PROJECT 1: Germ Cell Differentiation from Human iPSCs and hEScs

A.意义 A1.不孕从历史上看，不孕夫妇的生活质量已经大大降低了损失的， 有机会怀孕。然而，近年来，新的临床干预措施，如胞浆内精子， 注射大大改变了一些夫妇的前景，特别是那些严重的形式， 不孕症[23]。在临床成功的同时，也有突破性的科学进步 包括人类基因组的测序，人类胚胎干细胞（hESC）系的衍生，以及 将成人体细胞重编程为多能性[24-28]。总之，这些进步现在使我们能够 克服人类发展研究中两个历史上不可逾越的局限性： 早期人类发展到探索和基因组在发展过程中的遗传困难。 A2.需要研究人类生殖细胞的发育。10-15%的夫妇是不育的，但很少有人知道 生殖细胞生成不良的男性和女性的潜在病理。在此，我们建议扩大我们的 为了允许对人类生殖细胞发育进行遗传分析， 基础和临床研究及应用。人类生殖细胞发育有几个独特的方面 值得我们投资首先，人类生殖细胞发育所需的基因和剂量不同于 小鼠的那些，包括常染色体和性染色体基因和剂量[6，29 -34]。第二，人类是 在物种中罕见的是，不育是常见的，一半的病例与生殖细胞发育缺陷有关[35]。 此外，相对于其他物种，与减数分裂错误相关的病理在人类中很多， 减数分裂染色体分离错误发生在多达5-30%的人类生殖细胞中，这取决于 年龄与性别[36]这与酵母中的1/10000细胞、苍蝇中的1/1000细胞和 对小鼠随着最近的进展，我们现在有能力纳入新的战略，以检查 人类生殖细胞发育的细节。这将使我们能够充分受益于以下方面的丰富数据： 果蝇和小鼠等模型系统，从而开始了解人类生殖细胞的复杂遗传学 细胞形成和分化。在寻求了解生殖细胞生物学，我们也承认， 对生殖系有贡献的能力是区分多能干细胞的基本特性。为 例如，Han和他的同事最近证明，通过在常用的4个因子上增加第5个因子， 因子混合物的重编程，有助于生殖系的能力显着增加[37]。这是在 尽管事实上来源于5因子重编程的IPSC与标准的IPSC没有区别， 或mESC的基因表达和多能性标记。因此，这里提出的研究使我们能够 解决有关我们的生殖系起源，功能和病理学的基本问题，并奠定了 为设计合理的治疗和诊断奠定基础，以根据数据做出临床决策 从模式生物、人类遗传学研究和人类生殖细胞的直接实验分析中获得。 它还有助于多能干细胞生物学和再生医学的相关领域。

A. Significance A1. Infertility. Historically, the quality of life of infertile couples has been greatly diminished by the loss of opportunity to conceive. However, in recent years, novel clinical interventions such as intracytoplasmic sperm injection have dramatically changed the outlook for some couples, particularly those with severe forms of infertility[23]. In parallel with clinical successes, there have also been ground-breaking scientific advances including sequencing of the human genome, derivation of human embryonic stem cell (hESC) lines, and reprogramming of adult human somatic cells to pluripotency[24-28]. Together, these advances now allow us to overcome two historically-insurmountable limitations in studies of human development: the Inaccessibility of early human development to exploration and the genetic-intractability of the genome during development. A2. Need to study human germ cell development. 10-15% of couples are infertile, yet little is known of underlying pathologies in men and women with poor germ cell production. Here, we propose to extend our previous studies in order to permit genetic analysis of human germ cell development and thus, enable novel basic and clinical studies and applications. There are several unique aspects to human germ cell development that merit this investment. First, genes and dosages required for human germ cell development differ from those of mice, including both autosomal and sex chromosomal genes and dosages[6,29-34]. Second, humans are rare among species in that infertility is common, with half of all cases linked to faulty germ cell development[35]. Moreover, pathologies associated with meiotic errors are numerous in humans relative to other species, with errors in meiotic chromosome segregation occurring in as many as 5-30% of human germ cells depending on sex and age[36]. This is in contrast to frequencies of 1/10000 cells in yeast, 1/1000 cells in flies, and 1/100 cells in mice. With recent advances, we now have the ability to incorporate new strategies in order to examine the specifics of human germ cell development. This will allow us to derive full benefit from the wealth of data from model systems such as the fly and the mouse, to begin to understand the complex genetics of human germ cell formation and differentiation. In seeking to understand germ cell biology, we also acknowledge that the ability to contribute to the germ line is a fundamental property that distinguishes pluripotent stem cells. For example, Han and colleagues recently demonstrated that by addition of a 5th factor to the commonly-used 4 factor mixture for reprogramming, ability to contribute to the germ line was significantly increased[37]. This was in spite of the fact that IPSCs derived from 5 factor-reprogramming were indistinguishable from standard iPSCs or mESCs in gene expression and markers of pluripotency. Thus, the research proposed here allows us to address fundamental questions regarding our germ line origins, function, and pathology and lays the groundwork for designing rational therapeutics and diagnostics to inform clinical decisions based on data obtained from model organisms, human genetic studies and direct experimental analysis of human germ cells. It also contributes to the related field of pluripotent stem cell biology and regenerative medicine.