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（ESC）和胚胎生殖细胞（EGCs）具有无限自我更新以及能够产生人体中存在的大多数（如果不是所有）细胞类型的独特能力。鉴于这些特性，只有少数几个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来阐明各种ESC系和其他细胞类型之间使用转录组比较所涉及的机制。这些研究为这一提议提供了依据。然而，该项目提出了一种新的模型，通过研究参与原始生殖细胞（PGCs）转化为EGCs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ESC不同，EGCs起源的特定细胞类型是已知的，并且这些细胞，PGCs可以分离用于进一步研究。具体而言，在Aim 1中，将在从细胞培养物中分离的人PGC转化为多能EGCs的连续时间点进行基因组比较，而Aim 2将研究PGC与未分化EGCs和ESC（WA 01）以及经历分化的那些中的基因组改变。还将来自PGCs的数据与仅通过其衍生EGCs的功能能力区分的饲养细胞亚系之间的数据进行比较。这种比较将通过关注PGC和饲养细胞之间的互补系统来帮助优先考虑候选基因，从而为鉴定感兴趣的基因提供额外的信息。使用该模型，将使用以下优先级选择候选基因：（1）与从支持EGC衍生的小鼠胚胎饲养层鉴定的配体互补的跨膜蛋白;（2）与其它物种中的多能性相关的基因;（3）含有可能响应于已知多能性因子如Oct 4、Nanog或Sox 2的序列元件的那些基因;以及（4）最后，显示在多能或多能细胞中相关的途径。将使用标准定量核酸和蛋白质分析验证所选候选物的表达。在目标3中，除了使用生长因子及其抑制剂的细胞培养操作之外，还将通过条件性敲入或敲低基因方法来执行功能性作用。重要的是，这一提议证明了获得足够数量的人类起源的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ESC的共同起源。此外，PGC-EGC转化模型提供了有效的体外功能测定以测试候选因子，这将优化EGC的培养并提供对参与PGC向EGC的多能转化的基因的了解，这将有助于填补我们对多能干细胞衍生和维持的理解中的空白。公共卫生相关性：多能干细胞具有无限自我更新和产生人体中存在的大多数（如果不是所有）细胞类型的独特能力，因此在治疗人类疾病方面具有巨大的前景。然而，尽管证明了它们在基于细胞的疗法中的潜在用途，但关于它们作为未分化细胞的生长的调节知之甚少-这是最大化胚胎和成体干细胞利用的关键问题。为此，以下研究将使用一种探索性方法，涉及多能干细胞和单能祖细胞群体之间的基因组比较，以帮助确定其向多能状态发展的机制。

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.