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）具有分化为几乎任何细胞类型的能力，对推进人类疾病、再生生物学和开发更有效的治疗方法的研究具有很大的希望。然而，目前可用于引导胚胎干细胞分化为特定细胞类型并产生足够数量所需细胞的方法效率低、成本高且复杂。此外，使用胚胎干细胞培育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，而且，在传播成功的情况下，所产生的嵌合动物会产生来自宿主和引入的胚胎干细胞的杂交后代。然而，与此同时，已经确定的是，将小鼠胚胎干细胞（mES）引入小鼠桑葚胚期或囊胚期胚胎，然后植入假妊娠小鼠，引入的胚胎干细胞可以显著促进胚胎的所有组织，包括种系。此外，在小鼠中，研究表明，胚胎在胚胎发生过程中通常可以进行发育补偿，并在非生命必需的细胞群体被基因操作阻断或消融时存活下来。与此相关的是，研究还表明，由此产生的开放发育“生态位”通常可以由引入早期小鼠胚胎的胚胎干细胞填充，这些细胞分化以补偿和填补消融的宿主种群。因此，创造胚胎龛并使其优先定植胚胎干细胞的能力代表了一个独特的机会，可以开发工具，使嵌合体内的特定细胞类型（包括生殖系）能够从胚胎干细胞中有效地发育。我们建议开发和实施一种新的方法，以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，显着提高种系传播效率和产生的动物数量。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，在嵌合动物中开发和实施小鼠生殖系的有效定植，在宿主胚胎中，通过选择性地在发育早期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一组具有遗传多样性的新型15个mES和iPS细胞系的种系能力；3)利用体外受精（IVF）快速、特异性地、同步地扩增来自嵌合体的mES细胞衍生的种系后代。这种方法的建立将有助于嵌合体内特定组织的选择性定植。这将对研究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。

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.