In vitro modelling of pregastrulation embryogenesis using pluripotent stem cells

在哺乳动物发育之初，多能细胞扩张、分化和自我组织，为发育中的生物体奠定基础。一个关键的步骤是形成双层盘，其两个组成层的上胚层和下胚层。外胚层是一种多能组织，随后产生发育生物体的组织和器官。下胚层主要参与胚外组织的形成，但在轴的形成和上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非常重要。作为从个体多能细胞转化为组织和器官的有组织的集体结构的典型例子，该过程也是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认识都是使用小鼠胚胎获得的。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，它对许多其他物种（包括人类）的应用是有限的。在我提议的研究计划中，我将开发新的技术来在体外重现这些过程，并利用它们来解决有关双层椎间盘形成机制的未回答的问题。我推测，形成的双层光盘可以概括在微流体培养的多能干细胞。在目标一中，我将建立这些文化体系。在第二个目标中，我将确定形成上胚层和下胚层的细胞相互分离的机制。目前的建议侧重于使用小鼠多能干细胞，以便与现有的大量小鼠发育数据集进行比较。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。我的研究计划将导致有关早期发育中下胚层形成过程的新知识;提供一个独特的跨学科培训环境，涵盖微流体，发育生物学，组织工程和创业精神;并产生为科学界提供新研究能力的技术。我的AggreWell系统成功地被移植给了StemCell Technologies Inc，这是一家总部位于温哥华的加拿大公司，它现在已经将这项技术推广到世界各地，这将成为我在这里开发的系统广泛使用的典范。**我感谢专家组对2012年秋季竞赛中的一项提案进行了实质性的审查，这使我能够在目前的提交中做出重大改进：研究人员的卓越（强）：自从上一次申请以来，我在《自然通讯》中获得了额外的第二作者身份，一本书的章节是通讯作者，我实验室的第一份同行评议的出版物正在出版，实习生GR是第一作者。我的H指数从10上升到11。建议的优点（中等）：根据建议，我将建议的重点放在小鼠开发上;包括充分利用该系统筛选潜力的实验;建议进行详细的生物学研究，以测试我的工作假设;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有关材料和供应品的更多详细信息。对于高素质人才的培养（中等）：我现在培养两个博士。学生（谁共同撰写了新闻出版物），一个新来的硕士学生，还培训了三个本科暑期学生（两个全职和一个共同监督）。

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).