Derivation and Characterization of Germ Cells from Embryonic Stem Cells

描述（由申请人提供）：该提案的目的是从两个联邦批准的人胚胎干细胞（hESCs），称为UC01和UC06 （HSF-1和HSF-6）中产生生殖细胞，以评估BLIMP1介导的转录抑制和组蛋白H4精氨酸3 （H4R3me2）二甲基化在人生殖细胞形成中的作用。了解生殖细胞发育的分子调控对改善人类健康非常重要，因为生殖细胞异常可能导致；不孕不育，影响了美国10%的育龄人口；生殖细胞肿瘤，这是折磨15至35岁男性的最常见的癌症类型；出生缺陷，这发生在生殖系发育异常的父母所生的孩子身上。在这项提议中，我们的目的是评估在人类胎儿生殖细胞形成的初始阶段转录抑制的缺陷是否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。特异性目的1涉及确定人类胚胎妊娠6-9周人类生殖细胞的转录特征。这将通过FACS从人胎儿性腺分离的生殖细胞表达谱来实现。这一特异性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，鉴定能够区分hESC和生殖细胞的其他细胞表面标记物，并作为评估转录抑制因子在体外生殖细胞发育中的作用的基础。在特异性目标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，在小鼠模型中，BLIMP1对生殖细胞的形成至关重要。在这个特定的目标中，我们的目标是在hESCs中敲低和过表达BLIMP1，并检测人类生殖细胞的形成。本实验将确定转基因胚胎干细胞与对照胚胎干细胞的生殖细胞产量是否减少。我们还将从分化的hESCs中分离和FACS人类生殖细胞，并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ESCs研究生殖细胞形成的阳性对照和验证，我们将把我们的结果与野生型和Blimp1零突变小鼠ESCs在体外形成生殖系的能力进行比较。最后，在Specific Aim 3中，我们将评估PRMT5和Histone H4R3me2在人类生殖细胞衍生中的功能，并利用染色质免疫沉淀（Chromatin Immunoprecipitation, ChIP）和芯片技术在全基因组水平上鉴定ESCs和生殖细胞启动子处的BLIMP1、PRMT5和Histone H4R3二甲基化的结合位点。PRMT5是一种鱼精蛋白精氨酸甲基转移酶，它与BLIMP1相互作用，在生殖细胞形成过程中需要抑制的位点介导组蛋白H4R3的二甲基化。启动子共享BLIMP1、PRMT5和Histone H4R3me2的基因将在未来的提案中进行分析。这一建议构成了可以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。公共卫生相关性：这项建议的结果对于揭示导致人类不孕症的分子机制将是重要的。

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.