Engrailed 2 enhances neuronal differentiation of bone marrow mesenchymal stem cells to promote spinal cord injury repair.

Bone marrow mesenchymal stem cells (BMSCs) hold promise for spinal cord injury (SCI) repair, yet their limited neuronal conversion efficiency restricts therapeutic outcomes. Therefore, this study investigated the role of En2 in regulating the neuronal differentiation of BMSCs and its therapeutic potential in SCI. Here, we demonstrate that Engrailed 2 (En2) markedly enhances the neuronal differentiation of BMSCs and promotes functional recovery after SCI. En2 overexpression increases TUJ1⁺ and MAP2⁺ neuronal populations in vitro, while transcription factor prediction and experimental validation identify ASCL1 as a key downstream target. Silencing ASCL1 significantly impairs En2-induced neuronal differentiation and reduces nuclear β-catenin levels, indicating that En2 regulates BMSC neurogenesis through the ASCL1/β-catenin axis. In vivo, En2-overexpressing BMSCs delivered via HAMA hydrogel survive, differentiate into neurons, improve tissue preservation, and enhance locomotor recovery in rats with SCI, as confirmed by histology, immunofluorescence, BBB scoring, and CatWalk analysis. These findings reveal a previously unrecognized regulatory mechanism whereby En2 drives the neuronal commitment of BMSCs and highlight a promising gene-enhanced stem cell strategy for spinal cord injury repair.