A gastruloid-based model system to study human and non-human primate primordial germ cell specification and reprogramming

原始生殖细胞(PGC)是胚胎的前体细胞，可产生精子或卵子。在人类胚胎植入后的第二周左右，PGCs的规格开始于胚胎发育的早期，与原肠形成的开始同步。了解PGCs是如何发展的，将为未来的医学应用提供令人兴奋的前景，并解决关键的发展问题。然而，由于伦理方面的考虑，使用人类胚胎研究PGCs是不可能的。因此，我的目标是利用类似胚胎的体外模型。这些所谓的胃泌素是由三维聚集体中的多能干细胞自组织产生的，从而经历了早期的关键发育事件。半乳糖由于其真实的细胞结构、组织和时间控制的细胞命运转变，为研究灵长类动物的早期胚胎发生提供了前所未有的潜力。此外，小鼠原肠样细胞可以在相对于体细胞组织样结构的时空有组织的位置产生PGC样细胞。我的目标是研究人类和非人灵长类(NHP)原肠动物的PGC规格和重编程。并行使用人类和猕猴细胞将打开用现有的体内NHP数据验证体外获得的数据的可能性。此外，比较分析将使我能够剖析NHP和人类PGC规范以及早期差异之间的潜在异同。在建立并鉴定了灵长类动物特有的胃液系统后，我将进行有条件的功能丧失实验，以解决这个问题：多能性因素在PGC命运决定和重新编程中起什么作用？为此，我将使用一种可诱导的降解系统，在原肠形成的不同时间点快速、全面地耗尽候选转录因子，并评估其对PGC种群的影响。总之，该项目将构成一个明确的体外系统，用于研究灵长类动物的PGC规范和重编程。本申请中提出的组合工具集可用于药物毒性测试、先天性疾病建模，并有助于动物实验中的3R(缩减、改进和替换)。

Primordial germ cells (PGCs) are embryonic precursor cells, which give rise to sperm or eggs. PGCs specification starts during early embryonic development, concurrent with the beginning of gastrulation, at around week two in the human embryo post-implantation. Understanding how PGCs develop will provide exciting prospects for future medical applications and address key developmental questions. However, it is not possible to study PGCs using human embryos due to ethical concerns. Thus, I aim to make use of embryo-like in vitro models. These so-called gastruloids are generated by self-organization of pluripotent stem cells in three-dimensional aggregates, which thereby undergo early key developmental events. Gastruloids offer an unprecedented potential to study primate early embryogenesis due to their realistic cytoarchitecture, organization, and temporally controlled cell fate transitions. In addition, mouse gastruloids can give rise to PGC-like cells in a spatiotemporally organized location relative to somatic tissue-like structures. I aim to examine PGC specification and reprogramming in human and non-human primate (NHP) gastruloids. Using both human and macaque cells in parallel will open up the possibility to validate data obtained in vitro with available NHP in vivo data. Furthermore, comparative analysis will allow me to dissect potential similarities and differences between NHP and human PGC specification and early differentiation. After establishing and characterizing the primate-specific gastruloid system, I will perform conditional loss-of-function experiments to address the question: What is the role of pluripotency factors during PGC fate determination and reprogramming? For this, I will use an inducible degron system for rapid and comprehensive depletion of candidate transcription factors at different time points during gastruloid formation and evaluate the impact on the PGC population. Altogether, this project will constitute a defined in vitro system to study PGC specification and reprogramming in primates. The combined set of tools proposed in this application can be applied for drug toxicity testing, congenital disease modeling and contribute to the 3Rs (reduce, refine, and replace) in animal experimentation.