Derivation and Characterization of Germ Cells from Embryonic Stem Cells

描述（由申请人提供）：本提案的目的是从两种联邦批准的称为UC 01和UC 06（HSF-1和HSF-6）的人胚胎干细胞（hESC）系中产生生殖细胞，以评价BLIMP 1介导的转录抑制和组蛋白H4精氨酸3（H4 R3 me 2）二甲基化对人生殖细胞形成的作用。了解生殖细胞发育的分子调控对改善人类健康很重要，因为异常的生殖细胞可能导致：影响美国10%的育龄人口的不育症、折磨15至35岁男性的最常见癌症类型生殖细胞肿瘤、以及发生在具有异常生殖系发育的父母所生子女中的出生缺陷。在这个建议中，我们的目的是评估是否在人类胎儿生殖细胞形成的初始阶段的转录抑制缺陷导致生殖细胞发育受损。转录抑制的作用将在三个特定目的中进行评估。具体目标1涉及确定人胚胎妊娠第6-9周的人生殖细胞的转录特征。这将通过FACS分析从人胎儿性腺中分离的生殖细胞的表达谱来实现。从这个特定的目的的结果将被用来进一步澄清生殖细胞形成的阶段获得的hESC分化，以确定额外的细胞表面标志物，可以区分hESC从生殖细胞，并作为一个基础，在体外生殖细胞发育中的转录抑制因子的作用进行评估。在具体目标2中，我们将评估转录抑制因子BLIMP 1在调节人类生殖细胞形成中的作用。已知BLIMP 1对于鼠模型中的生殖细胞形成是必需的。在这个特定的目标中，我们的目标是在hESC中敲低和过表达BLIMP 1，并测定人生殖细胞的形成。该实验将确定生殖细胞的产量是否在遗传修饰的ESC中相对于对照ESC降低。我们还将流式细胞仪和分离人生殖细胞分化的hESC和评估生殖细胞特异性基因的转录，以及与体细胞分化相关的基因。作为使用ESC研究生殖细胞形成的阳性对照和验证，我们将比较我们的结果与野生型和Blimp 1无效突变小鼠ESC在体外形成生殖系的能力。最后，在特定目标3中，将评价PRMT 5和组蛋白H4 R3 me 2在人生殖细胞衍生中的功能，并将使用染色质免疫沉淀（ChIP）和芯片在全基因组水平上鉴定BLIMP 1、PRMT 5和组蛋白H4 R3在ESC和生殖细胞中启动子处的二甲基化的结合位点。PRMT 5是鱼精蛋白精氨酸甲基转移酶，其与BLIMP 1相互作用以介导组蛋白H4 R3在需要被抑制以发生生殖细胞形成的位点处的二甲基化。具有BLIMP 1、PRMT 5和组蛋白H4 R3 me 2共同启动子的基因将在未来的提案中进行分析。这一提议构成了第一步，从中可以评估正常人类生殖细胞发育所必需的其他下游靶标。公共卫生相关性：这项提案的结果对于解开导致人类不育的分子机制非常重要。

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.