Reconstitution of the Spinal Cord Injury Microenvironment in Adult Neural Stem Cell-Derived Organoids

Abstract

Following spinal cord injury, endogenous neural stem cells (NSCs) derived from ependymal cells become activated but fail to functionally regenerate the tissue, largely because the injury microenvironment constrains their differentiation toward glial fates. Dissecting how specific niche components drive these outcomes has remained challenging in vivo, and current neural organoid models predominantly recapitulate embryonic neurodevelopment rather than the adult injury context. Here we describe neuroids - a modular organoid system built from injury-activated adult spinal cord ependymal NSCs that spontaneously differentiate into neurons, astrocytes, and to some degree oligodendrocytes within a self-organised 3D structure. Using a bottom-up approach, we reconstruct the injury niche by incorporating meningeal fibroblasts and primary adult microglia, individually and in combination. Fibroblasts accumulate in the organoid core, deposit extracellular matrix (ECM), and trigger reactive astrocyte responses mirroring in vivo scar organisation, while microglia integrate throughout, adopt heterogeneous activation states, and remain functionally active. Their combined incorporation further enhances ECM deposition and promotes oligodendrocyte lineage commitment, suggesting cooperative niche interactions. Single-nucleus multiome profiling and trajectory inference show that these injury-like conditions shift NSC differentiation away from neuronal programs toward proliferative and astroglial states, recapitulating NSC behaviour after injury in vivo. Ligand-receptor analysis implicates microglia-derived TGF{beta}, WNT, and ECM-associated signals as candidate drivers of this gliogenic bias. Together, neuroids provide a tractable platform to study how the adult injury niche regulates endogenous NSC fate, and to identify strategies that simultaneously redirect these cells toward regeneration while targeting the fibrotic scar - two barriers that together prevent functional recovery after spinal cord injury.