MRC London Institute of Medical Sciences (LMS), London, UK.
Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, London, UK.
Methods Mol Biol. 2021;2214:59-73. doi: 10.1007/978-1-0716-0958-3_5.
Primordial germ cells (PGCs) are the embryonic precursors of the gametes. Despite decades of research, in vitro culture of PGCs remains a major challenge and has previously relied on undefined components such as serum and feeders. Notably, PGCs cultured for extended periods do not maintain their lineage identity but instead undergo conversion to form pluripotent stem cell lines called embryonic germ (EG) cells in response to LIF/STAT3 signaling. Here we report both established and new methodologies to derive EG cells, in a range of different conditions. We show that basic fibroblast growth factor is not required for EG cell conversion. We detail the steps taken in our laboratory to systematically remove complex components and establish a fully defined protocol that allows efficient conversion of isolated PGCs to pluripotent EG cells. In addition, we demonstrate that PGCs can adhere and proliferate in culture without the support of feeder cells or serum. This may well suggest novel approaches to establishing short-term culture of PGCs in defined conditions.
原始生殖细胞（PGCs）是配子的胚胎前体。尽管经过了几十年的研究，PGCs 的体外培养仍然是一个主要挑战，以前依赖于血清和饲养细胞等未定义的成分。值得注意的是，经过长时间培养的 PGC 不会保持其谱系身份，而是会响应 LIF/STAT3 信号转导转化为多能性干细胞系，称为胚胎生殖（EG）细胞。在这里，我们报告了一系列不同条件下衍生 EG 细胞的已建立和新方法。我们表明，碱性成纤维细胞生长因子对于 EG 细胞的转化不是必需的。我们详细介绍了我们实验室在系统去除复杂成分方面所采取的步骤，并建立了一个完全定义的方案，允许高效地将分离的 PGC 转化为多能性 EG 细胞。此外，我们证明 PGC 可以在没有饲养细胞或血清支持的情况下在培养中粘附和增殖。这可能为在定义条件下建立 PGC 的短期培养提供新的方法。
