Title: Improving in vitro induction efficiency of human primordial germ cell-like cells using N2B27 or NAC-based medium

Primordial germ cells (PGCs), the precursors of oocytes or spermatozoa, are highly pluripotent. In recent years, the in vitro induction of human primordial germ cell-like cells (hPGCLCs) has advanced significantly. However, the stability and efficacy of obtaining hPGCLCs in vitro still require further improvement. In the current study, we identified a novel induction system by using Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) as the basal medium supplemented with B27 and N2 (referred to as N2B27) in combination with four cytokines: bone morphogenetic protein 4 (BMP4), stem cell factor (SCF), epidermal growth factor (EGF), and leukemia inhibitory factor (LIF). The hPGCLCs induced under these conditions closely resemble PGCs from 4 to 5-week-old embryos at the transcriptome level. Compared with traditional GK15 (GMEM supplemented with 15% Knockout™ SR)-based induction conditions, the N2B27 system significantly increased the speed and efficacy of hPGCLC induction. RNA sequencing analysis revealed that this improvement resulted from an increased cell capacity to cope with hypoxic stress and avoid apoptosis. The N2B27 medium promoted an increase in mitochondrial activity, enabling cells to better cope with hypoxic stress while also reducing the production of reactive oxygen species. Moreover, by gradient concentration experiments, we demonstrated that addition of the common antioxidant N-acetyl-L-cysteine at an optimized concentration further enhanced the efficiency of PGCLC induction under GK15 conditions. Thus, our study established an optimized induction system that enhances the efficiency of hPGCLC differentiation by improving cellular resilience to hypoxic stress and apoptosis.