MODELING NEUROFIBROMATOSIS TYPE 1 (NF1) GLIOMAGENESIS USING HUMAN IPSC-DERIVED 3D ORGANOIDS AND NEURAL PROGENITOR SYSTEMS

Neurofibromatosis type 1 (NF1) is a tumor predisposition syndrome caused by mutations in the NF1 gene, which encodes the Ras GTPase-activating protein neurofibromin. Loss of heterozygosity (LOH) in neural progenitor cells drives Ras pathway hyperactivation, leading to the development of low-grade optic pathway gliomas (OPGs). Despite their clinical prevalence, the human-specific cellular mechanisms that initiate these tumors remain difficult to study in vivo. In this study, we established a suite of patient-derived human iPSC models, including cerebral organoids, neurospheres, and assembloids, to investigate early NF1-associated gliomagenesis. By transitioning organoid-derived cells into neurosphere cultures at defined developmental stages, we enriched for and characterized progenitor populations. We employed single-cell RNA sequencing and proteomic profiling to evaluate the cellular heterogeneity and developmental trajectories of these NF1-deficient populations.To examine regional interactions, we integrated retinal, thalamic, and cortical organoids within microfluidic platforms. This multi-organoid system facilitates the study of long-range axon outgrowth and connectivity between specific neural regions via microchannels, providing a controlled environment to assess compartment-specific axonal phenotypes, relevant to the optic glioma pathway. Our findings indicate that NF1 neurospheres exhibit altered proliferative and differentiation profiles compared to isogenic controls, reflecting dysregulated progenitor states. Furthermore, the multi-organoid microfluidic system allows for the observation of compartment-specific axonal phenotypes. Together, these complementary platforms provide a human-relevant framework for studying the early stages of NF1-associated OPGs and may serve as a tool for evaluating targeted therapeutic interventions.