In vitro modelling of pregastrulation embryogenesis using pluripotent stem cells

在哺乳动物发育的初期，多能细胞扩张、分化和自我组织，为发育中的有机体奠定基础。一个关键的步骤是形成双层盘，它有两层组成，即上胚层和下胚层。上胚层是一种多能组织，随后形成发育中的有机体的组织和器官。胚下母细胞主要参与胚外组织的形成，但在胚轴形成和外胚层分化中起关键作用。因此，胚胎发育这一阶段的分子机制对发育生物学领域具有重要意义。作为个体多能细胞向组织器官有组织的集体结构转变的典型例子，这一过程也引起了组织工程领域的极大兴趣。我们对这一发育阶段的大部分现有理解都是通过小鼠胚胎获得的。虽然这是一种强大的方法，但它可能是一种劳动密集型和昂贵的方法，而且出于伦理和实际原因，它在许多其他物种--包括人类--的应用受到限制。**在我提出的研究计划中，我将开发新的技术来概括这些体外过程，并利用它们来解决有关双椎板形成机制的未解问题。我推测，在多能干细胞的微流控培养中，双层盘的形成可以重现。在目标一中，我将建立这些文化体系。在目标二中，我将确定注定形成上胚层和次胚层的细胞彼此分离的机制。目前的建议侧重于使用小鼠多能干细胞，以便与现有的关于小鼠发育的大型数据集进行比较。在未来，我的研究计划将把这项研究扩展到模拟其他物种和胚胎发育的后续阶段。**我的研究计划将产生关于早期发育中低胚层形成过程的新知识；提供横跨微流体学、发育生物学、组织工程学和企业家精神的独特跨学科培训环境；并产生为科学界提供新研究能力的技术。我的AggreWell系统被成功地翻译到Stemcell Technologies Inc.，这是一家总部位于温哥华的加拿大公司，现在已经在世界各地提供了这项技术，这将成为我在这里开发的系统广泛使用的典范。**我感谢评审团对2012年秋季比赛中之前的一份提案进行了实质性审查，这使我在本次提交的作品中做出了重大改进：研究员的卓越(S)(斯特朗)：自从上一份申请以来，我在《自然通讯》上多了一位第二作者身份，作为通讯作者的一本书的章节，我实验室的第一本同行评议出版物正在出版中，学员GR是第一作者。我的H指数已从10攀升至11。建议的优点(中等)：按照建议，我将重点放在小鼠的发育上；包括充分利用该系统筛查潜力的实验；拟议的详细生物学研究以验证我的工作假设；将直接提供培训，而不是通过培训设施；并在预算理由中提供了有关材料和用品的更多细节。对于高素质人才的培训(中等)：我现在正在培训两名博士生(他们与人合著了一份报刊)，一名新来的理学硕士。还培训了三名本科生暑期学生(两名全职学生和一名联合督导)。

At the beginning of mammalian development, pluripotent cells expand, differentiate and self-organize, laying the foundations of the developing organism. A pivotal step is the formation of the bilaminar disc, with its two constituent layers the epiblast and the hypoblast. The epiblast is a pluripotent tissue which subsequently gives rise to the tissues and organs of the developing organism. The hypoblast contributes primarily to extraembryonic tissues, but plays a key role in axis formation and the differentiation of the epiblast. The molecular mechanisms that underlie this stage of embryogenesis are therefore of great importance to the field of developmental biology. As the quintessential example of the transformation from individual pluripotent cells to the organized collective structures of tissues and organs, this process is also of great interest to the field of tissue engineering. Much of our existing understanding of this stage of development has been obtained using mouse embryos. While a powerful approach, it can be labor intensive and expensive, and for both ethical and practical reasons its application to many other species - including humans - is limited.**In my proposed research program, I will develop new technologies to recapitulate these processes in vitro, and employ them to resolve unanswered questions about mechanisms of bilaminar disc formation. I hypothesize that the formation of the bilaminar disc can be recapitulated in microfluidic cultures of pluripotent stem cells. In Objective One, I will establish these culture systems. In Objective Two, I will determine the mechanisms by which the cells destined to form the epiblast and hypoblast segregate themselves from one another. The present proposal focuses on the use of mouse pluripotent stem cells, to allow comparison with the large existing data set on mouse development. In the future, my research program will extend this research to model other species and subsequent stages of embryogenesis.**My research program will result in new knowledge about the process of hypoblast formation in early development; provide a unique interdisciplinary training environment spanning microfluidics, developmental biology, tissue engineering and entrepreneurship; and generate technologies that provide new research capabilities to the scientific community. The successful translation of my AggreWell system to StemCell Technologies Inc, a Canadian company based in Vancouver which has now made the technology available around the world, will serve as the model for making the systems I develop here widely available.**I thank the panel for its substantive review of a previous proposal in the Fall 2012 competition, which allowed me to make significant improvements in the present submission: Excellence of the Researcher(s) (Strong): Since the previous application I have an additional second-authorship in Nature Communications, a book chapter as corresponding author, and the first peer-reviewed publication from my laboratory is in press with trainee GR as first author. My H-index has climbed from 10 to 11. For Merit of the Proposal (Moderate): As recommended, I have focussed the proposal specifically on mouse development; included experiments to take full advantage of the screening potential of this system; proposed detailed biological studies to test my working hypotheses; will provide training directly, rather than via training facilities; and have provided more details on the materials and supplies in the Budget Justification. For Training of Highly Qualified Personnel (Moderate): I am now training two Ph.D. students (who have co-authored an in-press publication), one newly arrived M.Sc. student, and also trained three undergraduate summer students (two full- and one co-supervised).