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

人类多能干细胞衍生的原始生殖细胞样细胞（hPGCLCs）目前是体外模拟早期人类生殖细胞发育过程的最佳模型，并允许实验设置更深入地了解生殖细胞规范和潜在的分子途径。原始生殖细胞的雄性命运是在睾丸形态发生过程中被诱导的。支持细胞吞噬生殖细胞并随后形成聚集体，这是精索形成的第一个标志。精索中的生殖细胞以男性特有的扩张和迁移模式作出反应，并实现转录和表观遗传变化。在这里，我们的目的是描述在CRU中鉴定的候选基因在早期PGC规范中的作用。利用在第一个资助期建立的方案，我们将通过CRISPR/Cas9系统产生在鉴定的候选基因中具有功能丧失（LoF）突变的人类iPSC系，并分析形态学特征、基因表达以及与体细胞的相互作用，以确定与生育能力受损相关的途径。作为第二个目标，我们将扩展在第一个资助期内建立的异种器官方法。重建的大鼠精管样结构在体外模拟睾丸形态发生，并允许在睾丸微环境中共同培养野生型或突变的pgclc。因此，我们期望描述与男性生殖细胞分化有关的关键因素和基因。作为表征pgclc中男性命运的终点，我们使用标记物应用成像和组织学终点来描述pgclc的男性导向分化、迁移和克隆扩增。此外，转录和甲基化的变化将在暴露于离体雄性微环境之前和之后确定。第三，我们将扩展和多样化体细胞睾丸微环境的产生和表征。为了延长观察期，将大鼠类器官移植到免疫缺陷小鼠体内。此外，我们将利用成人变性患者的人原代睾丸细胞体外生成精小管样结构，从而建立标准化的同源培养体系。我们的研究将产生新的方法和新的发现，以更好地了解人类生殖细胞的发育。随着对性别决定机制的关注，我们预见到描述可能影响男性不育的途径。该研究为性腺性别分化和生殖细胞与躯体组织相互作用的基本细胞和分子机制提供了新的见解。因此，这些数据具有直接和直接的临床意义，因为不正确的生殖细胞分化可能导致部分或全部生殖细胞丢失和不育。

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.