Engrailed 1 (En1) is a developmental transcription factor associated with skeletal formation; however, its functional role in mesenchymal stromal cell (MSC)-driven osteogenesis remains poorly understood. Here, we demonstrated that En1 is a critical regulator of adipose-derived MSCs differentiation into osteoblasts and bone repair induced by cell therapy using this MSC population. Using CRISPR-Cas9-based gain- and loss-of-function approaches in immortalized MSCs, we showed that En1 overexpression robustly enhanced osteoblastic differentiation, increasing RUNX2 protein level, alkaline phosphatase activity, and extracellular matrix mineralization, whereas En1 silencing produced the opposite effects. In a murine calvarial defect model, En1-overexpressing MSCs significantly improved bone formation and microarchitectural parameters, whereas En1-deficient MSCs impaired bone repair. Notably, temporal gene expression analyses revealed a dynamic, stage-dependent role for En1 during osteogenesis, consistent with the coordinated regulation of early commitment and later maturation. These effects were supported by consistent molecular, phenotypic, and in vivo outcomes. Collectively, our findings establish En1 as a key positive regulator of MSC-mediated osteogenesis and identify this transcription factor as a promising target for cell- and gene-based therapeutic strategies aimed at enhancing bone repair.
