Promoting residential cells, particularly endogenous neural stem and progenitor cells (NSPCs), for tissue regeneration represents a potential strategy for the treatment of spinal cord injury (SCI). However, adult NSPCs differentiate mainly into glial cells and contribute to glial scar formation at the site of injury. Gsx1 is known to regulate the generation of excitatory and inhibitory interneurons during embryonic development of the spinal cord. In this study, we show that lentivirus-mediated expression of Gsx1 increases the number of NSPCs in a mouse model of lateral hemisection SCI during the acute stage. Subsequently, Gsx1 expression increases the generation of glutamatergic and cholinergic interneurons and decreases the generation of GABAergic interneurons in the chronic stage of SCI. Importantly, Gsx1 reduces reactive astrogliosis and glial scar formation, promotes serotonin (5-HT) neuronal activity, and improves the locomotor function of the injured mice. Moreover, RNA sequencing (RNA-seq) analysis reveals that Gsx1induced transcriptome regulation correlates with NSPC signaling, NSPC activation, neuronal differentiation, and inhibition of astrogliosis and scar formation. Collectively, our study provides molecular insights for Gsx1-mediated functional recovery and identifies the potential of Gsx1 gene therapy for injuries in the spinal cord and possibly other parts of the central nervous system.

Although stem cell therapy holds enormous potential for treating debilitating injuries and diseases in the central nervous system, low survival and inefficient differentiation have restricted its clinical applications. Recently, 3D cell culture methods, such as stem cell-based spheroids and organoids, have demonstrated advantages by incorporating tissue-mimetic 3D cell-cell interactions. However, a lack of drug and nutrient diffusion, insufficient cell-matrix interactions, and tedious fabrication procedures have compromised their therapeutic effects in vivo. To address these issues, we developed a biodegradable nanomaterial-templated 3D cell assembly method that enables the formation of hybrid stem cell spheroids with deep drug delivery capabilities and homogeneous incorporation of 3D cell-matrix interactions. Hence, high survival rates, controlled differentiation, and functional recovery were demonstrated in a spinal cord injury animal model. Overall, our hybrid stem cell spheroids represent a substantial development of material-facilitated 3D cell culture systems and can pave the way for stem cell-based treatment of CNS injuries.

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