Defining factors associated with the conversion of primordial germ cells into plu

描述（由申请人提供）：多能干细胞，如胚胎干细胞（ESCs）和胚胎生殖细胞（EGCs）具有无限自我更新的独特能力，并且能够产生人体中存在的大多数（如果不是全部）细胞类型。鉴于这些特性，只有少数因素控制其生长为未分化细胞已被确定。事实上，已经报道了许多尝试通过在各种ESC系和其他细胞类型之间进行转录组比较来阐明涉及的机制。这些研究为这一建议提供了依据。然而，本项目提出了一个新的模型，通过研究参与原始生殖细胞（PGCs）向EGCs转化的基因来识别多能性的潜在因素。该模型的独特之处在于，与ESCs不同，EGCs的特定细胞类型起源是已知的，这些细胞，PGCs可以被分离出来进行进一步的研究。具体来说，在Aim 1中，基因组比较将在从细胞培养中分离的人类PGCs转化为多能性EGCs的顺序时间点进行，而Aim 2将研究PGCs与未分化的EGCs和ESCs （WA01）以及正在分化的PGCs的基因组变化。来自PGCs的数据也将与馈线子线之间的数据进行比较，馈线子线仅通过其导出EGCs的功能能力来区分。这种比较将通过关注PGCs和饲养细胞之间的互补系统来帮助确定候选基因的优先级，从而为鉴定感兴趣的基因提供额外的信息。使用该模型，将使用以下优先顺序选择候选基因：(1)跨膜蛋白，这些跨膜蛋白补充从小鼠胚胎喂养层中鉴定的支持EGC衍生的配体；(2)与其他物种多能性相关的基因；(3)含有可能对已知多能因子（如Oct4、Nanog或Sox2）有反应的序列元件；(4)最后，多能性或多能性细胞的相关通路。选择的候选表达将使用标准定量核酸和蛋白质分析进行验证。在目标3中，除了使用生长因子及其抑制剂进行细胞培养操作外，还将通过条件敲入或敲低基因方法来发挥功能作用。重要的是，这一建议证明了获得足够数量的人类早期生殖细胞和获得生物学相关数据的可行性。这些数据也可能表明EGCs和ESCs有共同的起源。此外，PGC-EGC转化模型提供了有效的体外功能分析，以测试候选因子，这些因子将优化EGCs的培养，并提供有关pgc向EGCs多能转化的基因的见解，这将有助于填补我们对多能干细胞衍生和维持的理解空白。公共卫生相关性：多能干细胞具有无限自我更新的独特能力，并能产生人体中存在的大多数（如果不是全部的话）细胞类型，因此在治疗人类疾病方面具有巨大的前景。然而，尽管它们在基于细胞的治疗中有潜在的应用，但对于它们作为未分化细胞的生长调节知之甚少-这是最大化胚胎和成体干细胞利用的关键问题。为此，下面的研究将采用探索性的方法，对多能干细胞和单能祖细胞群体进行基因组比较，以帮助确定多能干细胞向多能状态发展过程中的改变机制。

DESCRIPTION (provided by applicant): Pluripotent stem cells such as embryonic stem cells (ESCs) and embryonic germ cells (EGCs) have the unique abilities to both self renew indefinitely as well as being able to give rise to most, if not all cell types present in the human body. Given these properties, only a few factors that control their growth as undifferentiated cells have been identified. Indeed, many attempts have been reported to elucidate the mechanisms involved using transcriptome comparisons among various ESC lines and other cell types. These studies provide the basis for this proposal. However, this project proposes a novel model to identify potential factors in pluripotency by studying the genes involved in the conversion of primordial germ cells (PGCs) into EGCs. This model is unique in that unlike ESCs, the specific cell type of origin of EGCs is known and that these cells, PGCs can be isolated for further study. Specifically, in Aim 1 genomic comparisons will be performed among human PGCs isolated from cell culture at sequential time points in their conversion to pluripotent EGCs while Aim 2 will study genomic alterations in PGCs versus undifferentiated EGCs and ESCs (WA01) and those undergoing differentiation. Data from PGCs will also be compared to those made between feeder sublines which are distinguished only by their functional ability to derive EGCs. This comparison will provide additional information for identifying genes of interest by focusing on complementary systems between PGCs and feeder cells to help prioritize candidate genes. Using this model, candidate genes will be selected using the following prioritization (1) transmembrane proteins that complement ligands identified from mouse embryonic feeder layers which support EGC derivation; (2) genes associated with pluripotency in other species; (3) those containing sequence elements that may be responsive to known pluripotent factors such as Oct4, Nanog, or Sox2; and (4) finally, pathways shown to be relevant in pluripotent or multipotent cells. Expression of selected candidates will be validated using standard quantitative nucleic acid and protein analyses. In Aim 3, functional roles will then be performed by conditional knock-in or knock-down gene approaches in addition to cell culture manipulations using growth factors and their inhibitors. Importantly, this proposal demonstrates the feasibility of obtaining early germ cells of human origin in sufficient quantities and to obtain biologically relevant data. Data which may also suggest a common origin for EGCs and ESCs. Furthermore, the PGC-EGC conversion model provides for efficient in vitro functional assays to test candidate factors which will optimize the culturing of EGCs and provide insight into genes involved in the pluripotential conversion of PGCs to EGCs that will help fill gaps in our understanding of pluripotent stem cell derivation and maintenance. PUBLIC HEALTH RELEVANCE: With their unique abilities of unlimited self-renewal and to give rise to most, if not all cell types present in the human body, pluripotent stem cells have enormous promise for the treatment of human disease. However, despite demonstrations of their potential use in cell-based therapies, little is known regarding the regulation of their growth as undifferentiated cells -an issue critical for maximizing embryonic and adult stem cell utilization. For this purpose, the following study will use an exploratory approach involving genomic comparisons among pluripotent stem cells and an unipotent progenitor population to help identify mechanisms altered in the their progression toward the pluripotent state.