Abstract：
Direct reprogramming of somatic cells into functional cells still faces major limitations in terms of efficiency and achieving functional maturity of the reprogramed cells. While different approaches have been developed commonly based on exploiting biochemical signals, introducing appropriate mechanical cues that stimulate the reprogramming process is rarely reported. In this study, collagen-coated polyacrylamide (PAM) hydrogels with stiffness close to that of collagenous bone (40 kPa) were adopted to augment the direct reprogramming process of mouse fibroblasts to osteoblastic-like cells. The results suggested that culturing cells on a hydrogel substrate enhanced the overexpression of osteogenic transcription factors using nonviral vectors and improved the yield of osteoblast-like cells. Particularly, a synergistic effect on achieving osteogenic functionality has been observed for the mechanical cues and overexpression of transcriptional factors, leading to enhanced osteogenic transformation and production of bone mineral matrix. Animal experiments suggested that reprogramed cells generated on matrix hydrogels accelerated bone regeneration and stimulated ectopic osteogenesis. Mechanism analysis suggested the critical involvement of actomyosin contraction and mechanical signal-mediated pathways like the RhoA-ROCK pathway, leading to a synergistic effect on the key transcriptional processes, including chromatin remodeling, nuclear translocation, and epigenetic transition. This study provides insights into the mechanical cue-enhanced direct reprogramming and cell therapy.
摘要：
将体细胞直接重编程为功能细胞在效率和实现重编程细胞的功能成熟度方面仍然面临主要限制。虽然通常基于利用生化信号开发了不同的方法，很少报道引入刺激重编程过程的适当机械线索。在这项研究中,采用硬度接近胶原骨（40kPa）的胶原涂层聚丙烯酰胺（PAM）水凝胶，以增强小鼠成纤维细胞到成骨细胞样细胞的直接重编程过程。结果表明，使用非病毒载体在水凝胶基质上培养细胞可增强成骨转录因子的过表达，并提高成骨细胞样细胞的产量。特别是,已观察到机械线索和转录因子的过表达对实现成骨功能的协同作用，导致增强的成骨转化和骨矿物质基质的产生。动物实验表明，在基质水凝胶上产生的重编程细胞加速了骨再生并刺激了异位成骨。机制分析表明，肌动球蛋白收缩和机械信号介导的途径如RhoA-ROCK途径的关键参与，导致对关键转录过程的协同效应，包括染色质重塑,核易位,和表观遗传转变。这项研究提供了对机械提示增强的直接重编程和细胞治疗的见解。
