Single-cell RNA-seq offer new insights into the cell fate decision of the primordial germ cells.

The faithful production of primordial germ cells (PGCs) in vitro opens a wide range of novel applications in reproductive biology and medicine. However, the reproducibility of PGCs culture conditions across different laboratories or breeds remains a challenge. Therefore, it is necessary to research the molecular dynamics that lead to the gradual establishment of cultured PGCs lines network. Here, the results of single-cell RNA-seq indicated that the cell cycle drove cellular heterogeneity. The active populations engaged in PGC self-renewal and the characteristics of the aging cell fate have been identified. The active self-renewal populations presented a rising expression of DNA repair genes, couple with a high proportion of cells in G1/S phase and a low frequency of cells in G2 phase. Notably, Hippo, FoxO, AMPK and MAPK pathways are active within these populations. The combination of six activator or inhibitors, targeting these active pathways, resulted in a significantly higher proliferation rate of PGCs and an increased number of cells entering the G1 and S phases. Importantly, they greatly reduced the establishment time to a minimum of 26 days and increased the efficiency of male PGC line establishment to 59 % in FS medium. Our results provided several new insights into the PGCs.