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

与原始生殖细胞转化为 plu 相关的定义因素

• 批准号: 7532430
• 负责人: Candace Lynn Kerr
• 金额: $ 24.6万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7532430
• 关键词: Activities of Daily Living; Animals; Biological Assay; Candidate Disease Gene; Cell Maintenance; Cell Proliferation; Cell Therapy; Cells; Characteristics; Cultured Cells; Data; Derivation procedure; Development; ES Cell Line; Elements; Embryo; Experimental Models; Gene Expression; Gene Expression Profile; Genes; Genomics; Germ Cells; Growth; Growth Factor; Human; Human body; In Vitro; Integral Membrane Protein; Knock-in Mouse; Laboratories; Lentivirus Vector; Ligands; Maintenance; Microarray Analysis; Modeling; Molecular Profiling; Mus; Nature; Nucleic Acids; Pathway interactions; Pluripotent Stem Cells; Population; Process; Property; Proteins; Public Health; Purpose; RNA Interference; Regulation; Reporting; Research Project Grants; Role; Standards of Weights and Measures; Stem cells; Structure of primordial sex cell; System; Testing; Time; Undifferentiated; WA01 cell line; adult stem cell; base; cell type; embryonic stem cell; human disease; inhibitor/antagonist; insight; interest; knock-down; multipotent cell; novel; novel strategies; pluripotency; progenitor; receptor; self-renewal

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.

描述(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)，具有无限期自我更新的独特能力，并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，只有少数几个因素控制了它们作为未分化细胞的生长。事实上，据报道，已经有许多尝试通过在不同的ESC系和其他细胞类型之间进行转录组比较来阐明所涉及的机制。这些研究为这项建议提供了基础。然而，该项目提出了一个新的模型，通过研究原始生殖细胞(PGCs)转化为EGCs的相关基因来确定潜在的多能性因素。这个模型的独特之处在于，与ESCs不同，EGCs起源的特定细胞类型是已知的，这些细胞即PGCs可以被分离出来进行进一步研究。具体地说，在目标1中，将在从细胞培养中分离出的人PGCs转化为多能EGCs的连续时间点之间进行基因组比较，而目标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的数据也将与馈线亚线之间的数据进行比较，后者仅通过其推导EGC的功能能力来区分。这种比较将通过关注原生殖细胞和饲养层细胞之间的互补系统来帮助确定候选基因的优先顺序，从而为识别感兴趣的基因提供额外的信息。利用这个模型，候选基因的选择将使用以下优先顺序：(1)补充从支持EGC派生的小鼠胚胎饲养层中鉴定的配体的跨膜蛋白；(2)在其他物种中与多能性相关的基因；(3)那些包含可能对已知的多能性因子(如Oct4、Nanog或Sox2)做出反应的序列元件；以及(4)最后，被证明在多能性或多能性细胞中相关的通路。将使用标准的定量核酸和蛋白质分析来验证选定候选基因的表达。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，还将通过有条件的敲入或敲除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并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有共同的来源。此外，PGC-EGC转换模型提供了有效的体外功能分析来测试候选因素，这些候选因素将优化EGCs的培养，并提供对PGCs向EGCs多潜能转化所涉及的基因的洞察，这将有助于填补我们对多潜能干细胞来源和维护的理解空白。与公共卫生相关：多能干细胞具有无限自我更新的独特能力，能够产生人体内存在的大多数(如果不是全部)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的潜在用途得到了证明，但人们对它们作为未分化细胞的生长调控知之甚少--这是最大限度地利用胚胎和成人干细胞的关键问题。为此，下面的研究将使用一种探索性的方法，包括在多能干细胞和单能祖细胞群体之间进行基因组比较，以帮助确定它们向多能状态发展过程中发生变化的机制。