Kim Seung Hyun L, Cho Sungwoo, Kim Seoyeon, Kwon Janet, Lee Jaeyoung, Koh Rachel H, Park Ju Hyun, Lee Hwajin, Park Tai Hyun, Hwang Nathaniel S
Interdisciplinary Program in Bioengineering, Seoul National University, Seoul, 08826, Republic of Korea.
Department of Medicine, Standford University, 450 Serra Mall, Standford, 94305, USA.
Biomater Res. 2022 Jul 14;26(1):33. doi: 10.1186/s40824-022-00280-8.
The number of patients suffering from osteoporosis is increasing as the elderly population increases. The demand for investigating bone regeneration strategies naturally arises. One of the approaches to induce bone regeneration is somatic cell transdifferentiation. Among the transcriptional regulators for transdifferentiation, octamer-binding transcription factor 4 (OCT4) is famous for its role in the regulation of pluripotency of stem cells. Bone morphogenetic protein 4 (BMP4) is another factor that is known to have a significant role in osteogenic differentiation. Previous studies have achieved transdifferentiation of cells into osteoblasts using viral and plasmid deliveries of these factors. Although these methods are efficient, viral and plasmid transfection have safety issues such as permanent gene incorporations and bacterial DNA insertions. Herein, we developed a cell penetrating protein-based strategy to induce transdifferentiation of endothelial cells into osteoblasts via nuclear delivery of OCT4 recombinant protein combined with the BMP4 treatment. For the nuclear delivery of OCT4 protein, we fused the protein with 30Kc19, a cell-penetrating and protein stabilizing protein derived from a silkworm hemolymph of Bombyx mori with low cytotoxic properties. This study proposes a promising cell-based therapy without any safety issues that existing transdifferentiation approaches had.
OCT4-30Kc19 protein with high penetrating activities and stability was synthesized for a protein-based osteogenic transdifferentiation system. Cells were treated with OCT4-30Kc19 and BMP4 to evaluate their cellular penetrating activity, cytotoxicity, osteogenic and angiogenic potentials in vitro. The osteogenic potential of 3D cell spheroids was also analyzed. In addition, in vivo cell delivery into subcutaneous tissue and cranial defect model was performed.
OCT4-30Kc19 protein was produced in a soluble and stable form. OCT4-30Kc19 efficiently penetrated cells and were localized in intracellular compartments and the nucleus. Cells delivered with OCT4-30Kc19 protein combined with BMP4 showed increased osteogenesis, both in 2D and 3D culture, and showed increased angiogenesis capacity in vitro. Results from in vivo subcutaneous tissue delivery of cell-seeded scaffolds confirmed enhanced osteogenic properties of transdifferentiated HUVECs via treatment with both OCT4-30Kc19 and BMP4. In addition, in vivo mouse cranial defect experiment demonstrated successful bone regeneration of HUVECs pretreated with both OCT4-30Kc19 and BMP4.
Using a protein-based transdifferentiation method allows an alternative approach without utilizing any genetic modification strategies, thus providing a possibility for safer use of cell-based therapies in clinical applications.
随着老年人口的增加，骨质疏松症患者的数量也在上升。自然而然地就产生了对研究骨再生策略的需求。诱导骨再生的方法之一是体细胞转分化。在转分化的转录调节因子中，八聚体结合转录因子4（OCT4）因其在干细胞多能性调节中的作用而闻名。骨形态发生蛋白4（BMP4）是另一个已知在成骨分化中起重要作用的因子。先前的研究通过病毒和质粒递送这些因子实现了细胞向成骨细胞的转分化。尽管这些方法很有效，但病毒和质粒转染存在安全问题，如永久基因整合和细菌DNA插入。在此，我们开发了一种基于细胞穿透蛋白的策略，通过OCT4重组蛋白的核递送结合BMP4处理来诱导内皮细胞向成骨细胞的转分化。为了实现OCT4蛋白的核递送，我们将该蛋白与30Kc19融合，30Kc19是一种源自家蚕血淋巴的具有细胞穿透和蛋白稳定作用且细胞毒性低的蛋白。本研究提出了一种有前景的基于细胞的治疗方法，不存在现有转分化方法所具有的任何安全问题。
合成具有高穿透活性和稳定性的OCT4 - 30Kc19蛋白，用于基于蛋白的成骨转分化系统。用OCT4 - 30Kc19和BMP4处理细胞，以评估它们在体外的细胞穿透活性、细胞毒性、成骨和血管生成潜力。还分析了3D细胞球体的成骨潜力。此外，进行了体内细胞向皮下组织和颅骨缺损模型的递送。
OCT4 - 30Kc19蛋白以可溶且稳定的形式产生。OCT4 - 30Kc19有效地穿透细胞并定位在细胞内区室和细胞核中。用OCT4 - 30Kc19蛋白与BMP4联合递送的细胞在二维和三维培养中均显示出成骨增加，并且在体外显示出血管生成能力增强。体内皮下组织递送接种细胞的支架的结果证实，通过OCT4 - 30Kc19和BMP4处理，转分化的人脐静脉内皮细胞（HUVECs）的成骨特性增强。此外，体内小鼠颅骨缺损实验证明，用OCT4 - 30Kc19和BMP4预处理的HUVECs成功实现了骨再生。
使用基于蛋白的转分化方法提供了一种无需利用任何基因修饰策略的替代方法，从而为在临床应用中更安全地使用基于细胞的治疗方法提供了可能性。
