Cellular and Tissue Rejuvenation through Transcriptional Reprogramming

摘要 生物体和细胞再生是对抗衰老的令人兴奋的新方法，最近的突破 把它们带到了衰老研究的前沿。例如，在年轻血液中发现了系统性因素 恢复小鼠的各种组织和脑功能，并用四个干细胞进行部分重新编程 转录因子(山中因子)使组织和细胞恢复活力，并延长小鼠的寿命。 这些发现表明，“年轻的”和“老的”可以被描述为不同的状态，而“老”的状态 可以通过转录重编程将其逆转回“年轻”状态。 我们假设可能存在许多通过转录来恢复人类细胞活力的方法。 重新编程，其中一些解决方案可能比山中因子更安全、更有效。在最近的一个 在NIH/NIA R21拨款的支持下，LI实验室开发了一种系统的方法来测试这一点 假设并找到解决方案。使用复制衰老的人类细胞培养模型 Hayflick(连续传代的人成纤维细胞)，我们开发了一种高通量筛选，使用 扰动-序列以识别潜在的返老还童因子--那些在老年人中过度表达或抑制的因子 细胞，能够将全球基因表达程序从旧状态重新编程为新状态 州政府。我们鉴定了四种转录因子/染色质修饰物(E2F3，EZH2，STAT3，ZFX)，当过度表达或 单独抑制，能够使体外老化的人成纤维细胞恢复活力。 在此，我们建议进一步测试这四种因子对老化的人成纤维细胞的回春作用。 在它们的自然组织环境中和在小鼠的肝脏中。我们还将开发新的技术来筛选 寻找更有效的抗衰老因子组合，并在老化的人成纤维细胞和小鼠肝脏中测试它们。 如果成功，这项拟议的研究将确定能够在体外和体内衰老的TFS/Tf组合 人成纤维细胞和小鼠肝脏。这将为转基因小鼠的研究和翻译奠定基础 人类疗法。该方案中开发的方法可以推广到识别组合 产生任何所需细胞表型的转录程序，例如，细胞状态的逆转 在疾病的细胞培养模型中从疾病到正常。

Summary Organismal and cell rejuvenation are exciting new approaches to counteract aging, and recent breakthroughs have brought them to the forefront of aging research. For examples, systemic factors in young blood was found to rejuvenate various mouse tissues and brain function, and partial reprogramming with four stem cell transcription factors (TFs) (Yamanaka factors) rejuvenate tissues and cells and extend the lifespan of mice. These discoveries demonstrate that “young” and “old” can be described as different states, and the “old” state can be reversed back into a “young” state through transcriptional reprogramming. We hypothesized that there might exist many solutions to human cell rejuvenation through transcriptional reprogramming, and some of the solutions may be safer and more potent than Yamanaka factors. In a recently completed project supported by an NIH/NIA R21 grant, the Li lab developed a systematic approach to test this hypothesis and to find the solutions. Using a human cell culture model of replicative aging employed by Hayflick (continuously passaged human fibroblast cells), we developed a high throughput screen using Perturb-seq to identify the potential rejuvenating TFs -- those that when over-expressed or repressed in old cells, are capable of reprogramming the global gene expression program from the old state back to a younger state. We identified four TFs/chromatin modifier (E2F3, EZH2, STAT3, ZFX) that when over-expressed or repressed individually, are able to rejuvenate human fibroblast cells aged in vitro. Here we propose to further test the rejuvenating effect of these four factors in human fibroblast cells aged in vivo in their natural tissue environment, and in mouse liver. We will also develop new technologies to screen for more potent rejuvenating TF combinations and test them in aged human fibroblast cells and in mouse liver. If successful, this proposed study will identify TFs/TF combinations that can rejuvenate in vitro and in vivo aged human fibroblast cells and mouse liver. This will set the stage for transgenic mouse study and translation to human therapies. The methodologies developed in this proposal can be generalized to identify combinatorial transcriptional programs that produce any desired cellular phenotypes, e.g., the reversion of the cellular state from disease to normal in cell culture models of diseases.