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

描述（由申请人提供）：胚胎干细胞（ES）的使用，具有分化为几乎任何细胞类型的能力，为推进人类疾病，再生生物学和更有效治疗方法的开发研究带来了巨大的希望。然而，目前用于指导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、成本高且复杂。此外，使用ES细胞产生遗传修饰的动物是低效的，主要是因为生殖系传播的不可预测性，并且在实现传播的情况下，所得嵌合动物产生从宿主和引入的ES细胞两者父系衍生的后代的混合物。 然而，与此同时，已经确定的是，当将小鼠胚胎干（mES）细胞引入小鼠桑椹胚或胚泡期胚胎中，然后植入假孕小鼠中时，引入的ES细胞可以显著地促进胚胎的所有组织，包括种系。此外，在小鼠中，已经表明胚胎通常可以在胚胎发生期间进行发育补偿，并且当对生命不重要的细胞群体被遗传操作阻断或消融时存活。与此相关的是，也已经表明，所产生的开放的发育“生态位”通常可以由引入非常早期的小鼠胚胎中的ES细胞填充，这些细胞分化以补偿和填充消融的宿主群体。因此，产生胚胎小生境并导致其优先ES细胞定植的能力代表了开发工具的独特机会，所述工具使得能够从ES细胞有效开发嵌合体内的特定细胞类型，包括种系。我们建议开发和实施一种新的方法，将促进干细胞定向分化为特定的细胞类型，并作为该方法的实用性的证明，显着提高生殖系传播效率和动物的数量。为了建立这一工具，我们将：1）通过将mES或诱导多能干（iPS）细胞引入宿主胚胎中来开发和实施小鼠生殖系在嵌合动物中的有效定殖，其中通过在发育早期选择性地消融宿主胚胎生殖细胞来打开生态位; 2）应用这种优先嵌合方法来测试遗传多样性的一组新的15个mES和iPS细胞系的种系能力;和3）使用体外受精（IVF）从所得嵌合体快速、特异性和同步扩增mES细胞衍生的生殖系后代。该方法的建立将有助于嵌合体内特定组织的选择性定殖。这将对发育中的细胞和组织内特定基因功能的检查以及人类再生生物学产生深远的影响。 公共卫生相关性：干细胞具有产生几乎任何细胞类型的能力，有可能大大推进生物医学研究和改进疾病治疗的发展，包括再生生物学。小鼠模型是研究干细胞、控制其分化和理解基因功能的至关重要的工具-这是在人类使用基于干细胞的治疗之前所必需的研究。我们建议开发一种新的方法，使研究人员能够“驱动”干细胞到给定的所需细胞类型，这也将显着促进小鼠的产生，用于研究特定的人类疾病。

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.