Cellular and Tissue Rejuvenation through Transcriptional Reprogramming

总结 器官和细胞再生是对抗衰老的令人兴奋的新方法，最近的突破 把他们带到了衰老研究的前沿。例如，发现年轻血液中的全身因素 恢复各种小鼠组织和大脑功能，并用四种干细胞进行部分重编程， 转录因子（TF）（Yamanaka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”和“老年”可以被描述为不同的状态，而“老年”状态 可以通过转录重编程逆转回到“年轻”状态。 我们假设，可能存在许多解决人类细胞再生的方法， 重编程，一些解决方案可能比Yamanaka因素更安全，更有效。的情形发生在每一 在NIH/NIA R21基金支持下完成的项目中，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。使用复制性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， Perturb-seq识别潜在的恢复活力的TF-那些在老年人中过度表达或被抑制的TF。 细胞，能够将全球基因表达程序从旧状态重新编程回到年轻状态。 状态我们鉴定了四种TF/染色质修饰剂（E2 F3、EZH 2、STAT 3、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再生。 在这里，我们建议进一步测试这四种因子在人成纤维细胞中的返老还童作用。 在它们的天然组织环境中以及在小鼠肝脏中。我们还将开发新技术来筛选 寻找更有效的再生TF组合，并在老年人成纤维细胞和小鼠肝脏中进行测试。 如果成功的话，这项拟议的研究将确定TF/TF组合，可以在体外和体内老化 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， 人类疗法在这个建议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 从疾病到正常的细胞培养模型。

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.