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

体外将体细胞重编程为多能 ES 样细胞

• 批准号: 8100897
• 负责人: RUDOLF JAENISCH
• 金额: $ 31.16万
• 依托单位: WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8100897
• 关键词: Address; Bone Marrow Cells; Cell Differentiation process; Cell Fraction; Cell Line; Cell Nucleus; Cell Therapy; Cells; Chromatin; Cytoplasm; DNA; Degenerative Disorder; Drug resistance; Embryo; Epigenetic Process; Ethics; Event; Fibroblasts; Gene Targeting; Gene Transfer; Generations; Genes; Goals; Human; Human Cloning; Immune; In Vitro; Insertional Mutagenesis; Kinetics; Laboratories; Mediating; Medicine; Methods; Modeling; Molecular; Molecular Profiling; Neurons; Oncogenes; Parkinson Disease; Patients; Pharmaceutical Preparations; Population; Precipitation; Process; Protocols documentation; Regenerative Medicine; Resistance; Retroviridae; Scheme; Screening procedure; Sickle Cell Anemia; Site; Solutions; Somatic Cell; Sorting - Cell Movement; Staging; Stem cells; System; Technology; Therapeutic; Transgenic Mice; Transgenic Organisms; Work; X Chromosome; base; c-myc Genes; cell type; clinical application; demethylation; egg; embryonic stem cell; genetic element; genetic manipulation; genome-wide; high throughput screening; improved; induced pluripotent stem cell; keratinocyte; nuclear transfer; pluripotency; public health relevance; small molecule; transplantation medicine; vector

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.

描述（由申请人提供）：胚胎干细胞被认为具有重生医学的重要潜力。将核转移用于生成“定制” ES细胞的生成为患者特异性治疗提供了可能。但是，由于人类克隆的技术问题以及对使用人类胚胎的严重伦理异议，胚胎干细胞和核转移技术是高度争议的。干细胞场的关键问题是在不使用鸡蛋的情况下完成培养皿中的体细胞重编程为多能细胞。来自我们和其他实验室的新结果表明，通过逆转录病毒介导的Oct4，Sox2，C-Myc和Klf4转移到成纤维细胞中，可以实现重编程到多能性，而无需将体细胞核暴露于卵细胞质。重编程的细胞与正常ES细胞在分子和生物学上是无法区分的。该提案有4个目标：1。我们将定义重编程的分子机制。 2。我们将建立高吞吐量筛选，以识别小分子，以提高重编程效率并替代需要通过逆转录病毒介导的基因转移引入任何因素。 3。因为只有一小部分细胞被转换为多能状态，所以我们将定义重编程的目标细胞。另外，我们将评估是否可以重编程成纤维细胞以外的其他细胞类型。 4。目前的方案使用转基因供体细胞在培养中选择多能细胞，如果要为移植医学进行试验，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得出重编程的细胞的替代筛选方法。最后，我们将在帕金森氏病和镰状细胞疾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完成将体细胞的表观遗传状态变为多能状态的分子事件，以便最终可以用于治疗退行性疾病患者。体外重新编码体细胞为多能ES样细胞 公共卫生相关性：胚胎干细胞与核移植结合使用的希望是为许多退行性疾病提供患者特异性的细胞疗法，但是由于科学问题和道德异议，这种方法的任何临床应用都在极大争议。解决这些问题的一种潜在解决方案是体外重新编码可以用于“定制”疗法的多能ES细胞。该提案基于成纤维细胞成功的体外ES细胞的成功生成，并试图解决一些需要解决的关键问题，然后才能考虑使用该方法来实现医学的潜在应用。