Institute for the Advanced Study of Human Biology (ASHBi), Kyoto University, Kyoto, Japan.
Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Biol Reprod. 2021 Feb 11;104(2):344-360. doi: 10.1093/biolre/ioaa195.
Primordial germ cells (PGCs) are the founding population of the germ cell lineage that undergo a multistep process to generate spermatozoa or oocytes. Establishing an appropriate culture system for PGCs is a key challenge in reproductive biology. By a chemical screening using mouse PGC-like cells (mPGCLCs), which were induced from mouse embryonic stem cells, we reported previously that forskolin and rolipram synergistically enhanced the proliferation/survival of mPGCLCs with an average expansion rate of 20-fold. In the present study, we evaluated other chemicals or cytokines to see whether they would improve the current mPGCLC culture system. Among the chemicals and cytokines examined, in the presence of forskolin and rolipram, cyclosporin A (CsA) and fibroblast growth factors (FGFs: FGF2 and FGF10) effectively enhanced the expansion of mPGCLCs in vitro (50-fold on average). During the expansion by CsA or FGFs, mPGCLCs comprehensively erased their DNA methylation to acquire a profile equivalent to that of gonadal germ cells in vivo, while maintaining their highly motile phenotype as well as their transcriptional properties as sexually uncommitted PGCs. Importantly, these mPGCLCs robustly contributed to spermatogenesis and produced fertile offspring. Furthermore, mouse PGCs (mPGCs) cultured with CsA ex vivo showed transcriptomes and DNA methylomes similar to those of cultured mPGCLCs. The improved culture system for mPGCLCs/mPGCs would be instructive for addressing key questions in PGC biology, including the mechanisms for germ cell migration, epigenetic reprogramming, and sex determination of the germline.
原始生殖细胞（PGCs）是生殖细胞系的创始群体，经历多步过程产生精子或卵子。建立适当的 PGC 培养体系是生殖生物学的关键挑战。通过使用从小鼠胚胎干细胞诱导的 PGC 样细胞（mPGCLCs）进行化学筛选，我们之前报道过，福司可林和罗利普兰协同增强 mPGCLCs 的增殖/存活，平均扩增率约为 20 倍。在本研究中，我们评估了其他化学物质或细胞因子，以观察它们是否会改善当前的 mPGCLC 培养系统。在所检查的化学物质和细胞因子中，福司可林和罗利普兰存在的情况下，环孢素 A（CsA）和成纤维细胞生长因子（FGFs：FGF2 和 FGF10）可有效增强 mPGCLCs 的体外扩增（平均约 50 倍）。在 CsA 或 FGFs 的扩增过程中，mPGCLCs 全面消除了它们的 DNA 甲基化，获得了与体内性腺生殖细胞相当的特征，同时保持了它们作为未分化 PGC 高度迁移的表型以及转录特性。重要的是，这些 mPGCLCs 可有效地促进精子发生并产生可育后代。此外，用 CsA 体外培养的小鼠 PGC（mPGCs）显示出与培养的 mPGCLCs 相似的转录组和 DNA 甲基组。改进的 mPGCLCs/mPGCs 培养系统将有助于解决 PGC 生物学中的关键问题，包括生殖细胞迁移、表观遗传重编程和生殖系性别决定的机制。
