In Vitro Reprogramming of Somatic Cells into Pluripotent ES-like Cells

描述（由申请人提供）：胚胎干细胞被认为具有再生医学的巨大潜力。使用核移植来产生“定制”ES细胞为患者特异性治疗提供了可能性。然而，胚胎干细胞和核移植技术是非常有争议的，因为尚未解决的技术问题与克隆人，因为严重的道德反对使用人类胚胎。干细胞领域的关键问题是在不使用卵子的情况下在培养皿中完成体细胞到多能细胞的重编程。我们和其他实验室的新结果表明，通过逆转录病毒介导的Oct 4，Sox 2，c-myc和Klf 4转移到成纤维细胞中，可以在不将体细胞核暴露于卵细胞质的情况下实现多能性的重编程。重编程的细胞显示出与正常ES细胞在分子和生物学上无法区分。该提案有四个目标：1。我们将定义重编程的分子机制。2.我们将建立高通量筛选，以确定小分子，提高效率的重编程和替代需要引入任何因子的逆转录病毒介导的基因转移。3.因为只有一小部分细胞转化为多能状态，我们将定义重编程的靶细胞。此外，我们还将评估成纤维细胞以外的其他细胞类型是否可以重编程。4.目前的方案使用转基因供体细胞来选择培养物中的多能细胞，如果该方法被考虑用于移植医学，则这构成了主要障碍。我们将寻求建立可用于从非转基因小鼠的成纤维细胞中获得重编程细胞的替代筛选方法。最后，我们将在帕金森病和镰状细胞病模型中建立重编程细胞治疗潜力的原理证明。该项目的长期目标是了解将体细胞的表观遗传状态改变为多能状态的分子事件，以便该过程最终可用于治疗退行性疾病患者。体细胞体外重编程为多能ES样细胞 公共卫生相关性：胚胎干细胞与核移植相结合的前景是为许多退行性疾病提供患者特异性细胞治疗，但由于科学问题和伦理反对，这种方法的任何临床应用仍然存在很大争议。这些问题的一个潜在解决方案是在体外将体细胞重编程为可用于“定制”治疗的多能ES细胞。该建议是基于成功地在体外产生ES细胞从成纤维细胞，并寻求解决一些关键问题，需要制定出之前，该方法可以被认为是潜在的应用于医学。

DESCRIPTION (provided by applicant): Embryonic stem cells are thought to have a significant potential for regenerative medicine. The use of nuclear transfer for the generation of "customized" ES cells offers the possibility for patient-specific therapy. However, embryonic stem cell and nuclear transfer technology are highly controversial because of unresolved technical issues with human cloning and because of serious ethical objections to the use of human embryos. The key issue of the stem cell field is to accomplish reprogramming of a somatic into a pluripotent cell in the culture dish without the use of eggs. New results from our and from other laboratories demonstrate that reprogramming to pluripotency can be achieved without exposure of the somatic nucleus to the egg cytoplasm, by retrovirus-mediated transfer of Oct4, Sox2, c-myc and Klf4 into fibroblasts. The reprogrammed cells were shown to be molecularly and biologically indistinguishable from normal ES cells. This proposal has 4 aims: 1. We will define the molecular mechanism of reprogramming. 2. We will establish high throughput screens to identify small molecules that increase efficiency of reprogramming and substitute for the need to introduce any of the factors by retrovirus-mediated gene transfer. 3. Because only a small fraction of cells are converted to a pluripotent state we will define the target cells of reprogramming. Also, we will assess whether other cell types than fibroblasts can be reprogrammed. 4. The present protocols use transgenic donor cells for the selection of pluripotent cells in culture, which constitutes a major obstacle if the approach is ever to be contemplated for transplantation medicine. We will seek to establish alternative screening methods that could be used to derive reprogrammed cells from fibroblasts of non-transgenic mice. Finally, we will establish proof of principle of the therapeutic potential of reprogrammed cells in a model for Parkinson's and Sickle Cell Disease. The long-term goal of this project is to understand the molecular events that accomplish changing the epigenetic state of a somatic cell to a pluripotent state so the process can eventually be used for the treatment of patients with degenerative disease. In vitro Reprogramming of Somatic Cells into Pluripotent ES-Like Cells Public Health Relevance: The promise of embryonic stem cells in combination with nuclear transplantation is to provide patient-specific cell therapy for many degenerative diseases, but any clinical application of this approach remains highly controversial due to scientific problems and ethical objections. A potential solution to these problems is in vitro reprogramming of somatic cells to pluripotent ES cells that could be used for "customized" therapy. This proposal is based on the successful in vitro generation of ES cells from fibroblasts and seeks to solve some of the key problems that need to be worked out before the approach can be considered for the potential application to medicine.