PROJECT 1: Germ Cell Differentiation from Human iPSCs and hEScs

A.意义 A1.不孕不育。从历史上看，不育夫妇的生活质量因失去 怀孕的机会。然而，近年来，新的临床干预措施，如胞浆内精子 注射极大地改变了一些夫妇的前景，特别是那些患有严重形式的 不孕症[23]。在取得临床成功的同时，也取得了突破性的科学进步 包括人类基因组测序，人类胚胎干细胞(HESC)系的获得，以及 将成人体细胞重新编程为多能细胞[24-28]。总而言之，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： 从早期人类发育到探索，再到基因组在发育过程中的遗传难题。 A2.需要研究人类生殖细胞的发育。10%-15%的夫妇是不孕不育的，但人们对此知之甚少 生殖细胞产量低的男性和女性的潜在病理。在此，我们建议延长我们的 以前的研究是为了能够对人类生殖细胞发育进行遗传分析，从而使新的 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 值得这笔投资的东西。首先，人类生殖细胞发育所需的基因和剂量与 小鼠的，包括常染色体和性染色体基因和剂量[6，29-34]。第二，人类是 不孕不育在物种中很少见，所有病例中有一半与生殖细胞发育缺陷有关[35]。 此外，与其他物种相比，与减数分裂错误相关的病理现象在人类中也很多， 多达5%-30%的人类生殖细胞出现减数分裂染色体分离错误，这取决于 性别和年龄[36]。这与酵母中1/10000的细胞、果蝇中1/10000的细胞和1/100的细胞的频率形成了鲜明对比 在老鼠身上。随着最近的进展，我们现在有能力纳入新的战略，以便研究 人类生殖细胞发育的具体情况。这将使我们能够从丰富的数据中充分受益 建立苍蝇和老鼠等系统的模型，以开始了解人类细菌的复杂遗传学 细胞的形成和分化。在寻求了解生殖细胞生物学的过程中，我们也承认 对生殖系作出贡献的能力是区分多能干细胞的基本属性。为 例如，韩和他的同事最近证明了，通过在常用的4个因子的基础上增加5个因子 对于重新编程的混合因子，对生殖系的贡献能力显著增加[37]。这是最流行的 尽管来自5因素重编程的IPSCs与标准IPSCs没有区别 或mESCs在基因表达和多能性标记上的作用。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、功能和病理的基本问题，并提出 根据数据设计合理的治疗和诊断方法以指导临床决策的基础工作 从模型生物、人类遗传学研究和人类生殖细胞的直接实验分析中获得。 它还为多能干细胞生物学和再生医学的相关领域做出了贡献。

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.