Human iPSC-derived ovarian follicles as a model of female reproduction

项目总结 女性生殖的中心过程是在发育中的卵巢内形成卵母细胞，称为 卵子发生。这一过程对健康卵母细胞的形成和遗传和基因的正确传播至关重要。 表观遗传信息开始胚胎发育。卵巢发育和卵子发生的异常 是女性不孕不育和性发育障碍的主要原因，而且是 许多发育障碍的后代，如唐氏综合症和安杰曼综合征。 然而，人们对人类卵巢发育的基因调控知之甚少。这是在 与其他生物不同，如小鼠，转基因和基因敲除系，以及短的 生殖周期，使得这一领域有了许多研究。人卵巢体外器官模型的建立 发展将有助于填补这一空白，并使人们能够更好地了解人类卵巢 可能导致治疗不孕不育和预防发育障碍的发展。 卵巢发育涉及原始生殖细胞(PGCs)和体细胞(颗粒细胞)之间的相互作用 单元格)。颗粒细胞将PGC包裹在卵泡内，并支持它们分化为 卵原细胞，减数分裂过程中的进展，以及卵母细胞的发育。因此，这两种血统都是必需的 在体外模拟这一过程。现有的方法允许诱导多能干细胞(IPSCs)分化 转化为PGC样细胞，但这些细胞处于未成熟状态，保留了IPSCs的表观遗传学特征。为 卵子发生的体外模型要想成功，必须开发出改进的方法来产生成熟卵子 IPSCs的生殖系细胞和颗粒细胞。 通过表达转录因子(TF)对细胞身份进行重新编程是一种强大的技术，可以 允许将体细胞重新编程为IPSCs，并将IPSCs定向分化为特定细胞 类型。丘奇实验室最近开发了计算工具来预测指定细胞身份的TF，如 以及用于组合Tf表达的筛选方法，以找到能够将IPSCs区分为细胞的集合 兴趣类型。目前建议的研究将确定能够促进PGC成熟的TF- 像细胞一样，并从IPSCs产生颗粒细胞。结果将由单个单元格评估 转录学和表观遗传学分析，以及关键表型的功能验证。这项研究将 更好地理解卵巢发育的基因调控，导致体外培养 人类卵子发生的模型。

PROJECT SUMMARY The central process of female reproduction is the formation of oocytes within the developing ovary, known as oogenesis. This process is crucial for the formation of healthy oocytes and proper transmission of genetic and epigenetic information to begin embryonic development. Abnormalities in ovarian development and oogenesis are a leading cause of female infertility and disorders of sexual development, and furthermore are the cause of many developmental disorders in the subsequent generation, such as Down syndrome and Angelman syndrome. However, relatively little is known about the genetic regulation of human ovarian development. This is in contrast to other organisms such as the mouse, where transgenic and knockout lines, and a short reproductive cycle, have allowed much research in this area. An in vitro organoid model of human ovarian development would help fill this gap, and enable an improved understanding of human ovarian development that could lead to treatments for infertility and prevention of developmental disorders. Ovarian development involves interactions between primordial germ cells (PGCs) and somatic cells (granulosa cells). The granulosa cells enclose the PGCs within ovarian follicles, and support their differentiation into oogonia, progression through meiosis, and development as oocytes. Therefore, both lineages will be required to model this process in vitro. Existing methods allow differentiation of induced pluripotent stem cells (iPSCs) into PGC-like cells, but these cells are in an immature state, retaining epigenetic characteristics of iPSCs. For an in vitro model of oogenesis to be successful, improved methods must be developed to generate mature germline cells and granulosa cells from iPSCs. Reprogramming of cellular identity by expression of transcription factors (TFs) is a powerful technique that can allow both reprogramming of somatic cells into iPSCs, and directed differentiation of iPSCs to specific cell types. The Church lab has recently developed computational tools to predict TFs that specify cell identity, as well as screening methods for combinatorial TF expression to find sets that can differentiate iPSCs to a cell type of interest. The currently proposed research will identify TFs that can promote maturation of PGC- like cells and produce granulosa cells from iPSCs. Results will be evaluated by single-cell transcriptomic and epigenetic profiling, and by functional validation of key phenotypes. This research will provide an improved understanding of the genetic regulation of ovarian development, leading to an in vitro model of human oogenesis.