Optimizing In Vitro hPGCLC Induction with N2B27 Medium and Antioxidants for Enhanced Efficiency

Primordial germ cells (PGCs), the precursors of oocytes or spermatozoa, exhibit high pluripotency. In recent years, significant progress has been made in the in vitro induction of human primordial germ cell-like cells (hPGCLCs). However, the stability and efficiency of obtaining hPGCLCs in vitro still need improvement.In this study, we identified a novel induction system using N2B27, in combination with four cytokines: BMP4, SCF, EGF, and LIF. The hPGCLCs induced under these conditions closely resemble PGCs from 4,5-week embryos at the transcriptome level. Compared to traditional GK15-based induction conditions, the N2B27 system significantly enhances both the speed and efficiency of hPGCLC induction. RNA-seq analysis showed that the improvement is driven by the cells' enhanced capacity to cope with hypoxic stress and avoid apoptosis. N2B27 medium promotes higher mitochondrial activity, enabling cells to better cope with hypoxic stress, while also reducing the generation of reactive oxygen species (ROS). Additionally, we found that the addition of N-acetyl-L-cysteine (NAC), a common antioxidant, at an optimized concentration further increase the efficiency of PGCLC induction under GK15 conditions, as demonstrated through gradient concentration experiments.