Ding Xinbao, Li Liangdao, Gao Jingyi, Yi Dain, Schimenti John C
Cornell University, College of Veterinary Medicine, Department of Biomedical Sciences, Ithaca, NY 14853.
bioRxiv. 2024 Feb 15:2024.02.15.580543. doi: 10.1101/2024.02.15.580543.
Primordial germ cells (PGCs) are the founder cells of the germline. The ability to generate PGC-like cells (PGCLCs) from pluripotent stem cells has advanced our knowledge of gametogenesis and holds promise for developing infertility treatments. However, generating an ample supply of PGCLCs for demanding applications such as high-throughput genetic screens has been a limitation. Here, we demonstrated that simultaneous overexpressing 4 transcriptional factors - and three PGC master regulators , and - in suspended mouse epiblast like cells (EpiLCs) and formative embryonic stem cells (ESCs) results in efficient and cost-effective production of PGCLCs. The overexpression of enhances the PGC regulatory network and suppresses differentiation of somatic lineages, enabling a significant improvement in the efficiency of PGCLC production. Transcriptomic analysis reveals that differentiated PGCLCs exhibit similarities to PGCs and are more advanced compared to cytokine-induced PGCLCs. These differentiated PGCLCs could be sustained over prolonged periods of culture and could differentiate into spermatogonia-like cells . Importantly, the ability to produce PGCLCs at scale, without using costly cytokines, enables biochemical and functional genomic screens to dissect mechanisms of germ cell development and infertility.
原始生殖细胞（PGCs）是生殖系的起始细胞。从多能干细胞生成类原始生殖细胞（PGCLCs）的能力增进了我们对配子发生的了解，并为开发不孕症治疗方法带来了希望。然而，为诸如高通量基因筛选等苛刻应用生成充足的PGCLCs一直是一个限制因素。在此，我们证明，在悬浮的小鼠外胚层样细胞（EpiLCs）和定向胚胎干细胞（ESCs）中同时过表达4种转录因子——以及三种PGC主要调节因子、和——可实现高效且经济高效地生产PGCLCs。的过表达增强了PGC调节网络并抑制体细胞谱系的分化，从而显著提高了PGCLC的生产效率。转录组分析表明，分化的PGCLCs与PGCs表现出相似性，并且与细胞因子诱导的PGCLCs相比更为先进。这些分化的PGCLCs可以在长时间培养中维持，并可以分化为精原细胞样细胞。重要的是，可以在不使用昂贵细胞因子的情况下大规模生产PGCLCs，这使得生化和功能基因组筛选能够剖析生殖细胞发育和不孕症的机制。
