which fail to recapitulate complex three-dimensional architecture, cellular diversity, and tumour–brain interactions present in vivo. These limitations restrict accurate modelling of drug screening, treatment response, and toxicity to normal brain tissue.

Human induced pluripotent stem cell (iPSC)–derived cerebellar organoids provide a physiologically relevant human platform to model tumorigenic events and enable more predictive drug screening. In this study, we established a robust protocol for generating cerebellar organoids from human iPSCs and modelled oncogene-driven transformation toward medulloblastoma using two clinically relevant oncogenic combinations: MYC with OTX2, associated with Group 3/4–like disease, and MYC with GFI1, associated with Group 3 medulloblastoma.

Cerebellar differentiation was optimised and characterised using time-resolved expression of neural progenitor markers and lineage-specific markers corresponding to ventricular zone and rhombic lip populations. Oncogenes were initially introduced into organoids using electroporation-based delivery of PiggyBac transposon plasmids tagged with fluorescent reporter. Although stable oncogene expression was detectable for up to 50 days, this approach exhibited significant technical limitations, including heterogeneous expression, restricted plasmid distribution within mature organoids, and variable transfection efficiency, with successful modification achieved in only 30–60% of organoids.

To address these challenges, we are implementing a doxycycline-inducible PiggyBac transposase–mediated genomic integration strategy, enabling stable and controllable oncogene expression, real-time fluorescent monitoring, and puromycin selection, thereby improving reproducibility, consistency, and temporal control while preventing premature oncogene activation.

Overall, this dual-model platform enables physiologically relevant drug screening and therapeutic evaluation in developing and established medulloblastoma. Integration of cerebellar cells permits assessment of tumour response and normal brain toxicity, while expandable mini tumour organoids support scalable drug testing and personalised therapy development beyond conventional 2D models.