Abstract
Abstract During the development of the central nervous system, pools of progenitor cells undergo proliferation and subsequent differentiation into mature neurons, establishing a functional neuronal network. However, an imbalance in these processes, characterized by excessive proliferation and loss of differentiation, can lead to tumor formation in pediatric patients. In the developing cerebellum, medulloblastomas of the Sonic Hedgehog (SHH) group arise from excessive proliferation of granule neuron progenitor cells (GNPs) driven by SHH signaling, accompanied by hindered differentiation into mature granule neurons. Genetic predisposition accounts for nearly 40% of all pediatric SHH-medulloblastomas, but the development of humanized models to study these predisposition genes has been lacking. To address this gap, we created an ATOH1-driven EGFP reporter iPSC line to generate reliable cerebellar organoids focused on the development of the granule cell lineage. Our cerebellar organoid protocol yielded homogeneous organoids with robust activation of the ATOH1-EGFP reporter around day 30. Through bulk transcriptomics analysis at day 30, we observed pronounced expression of GNP markers, followed by increased expression of markers associated with mature granule neurons by day 60. Furthermore, by aligning the single-nuclei RNA sequencing (snRNAseq) data of day 60 cerebellar organoids with our human cerebellar development snRNAseq atlas, we identified various cerebellar cell types, including GNPs, mature granule neurons, Purkinje cells, interneurons, as well as glutamatergic and GABAergic neurons. Using this protocol, we examined the development of cerebellar organoids in models of hereditary predisposition to childhood cancer. Using a Gorlin syndrome model, we found that PTCH1HET organoids are larger in size than their isogenic control counterparts, while a model for ELP1HET hereditary predisposition showed no change in size compared with control organoids. These organoid models will allow us to uncover novel molecular mechanisms of hereditary predisposition using human cells.
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