Abstract
BACKGROUND: The inflammatory response following spinal cord injury (SCI) is a critical factor contributing to neural dysfunction. This response exacerbates neural damage in the injured area by facilitating the infiltration of inflammatory cells and the release of cytokines, thereby remarkably impeding neural repair and functional recovery. Spermatogonial stem cells (SSCs) have the potential for spinal cord nerve regeneration. Concurrently, olfactory ensheathing cells (OECs), a unique cell type with neurorepair potential, can effectively enhance nerve regeneration through the secretion of various neurotrophic factors, the suppression of inflammatory responses, and improvements in the injury microenvironment. However, research concerning OEC induction systems and the differentiation of SSCs into spinal cord neurons remains limited. This study aimed to investigate the synergistic effects of OECs and SSCs to develop a more effective and safe stem cell therapy strategy for SCI. METHODS: This study integrates in vitro and in vivo methodologies to investigate the mechanisms through which an efficient induction system facilitates the differentiation of SSCs into neurons. Initially, primary SSCs were cultured and characterized through immunofluorescence, and the molecular mechanisms underlying the induction system were examined via western blot and quantitative reverse transcription (qRT-PCR) methods. An inflammatory environment was subsequently developed in a lipopolysaccharide (LPS)-activated microglial model to evaluate the impact of the induction system on SSC differentiation. Finally, differentiated SSCs were transplanted into a traumatic SCI model, and functional recovery was assessed via the Basso, Beattie and Bresnahan (BBB) locomotor scale, as well as electrophysiology, including somatosensory evoked potential (SEP) and motor evoked potential (MEP) tests. RESULTS: In vitro experimental results demonstrated that the activation of the OEC induction system significantly enhanced the efficiency of the differentiation of SSCs into spinal cord neurons. Notably, in the inflammatory milieu, an optimal concentration of lipopolysaccharide (LPS) significantly augments both neuronal differentiation efficiency and survival rates. In addition, compared with those in the other experimental groups, the induced neuronal-like cells exhibited genuine neuronal characteristics. Importantly, JAK2/STAT3 pathway signaling plays a crucial role in this intricate cellular transformation. Suppression of the JAK2/STAT3 signaling pathway significantly improved differentiation efficiency and facilitated neural repair. In vivo experimental results revealed that the transplantation of differentiated spinal neurons into the lesioned spinal cord substantially improved sensory and motor functions, as evidenced by behavioral assessments (such as BBB scoring) and electrophysiological tests (MEP and SEP). CONCLUSION: This study developed an efficient induction system that facilitates the differentiation of SSCs into spinal cord neurons. The system also significantly improved neuronal differentiation and survival under LPS-induced inflammatory conditions. Crucially, the JAK2/STAT3 signaling pathway has been identified as a critical regulatory mechanism in this complex process. Intriguingly, inhibition of the JAK2/STAT3 pathway significantly increases neuronal differentiation efficiency. The transplantation of these differentiated cells resulted in the functional recovery of sensory and motor functions in the lesioned spinal cord. These findings provide a novel therapeutic strategy for stem cell-based treatment of spinal cord injuries and lay both a theoretical and practical foundation for clinical applications.