Despite the presence of neural stem cells (NSCs) in the adult mammalian central nervous system, their neuronal and oligodendroglial potential is largely suppressed following injury, particularly in the spinal cord (SC). Spinal cord injuries (SCIs) are therefore especially challenging to treat. In the SC, ependymal cells (ECs) lining the central canal function as a latent NSC population and become activated after injury. Although EC-derived NSCs can generate astrocytes, oligodendrocytes, and neurons in vitro, in vivo they almost exclusively differentiate into scar-forming astrocytes, with minimal oligodendrocyte production and no neuronal differentiation. The mechanisms within the injury environment that restrict EC-derived NSC fate remain poorly understood. Rather than solely interrogating endogenous immune responses in vivo, this project will adopt a modular, bottom-up approach to reconstruct the spinal cord injury niche in vitro using neural organoids derived from endogenous spinal cord NSCs. These organoids will provide a controllable system to study NSC activation and differentiation under defined injury-like conditions. By systematically incorporating key components of the SCI environment—such as fibroblasts, microglia, and injury-associated cytokines—I will dissect how individual and combinatorial niche signals regulate NSC fate decisions. To resolve the cellular states, gene regulatory programs, and cell–cell interactions within these engineered injury organoids, I will perform single-cell RNA sequencing and single-cell ATAC sequencing. These approaches will enable high-resolution cell type identification, analysis of transcriptional and chromatin accessibility changes associated with injury cues, and inference of ligand–receptor interactions that control NSC behaviour. Integration of single-cell transcriptomic and epigenomic datasets will allow reconstruction of gene regulatory networks underlying astrocytic versus regenerative differentiation trajectories. The scale and complexity of these single-cell multi-omics datasets necessitate access to a high-performance computing cluster for data processing, integration, and computational modelling. Computational analyses will include cell clustering, trajectory inference, regulatory network reconstruction, and systematic mapping of intercellular signalling pathways influencing NSC fate. By reconstituting the spinal cord injury environment in a controlled organoid system and interrogating it with single-cell multi-omics, this project will provide mechanistic insight into the niche signals that restrict or promote regenerative NSC outcomes. These findings will inform future strategies aimed at modulating the injured spinal cord environment to direct endogenous EC-derived NSCs toward oligodendrocyte and neuronal regeneration in mammals.