Derivation and Characterization of Germ Cells from Embryonic Stem Cells

胚胎干细胞生殖细胞的衍生和表征

• 批准号: 8235017
• 负责人: Amander Clark
• 金额: $ 32.35万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-05 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8235017
• 关键词: Address; Affect; Age; Animal Model; Arginine; Back; Binding Sites; Biology; Cell Differentiation process; Cell Lineage; Cell surface; Cells; Child; Chromatin; Colony-Forming Units Assay; Complex; Congenital Abnormality; Defect; Derivation procedure; Development; Disease; Evaluation; Event; Fetal Development; Fetus; Foundations; Future; Gene Expression Profile; Gene-Modified; Genes; Genetic Programming; Genetic Transcription; Genetic screening method; Germ Cells; Germ Lines; Germ cell tumor; Goals; Gonadal structure; Health; Histone H4; Histones; Human; In Vitro; Infertility; Knock-out; Laboratories; Lead; Lentivirus Vector; Mediating; Methods; Methylation; Modeling; Modification; Molecular; Molecular Profiling; Mus; PRDM1 gene; Parents; Pathway interactions; Pattern; Population; Postdoctoral Fellow; Pregnancy; Protamines; Regulation; Reporting; Repression; Role; Signal Transduction; Somatic Cell; Staging; Stem cells; Structure of primordial sex cell; Testing; Time; Transcription Repressor/Corepressor; UC01; UC06; Undifferentiated; United States; Validation; Work; arginine methyltransferase; base; cancer type; chromatin immunoprecipitation; embryonic stem cell; fetal; gene repression; genome-wide; homologous recombination; human embryonic stem cell; human embryonic stem cell line; imprint; improved; in vivo; male; mouse model; mutant; promoter; reproductive; research study; response; stem cell differentiation; tool

DESCRIPTION (provided by applicant): The objective of this proposal is to generate germ cells from two federally approved lines of human embryonic stem cells (hESCs) called UC01 and UC06 (HSF-1 and HSF-6) in order to evaluate the role of transcriptional repression mediated by BLIMP1 and dimethylation of Histone H4 Arginine 3 (H4R3me2) on human germ cell formation. Understanding the molecular regulation of germ cell development is important to improving human health as abnormal germ cells can result in; infertility, which affects 10% of the reproductive age population in the United States, germ cell tumors, which are the most common cancer type to afflict males between the ages of 15 and 35, and birth defects, which occur in children born to parents with abnormal germ line development. In this proposal, we aim to evaluate whether defects in transcriptional repression during the initial stages of germ cell formation in the human fetus result in compromised germ cell development. The role of transcriptional repression will be evaluated in three Specific Aims. Specific Aim 1 involves determining the transcriptional signature of human germ cells from week 6-9 of human fetal gestation. This will be achieved by expression profiling germ cells isolated from human fetal gonads by FACS. Results from this Specific Aim will be used to further clarify the stage of germ cell formation acquired with hESC differentiation, to identify additional cell surface markers that can distinguish hESCs from germ cells, and as a foundation for evaluating the role of transcriptional repressors in germ cell development in vitro. In Specific Aim 2, we will evaluate the effect of a transcriptional repressor, BLIMP1 for a role in regulating human germ cell formation. It is known that BLIMP1 is essential for germ cell formation in murine models. In this Specific Aim, our goal is to knockdown and over express BLIMP1 in hESCs and assay human germ cell formation. This experiment will determine whether the yield of germ cells is reduced in genetically modified verses control ESCs. We will also FACS and isolate human germ cells from differentiating hESCs and evaluate transcription of germ cell specific genes, as well as genes associated with somatic cell differentiation. As a positive control and validation for the use of ESCs to study germ cell formation, we will compare our results to wild type and Blimp1 null mutant murine ESCs for their ability to form germ line in vitro. Finally in Specific Aim 3, the function of PRMT5 and Histone H4R3me2 in human germ cell derivation will be evaluated, and the binding sites of BLIMP1, PRMT5 and dimethylation of Histone H4R3 at promoters in ESCs and germ cells will be identified at a genome-wide level using Chromatin Immunoprecipitation (ChIP) followed by chip. PRMT5 is a protamine arginine methyltransferase that interacts with BLIMP1 to mediate dimethylation of histone H4R3 at loci that need to be repressed in order for germ cell formation to occur. Genes with promoters that share BLIMP1, PRMT5 and Histone H4R3me2 will be analyzed in future proposals. This proposal constitutes the first step from which additional downstream targets essential for normal human germ cell development can be evaluated. PUBLIC HEALTH RELEVANCE: Results from this proposal will be important for unraveling molecular mechanisms that lead specifically to human infertility.

描述（由申请人提供）：本提案的目的是从联邦批准的两个人类胚胎干细胞 (hESC) 系 UC01 和 UC06（HSF-1 和 HSF-6）中生成生殖细胞，以评估生殖细胞的作用BLIMP1 介导的转录抑制和组蛋白 H4 精氨酸 3 (H4R3me2) 的二甲基化对人类生殖细胞形成的影响。了解生殖细胞发育的分子调控对于改善人类健康非常重要，因为异常的生殖细胞可能导致；不孕症（影响美国 10% 的育龄人口）、生殖细胞肿瘤（影响 15 岁至 35 岁男性的最常见癌症类型）以及出生缺陷（父母患有此类疾病的孩子所生的孩子）种系发育异常。在本提案中，我们的目的是评估人类胎儿生殖细胞形成初始阶段的转录抑制缺陷是否会导致生殖细胞发育受损。转录抑制的作用将在三个具体目标中进行评估。具体目标 1 涉及确定人类胎儿妊娠第 6-9 周的人类生殖细胞的转录特征。这将通过 FACS 对从人类胎儿性腺分离的生殖细胞进行表达谱分析来实现。该特定目标的结果将用于进一步阐明 hESC 分化获得的生殖细胞形成阶段，鉴定可区分 hESC 与生殖细胞的其他细胞表面标记，并作为评估生殖细胞中转录抑制因子的作用的基础体外发育。在具体目标 2 中，我们将评估转录抑制因子 BLIMP1 在调节人类生殖细胞形成中的作用。众所周知，BLIMP1 对于小鼠模型中生殖细胞的形成至关重要。在此具体目标中，我们的目标是在 hESC 中敲低并过度表达 BLIMP1，并分析人类生殖细胞的形成。该实验将确定转基因胚胎干细胞相对于对照胚胎干细胞的生殖细胞产量是否有所降低。我们还将进行 FACS 并从分化的 hESC 中分离人类生殖细胞，并评估生殖细胞特定基因以及与体细胞分化相关的基因的转录。作为使用 ESC 研究生殖细胞形成的阳性对照和验证，我们将我们的结果与野生型和 Blimp1 缺失突变鼠 ESC 进行体外形成生殖细胞系的能力进行比较。最后，在具体目标 3 中，将评估 PRMT5 和组蛋白 H4R3me2 在人类生殖细胞衍生中的功能，并将在全基因组范围内鉴定 ESC 和生殖细胞启动子处的 BLIMP1、PRMT5 和组蛋白 H4R3 二甲基化的结合位点。使用染色质免疫沉淀 (ChIP) 和芯片进行水平分析。 PRMT5 是一种鱼精蛋白精氨酸甲基转移酶，它与 BLIMP1 相互作用，介导组蛋白 H4R3 在需要被抑制以发生生殖细胞形成的位点上的二甲基化。具有共享 BLIMP1、PRMT5 和组蛋白 H4R3me2 的启动子的基因将在未来的提案中进行分析。该提案构成了评估正常人类生殖细胞发育所必需的其他下游靶点的第一步。公共健康相关性：该提案的结果对于揭示导致人类不育的分子机制非常重要。