Modelling the developing gonad –organoid- and iPSC-based approaches

基于类器官和 iPSC 的方法对发育中的性腺进行建模

• 批准号: 388866673
• 负责人: Professor Dr. Stefan Schlatt
• 金额: --
• 依托单位: Abteilung für Klinische und Operative Andrologie
• 依托单位国家: 德国
• 项目类别: Clinical Research Units
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/388866673?language=en
• 关键词: Modelling; developing; gonad; organoid; iPSC

Human pluripotent stem cell-derived primordial germ cell-like cells (hPGCLCs) currently represent the best available model to mimic early human germline developmental processes in vitro and allow experimental setups to deeper understand germ cell specification and underlying molecular pathways. The male fate in primordial germ cells is induced during testicular morphogenesis. Sertoli cells engulf germ cells and subsequently form aggregates as the first sign of seminiferous cord formation. Germ cells in seminiferous cords respond with a male-specific pattern of expansion and migration and implement transcriptional and epigenetic changes. Here, we aim to describe the role of candidate genes identified within the CRU in early PGC specification. Using protocols established during the first funding period, we will generate human iPSC lines with a loss of function (LoF) mutation in identified candidate genes via the CRISPR/Cas9 system, and analyze morphological features, gene expression, and interaction with somatic cells to identify pathways associated with impaired fertility. As second objective, we will extend our xeno-organoid approach, established within the first funding period. The reconstituting rat seminiferous tubule-like structures mimic testicular morphogenesis in vitro and allow co-culture of wildtype or mutated PGCLCs within a testicular microenvironment. We thereby expect to describe crucial factors and genes involved in male germ cell differentiation. As endpoints for characterization of male fate in PGCLCs, we apply imaging and histological endpoints using markers to describe male-directed differentiation, migration, and clonal expansion of PGCLCs. Additionally, changes in transcription and methylation will be determined before and after exposure to the ex vivo male microenvironment. Third, we will extend and diversify the generation and characterization of the somatic testicular microenvironments. To extend the observation period, the rat organoids will be subjected to xenografting into immunodeficient mice. Additionally, we will apply human primary testicular cells from adult transgender patients to generate seminiferous tubule-like structures in vitro and thus establish a standardized homologous culture system. Our studies will generate a new approach and new findings to better understand human germ cell development. With a focus on sex determining mechanisms, we foresee to describe pathways potentially affected in male infertility. The study provides novel insights into basic cellular and molecular mechanisms controlling gonadal sex-differentiation and interplay of germ cells with somatic tissues. The data have therefore immediate and direct clinical relevance since incorrect differentiation of germ cells may lead to partial or complete germ cell loss and infertility.

人多能干细胞衍生的原始生殖细胞样细胞（hPGCLC）目前代表了体外模拟早期人类生殖系发育过程的最佳模型，并允许实验装置更深入地了解生殖细胞的特化和潜在的分子途径。原始生殖细胞中的雄性命运在睾丸形态发生过程中被诱导。支持细胞吞噬生殖细胞，随后形成聚集体，这是生精索形成的第一个迹象。生精索中的生殖细胞以男性特有的扩张和迁移模式做出反应，并实现转录和表观遗传变化。在这里，我们的目标是描述的候选基因中确定的CRU在早期PGC规范的作用。使用在第一个资助期内建立的方案，我们将通过CRISPR/Cas9系统在已确定的候选基因中产生具有功能丧失（LoF）突变的人类iPSC系，并分析形态特征，基因表达以及与体细胞的相互作用，以确定与生育力受损相关的途径。作为第二个目标，我们将扩展我们在第一个资助期内建立的异种类器官方法。重建大鼠生精小管样结构模拟睾丸形态发生在体外，并允许在睾丸微环境中的野生型或突变PGCLC的共培养。因此，我们希望描述参与雄性生殖细胞分化的关键因素和基因。作为PGCLC中男性命运表征的终点，我们应用标记物描述PGCLC的男性定向分化、迁移和克隆扩增的成像和组织学终点。此外，将在暴露于离体雄性微环境之前和之后确定转录和甲基化的变化。第三，我们将扩展和多样化的体细胞睾丸微环境的产生和表征。为了延长观察期，将大鼠类器官异种移植到免疫缺陷小鼠中。此外，我们将应用成人变性患者的原代睾丸细胞在体外产生生精小管样结构，从而建立标准化的同源培养系统。我们的研究将产生一种新的方法和新的发现，以更好地了解人类生殖细胞的发育。随着性别决定机制的重点，我们预见到男性不育症的潜在影响的途径描述。这项研究为控制性腺性别分化的基本细胞和分子机制以及生殖细胞与体细胞组织的相互作用提供了新的见解。因此，这些数据具有直接和直接的临床相关性，因为生殖细胞的不正确分化可能导致部分或全部生殖细胞损失和不育。