Initiation Phase Cellular Reprogramming Rejuvenates DNA Damage Repair Defect In The Ercc1 Accelerated Aging Mouse Model

Unlike aging somatic cells, which exhibit a decline in molecular fidelity and eventually reach a state of replicative senescence, pluripotent stem cells can indefinitely replenish themselves while retaining full homeostatic capacity. The conferment of beneficial -pluripotency related traits via in vivo partial cellular reprogramming (IVPR) significantly extends lifespan and restores aging phenotypes in mouse models. Although the phases of cellular reprogramming are well characterized, details of the rejuvenation processes are poorly defined. Here we created the first reprogrammable accelerated aging mouse model with a DNA damage repair defect to better understand the effects of reprogramming on a key driver of aging. Using enhanced partial reprogramming by combining small molecules with the Yamanaka factors, we observed potent reversion of DNA damage, significant upregulation of multiple DNA damage repair processes, and restoration of the epigenetic clock is observed. In addition, we present evidence that TGFb inhibition of the ALK5 or ALK2 receptor is able to phenocopy some benefits including epigenetic clock restoration suggesting a role in the mechanism of rejuvenation during partial reprogramming. Fibroblasts from WT, ERCC1 d/-, OSKM, Oct4-GFP mice and crosses were isolated from the tail tip or skin and cultured. For reprogramming experiments, doxycycline was added to the medium for 2 or 4 days. For TGFB inhibition experiments, small molecule TGBF inhibitors were added to the medium.

与出现分子保真度下降、最终进入复制性衰老状态的衰老体细胞不同，多能干细胞（pluripotent stem cells）可实现无限自我更新，并维持完整的稳态能力。通过体内部分细胞重编程（in vivo partial cellular reprogramming, IVPR）赋予有益的多能性相关特征，可显著延长小鼠模型的寿命并逆转衰老表型。尽管细胞重编程的各个阶段已得到充分表征，但重编程介导的年轻化过程的具体机制仍有待明确。本研究构建了首个携带DNA损伤修复缺陷的可诱导重编程加速衰老小鼠模型，以更好地解析重编程对衰老关键驱动因素的影响。我们通过将小分子与山中伸弥因子（Yamanaka factors）联合使用，优化了部分重编程方案，观察到DNA损伤得到有效逆转，多种DNA损伤修复通路显著上调，同时表观遗传时钟（epigenetic clock）得以恢复。此外，我们发现抑制ALK5或ALK2受体的TGFβ信号通路，可复刻部分年轻化益处（包括表观遗传时钟恢复），这提示该通路在部分重编程的年轻化机制中发挥关键作用。我们从野生型（WT）、ERCC1缺陷型（ERCC1 d/-）、OSKM过表达型以及Oct4-GFP转基因小鼠及其杂交后代的尾尖或皮肤中分离成纤维细胞并进行体外培养。在重编程实验中，我们向培养基中添加多西环素（doxycycline），处理时长为2天或4天。在TGFβ抑制实验中，我们向培养基中添加小分子TGFβ抑制剂。