EAGER: Reprogramming to the Totipotent State

非技术段落将体细胞或分化细胞重新编程为类胚胎干细胞代表了干细胞生物学的一项重大技术突破。成纤维细胞中四种定义因子的过度表达表明，谱系承诺的细胞可以恢复到类似胚胎的基础状态。这些重新编程的细胞或诱导的多能干细胞(IPSCs)具有类似于自然发生的胚胎干细胞(ESCs)的属性；因此，它们具有在胚胎中产生所有组织类型和自我更新的非凡能力。然而，在受精和胚胎干细胞形成之间的一段狭窄的时间窗口内，存在着具有更大发育潜力的细胞。这些细胞被称为全能细胞，因为它们能够产生胚胎和非胚胎组织，如胎盘。目前，全能状态可以通过自然受精、克隆或对ESCs或IPSCs进行遗传操作来实现。这些方法受到低效率和许多尚未确定的分子障碍的阻碍。此外，全能细胞不能自我更新和繁殖，因此限制了大规模的应用。这笔急切的拨款将测试一种简单的单基因操作策略，将iPSCs或体细胞完全转化为具有自我更新能力的全能细胞。研究计划的成功将提供一种新的重新编程策略，为理解调节全能状态的分子体系和产生整个哺乳动物有机体的潜力铺平道路。利用这一新颖的技术创新，一年级和二年级的学生将从生物学入门课程中招募，学习干细胞生物学的基本技术。这一学生实践培训活动是更大战略的一部分，目的是最终提高少数族裔服务机构的留校率和毕业率，这些机构渴望获得一流的研究地位。全能细胞具有产生胚胎和胚胎外组织的非凡能力，如胎盘。因此，全能细胞是近4000种胎盘哺乳动物繁殖的决定性特征。在植入前胚胎发育期间，全能细胞在受精和胚泡内细胞团(ICM)形成之前短暂存在。除了自然受精，全能细胞还可以通过体细胞核移植(克隆)或通过消除ICM来源的胚胎干细胞(ESCs)或诱导多能干细胞(IPSCs)中的表观遗传调节因子来人工获得。然而，这些方法的效率相对较低，需要存在和/或消除各种因素，这些因素构成了全能状态的障碍。因此，利用确定的因素将多能干细胞或体细胞完全重新编程为全能状态的策略仍然是一个挑战。这笔急切的拨款解决了这一挑战，并将测试一种单基因操作策略，将iPSCs或体细胞完全重新编程为全能细胞。具体地说，通过这种方法获得的全能细胞将受到小鼠嵌合体试验的挑战。这种严格的方法将决定新获得的全能细胞是否具有同时生成胚胎和胚胎外组织的潜力。这项研究计划的成功完成将通过提供另一种可选的重新编程策略、洞察调节全能性和植入前胚胎发育的分子层次结构，从而深刻地影响科学界。作为这项拨款的更广泛影响的一部分，本科生将被招募到实验室从事干细胞生物学的基本方法。以一年级和二年级学生为目标是一项更大的战略的一部分，该战略旨在帮助争取一流研究地位的少数族裔服务机构保持和提高毕业率。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Non-Technical Paragraph Reprogramming somatic or differentiated cells into embryonic-like stem cells represents a major technical breakthrough in stem cell biology. Overexpression of four-defined factors in fibroblasts revealed that lineage-committed cells can revert to an embryonic-like ground state. These reprogrammed cells or induced pluripotent stem cells (iPSCs) have attributes similar to naturally-occurring embryonic stem cells (ESCs); and therefore, have the remarkable capacity to produce all tissue-types in the embryo and self-renew. However, cells with an even greater developmental potential exists during a narrow time window between fertilization and ESC formation. These cells are termed totipotent for their capacity to generate both embryonic and non-embryonic tissues, such as the placenta. Currently, the totipotent state can be achieved by natural fertilization, cloning, or genetic manipulation of ESCs or iPSCs. These approaches are impeded by low efficiency rates and a host of molecular barriers that have yet be identified. Furthermore, totipotent cells are unable to self-renew and propagate; thus, limiting large scale applications. This EAGER grant will test a simple, single-gene manipulation strategy to transform iPSCs or somatic cells entirely into totipotent cells that are capable of self-renewing. Success of the research plan will provide a novel reprogramming strategy that can pave the way to understand a molecular hierarchy regulating the totipotent state and the potential to generate the entire mammalian organism. Capitalizing on this novel technical innovation, freshmen and sophomore students will be recruited from introductory biology courses to learn basic techniques in stem cell biology. This hands-on-student training exercise is part of larger strategy to ultimately enhance retention and graduation rates at a minority-serving institution aspiring for tier-one research status. Technical Paragraph Totipotent cells have the remarkable capacity to generate both embryonic and extraembryonic tissues, such as the placenta. Totipotent cells are therefore a defining feature that contributed to the propagation of the nearly 4,000 placental mammals. During preimplantation embryonic development, totipotent cells are present transiently between fertilization and prior to formation of the inner cell mass (ICM) of the blastocyst. In addition to natural fertilization, totipotent cells can be acquired artificially by somatic nuclear transfer (cloning) or by eliminating epigenetic regulators in ICM-derived embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). However, the efficiency of these approaches is relatively low, and require the presence and/or elimination of a myriad of factors that establish barriers to the totipotent state. Strategies to fully-reprogram pluripotent stem or somatic cells to the totipotent state with defined factors therefore remain a challenge. This EAGER grant addresses this challenge, and will test a single-gene manipulation strategy that fully-reprograms iPSCs or somatic cells into totipotent cells. Specifically, totipotent cells acquired by this approach will be challenged with the mouse chimera assay. This rigorous approach will determine whether newly-acquired totipotent cells have the potential to generate both embryonic and extraembryonic tissues. Successful completion of this research program will profoundly impact the scientific community by providing an alternative reprogramming strategy, insight into a molecular hierarchy regulating totipotency and preimplantation embryonic development. As part of the Broader Impacts of this grant, undergraduate students will be recruited to the lab to engage in basic methodologies in stem cell biology. Targeting freshmen and sophomores is part of a larger strategy to help retain and enhance graduate rates at a minority-serving institution striving for tier-one research status.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.