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

原始生殖细胞（PGCs）是胚胎前体细胞，可产生精子或卵子。PGCs的特化在早期胚胎发育期间开始，与原肠胚形成的开始同时，在人类胚胎植入后的第二周左右。了解PGC如何发展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，由于伦理问题，不可能使用人类胚胎研究PGC。因此，我的目标是利用胚胎样体外模型。这些所谓的类胃体是由三维聚集体中的多能干细胞的自组织产生的，从而经历早期关键发育事件。类胃腺状体由于其逼真的细胞结构、组织和时间控制的细胞命运转变，为研究灵长类动物早期胚胎发生提供了前所未有的潜力。此外，小鼠类胃体可以在相对于体细胞组织样结构的时空组织位置产生PGC样细胞。我的目的是检查PGC规范和人类和非人类灵长类动物（NHP）gastruloids重编程。平行使用人和猕猴细胞将打开验证体外获得的数据与可用的NHP体内数据的可能性。此外，比较分析将使我能够剖析NHP和人类PGC特化和早期分化之间的潜在相似性和差异。在建立和表征灵长类动物特异性gastruloid系统后，我将进行条件性功能丧失实验来解决这个问题：多能性因子在PGC命运决定和重编程过程中的作用是什么？为此，我将使用一种诱导型降解决定子系统，在类胃体形成期间的不同时间点快速全面地耗尽候选转录因子，并评估对PGC群体的影响。总之，该项目将构成一个定义的体外系统，以研究灵长类动物中的PGC特化和重编程。本申请中提出的组合工具集可应用于药物毒性测试、先天性疾病建模，并有助于动物实验中的3R（减少、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.