Reprogramming of somatic cells by defined factors

核重编程定义了成体细胞的去分化为多能 胚胎细胞，具有巨大的治疗潜力，因为它允许生成 来自患者的基因匹配的细胞用于细胞治疗。到目前为止，重新编程一直是 通过将核移植到卵母细胞中以及胚胎细胞和 体细胞，这两种方法都有严重的技术或伦理限制。基于 最近发表的观察结果表明，我们已经产生了所谓的诱导多能干细胞 逆转录病毒过表达直接从成纤维细胞(IPS)获得的转录 因子Oct4、Sox2、c-myc和Klf4。与之前报道的iPS细胞不同，我们的 IPS细胞在表观遗传状态和发育过程中与ES细胞没有什么区别 潜力。这些发现提出了几个关键问题：(I)顺序是什么 伴随着核重编程的分子变化，(Ii)什么是动力学 重新编程以及是否需要细胞分裂，(Iii)不同的细胞类型在不同 分化阶段同样容易重新编程，以及(Iv)人类细胞是否可以 重新编程为iPS细胞？解决这些问题对于理解 核重新编程的分子性质，并可能导致允许 有效地将患者S的细胞重新编程为多能细胞。目前对 解决这些问题的是直接重新编程的低效率和无法 实时跟踪重新编程。我们将通过生成以下内容来解决这些问题 ？可重新编程的小鼠？其中每一个细胞都可以被可逆地诱导表达 在重新编程所必需的水平上的四个因素，以及通过尝试重新编程 人类细胞。因此，本提案的目标是确定(I)健壮性和 重新编程的动力学，(II)转录和表观遗传变化的等级 伴随着核重新编程，(Iii)不同类型的细胞对 四个因素，以及(四)人类重新编程的可行性。

Nuclear reprogramming defines the dedifferentiation of adult cells into pluripotent embryonic cells and has enormous therapeutic potential as it allows generating genetically matched cells from patients for cell therapy. Reprogramming has so far been achieved by nuclear transfer into oocytes and by cell fusion between embryonic cells and somatic cells, two approaches that have serious technical or ethical limitations. Based on recently published observations, we have generated so-called induced pluripotent stem (iPS) cells directly from fibroblasts by retroviral overexpression of the transcription factors Oct4, Sox2, c-myc and Klf4. In contrast to the previously reported iPS cells, our iPS cells were indistinguishable from ES cells in their epigenetic state and developmental potential. Several crucial questions were raised by these findings; (i) what is the sequence of molecular changes that accompany nuclear reprogramming, (ii) what is the kinetics of reprogramming and does it require cell division, (iii) are different cell types at different differentiation stages equally amenable to reprogramming, and (iv) can human cells be reprogrammed into iPS cells? Resolving these questions will be critical for understanding the molecular nature of nuclear reprogramming and may lead to strategies that allow efficient reprogramming of patient?s cells into pluripotent cells. The current limitations to solve these questions are the low efficiency of direct reprogramming and the inability to follow reprogramming in real time. We will tackle these questions by generating ?reprogrammable mice? in which every single cell can be reversibly induced to express the four factors at levels necessary for reprogramming, and by attempting to reprogram human cells. The goals of this proposal are thus to determine (i) the robustness and kinetics of reprogramming, (ii) the hierarchy of transcriptional and epigenetic changes that accompany nuclear reprogramming, (iii) the responsiveness of different cell types to the four factors, and (iv) the feasibility of human reprogramming.