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