Direct reprogramming of human dermal fibroblasts into functional osteoblasts using cell-permeable Oct4-30Kc19 and Cbfß-30Kc19

This study aimed to induce the direct reprogramming of human dermal fibroblasts (HDFs) into osteoblast-like cells using the recombinant fusion proteins Oct4-30Kc19 and Cbfß-30Kc19. Oct4 is a transcription factor known to play a crucial role in the direct reprogramming of various cell types, while Cbfß facilitates the differentiation of bone marrow-derived mesenchymal stem cells into osteogenic lineages. To enable intracellular delivery, both transcription factors were fused with 30Kc19, a bombyx mori-derived cell-permeable protein, and expressed using an Escherichia coli system. Oct4-30Kc19 and Cbfß-30Kc19 were successfully delivered into HDFs, and the treated cells exhibited calcium deposition, a hallmark of functional osteoblasts. RNA sequencing was performed on control, treatment, and positive control (osteoblast) groups, revealing that Oct4-30Kc19 and Cbfß-30Kc19 treatment shifted the gene expression profile of HDFs toward an osteoblast-like state. These findings suggest that recombinant fusion protein-based transcription factor delivery may serve as a potential strategy for direct cell reprogramming into osteogenic lineages. Overall design: Oct4-30Kc19 and Cbfß-30Kc19 were applied to induce the direct reprogramming of human dermal fibroblasts (HDFs) into osteoblasts. To achieve this, HDFs (Gibco) were cultured in osteogenic medium supplemented with Oct4-30Kc19 and Cbfß-30Kc19, and their gene expression profiles were analyzed by RNA sequencing (RNA-seq) and compared with those of human osteoblasts.

本研究旨在利用重组融合蛋白Oct4-30Kc19与Cbfβ-30Kc19，将人真皮成纤维细胞（human dermal fibroblasts, HDFs）直接重编程为成骨样细胞。Oct4是一类在多种细胞类型的直接重编程过程中发挥关键作用的转录因子；而Cbfβ可促进骨髓来源间充质干细胞向成骨谱系分化。为实现胞内递送，两种转录因子均与30Kc19——家蚕（Bombyx mori）来源的细胞穿透蛋白——融合，并通过大肠杆菌（Escherichia coli）表达系统进行重组表达。实验结果显示，Oct4-30Kc19与Cbfβ-30Kc19可成功递送至HDFs内，经处理的细胞出现了功能性成骨细胞的标志性特征——钙沉积。随后对对照组、处理组及阳性对照组（成骨细胞组）开展RNA测序（RNA-seq）分析，结果表明，Oct4-30Kc19与Cbfβ-30Kc19处理可使HDFs的基因表达谱向成骨样细胞状态偏移。上述发现提示，基于重组融合蛋白的转录因子递送策略，有望成为直接将细胞重编程为成骨谱系的潜在方案。整体实验设计：将Oct4-30Kc19与Cbfβ-30Kc19用于诱导人真皮成纤维细胞直接重编程为成骨细胞。具体操作中，将购自Gibco的HDFs置于添加了Oct4-30Kc19与Cbfβ-30Kc19的成骨培养基中培养，通过RNA测序（RNA-seq）分析其基因表达谱，并与人类成骨细胞的基因表达谱进行对比。