In vitro modelling of pregastrulation embryogenesis using pluripotent stem cells

在哺乳动物发育之初，多能细胞增殖、分化和自组织，为生物发育奠定了基础。一个关键的步骤是双层椎间盘的形成，它有两个组成层：外胚层和下胚层。外胚层是一种多能性组织，随后产生发育中的生物体的组织和器官。下胚层主要形成胚外组织，但在轴的形成和外胚层的分化中起关键作用。因此，胚胎发生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和器官有组织的集体结构转化的典型例子，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现有认识大多是通过小鼠胚胎获得的。虽然这是一种强大的方法，但它可能是劳动密集型和昂贵的，并且出于道德和实际原因，它在许多其他物种（包括人类）上的应用是有限的。**在我提出的研究计划中，我将开发新的技术来体外重现这些过程，并利用它们来解决关于双板椎间盘形成机制的未解之谜。我假设双层椎间盘的形成可以在多能干细胞的微流体培养中重现。在目标一中，我将建立这些文化体系。在目标二中，我将确定注定形成外胚层和下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，以便与小鼠发育的大量现有数据集进行比较。在未来，我的研究计划将扩展这项研究，以模拟其他物种和胚胎发生的后续阶段。**我的研究项目将对发育早期的次爆层形成过程产生新的认识；提供独特的跨学科培训环境，涵盖微流体，发育生物学，组织工程和创业；并产生为科学界提供新的研究能力的技术。我的AggreWell系统成功地翻译给了一家总部位于温哥华的加拿大公司StemCell Technologies Inc .，该公司现在已将该技术推广到世界各地，这将成为我在这里开发的系统广泛应用的典范。**我感谢评审小组在2012年秋季竞赛中对之前的提案进行了实质性的审查，这使我在目前的提交中做出了重大改进：卓越的研究员（Strong）：自上次申请以来，我在《自然通讯》上有了额外的第二作者，作为通讯作者的一本书章节，并且我实验室的第一篇同行评议出版物正在出版，实习生GR作为第一作者。我的h指数从10上升到了11。提案优点（中等）：根据建议，我将提案特别集中在鼠标开发上；纳入实验，充分利用该系统的筛选潜力；提出详细的生物学研究来验证我的工作假设；将直接提供培训，而不是通过培训设施；并在预算说明中提供了更多关于材料和用品的细节。培养高层次人才（中等）：我现在正在培养两名博士研究生（他们已经共同发表了一篇论文），一名新到的硕士研究生，还培养了三名本科生暑期生（两名全职和一名共同指导）。

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).