A New Tool for Directed Mouse ES Cell Differentiation and Germline Transmission

小鼠 ES 细胞定向分化和种系传递的新工具

• 批准号: 8105083
• 负责人: Michael Van Wiles
• 金额: $ 26.69万
• 依托单位: JACKSON LABORATORY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-07 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8105083
• 关键词: Ablation; Adult; Animals; Automobile Driving; Biochemical; Biological Models; Biology; Biomedical Research; Cell Line; Cells; Chimera organism; Chimerism; Complex; Development; Disease; ES Cell Line; Embryo; Embryonic Development; Evaluation; Fertilization in Vitro; Generations; Genes; Genetic; Genetically Modified Animals; Germ Cells; Government; Hand; Histocompatibility Testing; Human; Implant; In Vitro; Inbred Strains Mice; Inbreeding; Lead; Left; Life; Methods; Morula; Mus; Persons; Physical environment; Population; Public Health; Research; Research Personnel; Specific qualifier value; Staging; Stem Cell Research; Stem cells; Testing; Time; Tissues; Whole Organism; Work; base; blastocyst; cell type; effective therapy; embryo cell; embryo tissue; embryonic stem cell; gene function; genetic manipulation; human disease; human embryonic stem cell; human tissue; implantation; improved; in vivo; induced pluripotent stem cell; mature animal; mouse model; novel; novel strategies; offspring; public health relevance; regenerative; research and development; stem; stem cell biology; stem cell differentiation; stem cell population; tool; transmission process

DESCRIPTION (provided by applicant): The use of embryonic stem (ES) cells, with their capacity to differentiate to almost any cell type, holds great promise for advancing research on human diseases, regenerative biology, and the development of more effective treatments. However, currently available methods for directing the differentiation of ES cells to specific cell types and producing sufficient numbers of the desired cells are inefficient, costly, and complex. In addition, the generation of genetically modified animals using ES cells is inefficient, primarily because of the unpredictability of germline transmission, and, where transmission is achieved, the resulting chimeric animals give rise to a mixture of offspring derived paternally from both the host and the introduced ES cells. At the same time, however, it is well established that when mouse embryonic stem (mES) cells are introduced into a mouse morula or blastocyst stage embryo, and then implanted into a pseudopregnant mouse, the introduced ES cells can contribute significantly to all tissues of the embryo proper, including the germline. Additionally, in mice it has been shown that embryos can often developmentally compensate during embryogenesis and survive when populations of cells nonessential to life are blocked or ablated by genetic manipulation. Of relevance here, it has also been shown that the resulting open developmental 'niche' can often be populated by ES cells introduced into the very early mouse embryo, with these cells differentiating to compensate and fill in for the ablated host populations. Hence, the capability to create embryonic niches and to cause their preferential ES-cell colonization represents a unique opportunity to develop tools enabling the efficient development of specific cell types within chimeras, including the germline, from ES cells. We propose to develop and implement a novel approach that will facilitate the directed differentiation of stem cells to specific cell types, and as a demonstration of the approach's utility, significantly improve germline transmission efficiency and the numbers of animals produced. To establish this tool, we will: 1) Develop and implement the efficient colonization of the mouse germline in chimeric animals through the introduction of mES or induced pluripotent stem (iPS) cells into host embryos in which a niche will be opened by selectively ablating host embryo germ cells early in development; 2) Apply this preferential chimerism approach to test the germline capability of a genetically diverse, novel set of fifteen mES and iPS cell lines; and 3) Use in vitro fertilization (IVF) to rapidly, specifically, and synchronously expand mES cell-derived germline offspring from the resulting chimeras. Establishment of this method will facilitate the selective colonization of specific tissues within chimeras. This will have far-reaching consequences for the examination of specific gene function within developing cells and tissues and for human regenerative biology. PUBLIC HEALTH RELEVANCE: Stem cells, with their capacity to give rise to almost any cell type, have the potential to significantly advance biomedical research and the development of improved disease treatments including regenerative biology. Mouse models are critically important tools for the study of stem cells, control of their differentiation, and understanding gene function - research that is necessary prior to use of stem cell-based treatments in humans. We propose to develop a novel approach that will enable investigators to 'drive' stem cells to given desired cell types, which will also significantly facilitate the generation of mice for studying specific human diseases.

描述（由申请人提供）：使用胚胎茎（ES）细胞的使用，其能力与几乎任何细胞类型都有区分，对推进人类疾病的研究，再生生物学和开发更有效的治疗构成了巨大的希望。但是，当前可用的方法将ES细胞分化为特定细胞类型并产生足够数量的所需细胞的方法效率低下，昂贵且复杂。此外，使用ES细胞的转基因动物的产生效率低下，这主要是由于种系传播的不可预测性，并且在实现传播的情况下，产生的嵌合动物产生了从宿主和引入的ES细胞衍生出的后代的混合物。 然而，同时，可以很好地确定，当小鼠胚胎茎（MES）细胞被引入小鼠杂物或胚泡阶段胚胎中，然后植入伪久的小鼠中时，引入的ES细胞可以对包括生殖器在内的胚胎的所有组织有显着贡献。此外，在小鼠中，已经表明，胚胎在胚胎发生过程中通常可以在发育中补偿，并且当细胞的种群被遗传操作封闭或消融时，胚胎在胚胎发生过程中经常可以生存。在这里相关性，还显示出所得开放的发育“利基”通常会被引入非常早期小鼠胚胎的ES细胞填充，这些细胞有区分以补偿并填充融入的宿主群体。因此，创建胚胎生态位并引起其优先ES细胞定植的能力代表了开发工具的独特机会，从而使Chimeras中特定细胞类型有效地开发了来自ES细胞在内的嵌合体（包括种系）。我们建议开发和实施一种新型方法，以促进干细胞与特定细胞类型的定向分化，并证明该方法的实用性，可显着提高生殖线传递效率和产生的动物数量。为了建立该工具，我们将：1）通过引入MES或诱导的多能茎（IPS）细胞中的嵌合动物中小鼠种系的有效定殖进入宿主胚胎，在该胚胎中可以通过在发育早期有选择性地烧掉生物群来通过选择性地烧掉宿主胚胎生殖细胞来打开。 2）应用这种优先的嵌合方法来测试一组遗传多样，新的15个ME和IPS细胞系的种系能力； 3）使用体外受精（IVF）快速，具体，并同步从所得的嵌合体扩展了MES细胞衍生的种系后代。这种方法的建立将促进嵌合体内特定组织的选择性定植。这将对发育细胞和组织中的特定基因功能以及人类再生生物学的特定基因功能产生深远的影响。 公共卫生相关性：干细胞具有几乎所有细胞类型的能力，有可能显着推进生物医学研究并发展改善包括再生生物学在内的疾病治疗方法。小鼠模型是研究干细胞，控制其分化以及理解基因功能的至关重要的工具 - 在使用基于干细胞的人类治疗之前所必需的研究。我们建议开发一种新型方法，该方法将使研究人员能够“驱动”干细胞以给定所需的细胞类型，这也将显着促进小鼠的产生，以研究特定的人类疾病。