Transgene-Free Direct Osteogenic Reprogramming Using Cell-Permeable Octamer-Binding Transcription Factor 4/Core-Binding Factor β Fusion Proteins

Bone-related disorders, including fractures and osteoporosis, remain substantial clinical challenges, partly because of the limited availability of reliable osteogenic cell sources and complications associated with current therapies. To address these limitations, this study introduces a novel protein-based direct reprogramming platform for the conversion of human dermal fibroblasts into functional osteoblasts using only 2 transcription factors, octamer-binding transcription factor 4 (Oct4) and core-binding factor β (Cbfβ), fused to the silkworm-derived cell-penetrating protein, 30Kc19. Genetic fusion with 30Kc19 markedly improves the stability and cellular uptake of both Oct4 and Cbfβ, resulting in recombinant constructs (Oct4-30Kc19 and Cbfβ-30Kc19) that achieve high reprogramming efficiency with negligible cytotoxicity, outperforming plasmid DNA-based methods. The protein-induced osteoblasts (piOBs) exhibit a characteristic osteoblast morphology, express established osteogenic markers, and display a global transcriptomic profile that aligns with key features of primary human osteoblasts. Importantly, transplantation of piOBs into a murine calvarial defect model induces substantial new bone formation, demonstrating in vivo therapeutic efficacy. By leveraging the unique cell-permeable and protein-stabilizing properties of 30Kc19, this streamlined 2-factor system represents a potentially safer, more scalable, and clinically feasible strategy for regenerative therapies targeting bone diseases, circumventing the inherent risks associated with viral vectors and genomic integration.