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

描述(申请人提供)：胚胎干细胞(ES)的使用，具有向几乎任何细胞类型分化的能力，在推进人类疾病研究、再生生物学和开发更有效的治疗方法方面具有巨大的前景。然而，目前可用于引导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细胞产生转基因动物的效率很低，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，所产生的嵌合动物产生来自宿主和引入的ES细胞的父系后代的混合。然而，与此同时，众所周知，当小鼠胚胎干细胞(MES)被导入小鼠桑拿期或囊胚期胚胎，然后移植到假孕小鼠体内，被导入的ES细胞可以显著促进胚胎本身的所有组织，包括生殖系。此外，在小鼠身上，研究表明，胚胎通常可以在胚胎发生期间进行发育补偿，并在非生命必需的细胞群被基因操作阻断或消融时存活下来。与此相关的是，也有研究表明，由此产生的开放的发育‘生态位’通常可以由引入非常早期的小鼠胚胎的ES细胞填充，这些细胞分化以补偿和填补被消融的宿主群体。因此，创造胚胎生态位并导致其优先的胚胎干细胞定植的能力是开发工具的独特机会，能够从胚胎干细胞高效地发育嵌合体内的特定细胞类型，包括生殖系。我们建议开发和实施一种新的方法，这种方法将促进干细胞向特定细胞类型的定向分化，并作为该方法的实用性的证明，显著提高生殖系的传输效率和生产的动物数量。为了建立这一工具，我们将：1)通过将MES或诱导多能干细胞(IPS)引入宿主胚胎并在宿主胚胎中高效定植小鼠生殖系，在宿主胚胎中，将有选择地去除发育早期的宿主胚胎生殖细胞，从而打开一个利基；2)应用这种优惠的嵌合体方法来测试一套具有遗传多样性的、新颖的15个MES和iPS细胞系的生殖系能力；以及3)使用体外受精(IVF)来快速、特异地、同步地扩增由所产生的嵌合体产生的MES细胞衍生的生殖系后代。这种方法的建立将促进嵌合体中特定组织的选择性定植。这将对检测发育中的细胞和组织中的特定基因功能以及人类再生生物学产生深远的影响。 与公共卫生相关：干细胞具有产生几乎任何类型细胞的能力，有可能显著促进生物医学研究和改进疾病治疗方法的开发，包括再生生物学。小鼠模型是研究干细胞、控制干细胞分化和了解基因功能的关键工具--在人类使用基于干细胞的治疗之前，研究是必要的。我们建议开发一种新的方法，使研究人员能够将干细胞‘驱动’到给定的所需细胞类型，这也将极大地促进研究特定人类疾病的小鼠的产生。

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.