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(申请人提供)：胚胎干细胞被认为具有巨大的再生医学潜力。利用核移植来产生“定制的”胚胎干细胞，为针对患者的治疗提供了可能性。然而，胚胎干细胞和核移植技术极具争议性，因为克隆人类的技术问题尚未解决，而且使用人类胚胎存在严重的伦理反对。干细胞领域的关键问题是在不使用卵子的情况下，在培养皿中完成将体细胞重新编程为多能细胞。我们和其他实验室的新结果表明，通过逆转录病毒将Oct4、Sox2、c-myc和Klf4转移到成纤维细胞中，可以在不将体细胞核暴露在卵细胞质的情况下实现重新编程到多能性。重新编程的细胞在分子和生物学上与正常的ES细胞没有区别。这一建议有四个目的：1.我们将确定重编程的分子机制。2.我们将建立高通量筛选，以确定提高重编程效率的小分子，并取代通过逆转录病毒介导的基因转移引入任何因子的需要。3.由于只有一小部分单元被转换为多能状态，因此我们将定义重新编程的目标单元。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新编程。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，如果考虑将这种方法用于移植医学，这构成了一个主要障碍。我们将寻求建立可用于从非转基因小鼠的成纤维细胞中提取重编程细胞的替代筛选方法。最后，我们将在帕金森氏症和镰刀细胞病的模型中建立重新编程细胞治疗潜力的原则证据。该项目的长期目标是了解完成将体细胞的表观遗传状态改变为多潜能状态的分子事件，以便这一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针对患者的细胞治疗，但由于科学问题和伦理反对，这种方法的任何临床应用仍存在极大争议。这些问题的一个潜在解决方案是在体外将体细胞重新编程为多潜能的ES细胞，可用于“定制”治疗。这项建议是基于成功地从成纤维细胞体外生成ES细胞，并试图解决一些关键问题，这些问题需要解决，然后才能考虑将该方法用于医学。