Expanding highly homogenous population of human primordial germ cell like cells in long-term and feeder-free culture condition [RNA-Seq]

The culture methods for human primordial germ cell like cells (hPGCLCs) has not been established. Here we report the culture method to expand long-term culture hPGCLCs (LTC-hPGCLCs) representing migrating morphology and early hPGC transcriptome and methylome. Key components of the culture media are stem cell factor, STO-conditioned media and absence of fetal calf serum. In this condition, these LTC-hPGCLCs were highly homogenous population without feeder cells and expanded at least 150 days. They did not express senescent phenotype such as β-galactosidase activity and shortening of telomere length at the 150-day culture, indicating that the cell might be a perpetual cell line. LTC-hPGCLCs still have potent pluripotency indicating the phenotype that the cells become human embryonic germ cells which were distinguishable from human iPS cells in strong DLK1 expression and H19 biallelic expression. In summary this simple culture method and highly homogenous LTC-hPGCLCs would be a useful resource that supports investigations on human germline cells.

人类原始生殖细胞样细胞（human primordial germ cell-like cells，缩写hPGCLCs）的培养方法此前尚未建立。本研究报道了一种可长期扩增hPGCLCs（long-term culture hPGCLCs，缩写LTC-hPGCLCs）的培养方法，所获细胞具备迁移阶段的形态学特征，且保留早期人类原始生殖细胞的转录组与甲基化组特征。该培养液的关键组分为干细胞因子、STO条件培养液，且不含胎牛血清。在该培养体系下，所获LTC-hPGCLCs为无饲养层的高度均一细胞群，可连续扩增至少150天。培养至150天时，这些细胞未表现出β-半乳糖苷酶活性阳性、端粒长度缩短等衰老表型，提示该细胞可能为永生化细胞系。LTC-hPGCLCs仍具备较强的多能性，其表型与人类胚胎生殖细胞高度相似；与人类诱导多能干细胞（iPS细胞）相比，该细胞高表达DLK1且H19呈双等位基因表达，二者可据此明确区分。综上，该简便培养方法及所获高度均一的LTC-hPGCLCs，将成为支持人类生殖细胞相关研究的宝贵资源。