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

Abstract

Primordial germ cells (PGCs), the precursors of oocytes and 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 improvement. In the current study, we identified a novel induction system 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, stem cell factor, epidermal growth factor, and leukemia inhibitory factor. The hPGCLCs induced under these conditions closely resembled PGCs from 4- to 5-week-old embryos at the transcriptomic level. Compared with traditional GK15 (GMEM supplemented with 15% Knockout™ Serum Replacement)-based induction conditions, the N2B27 system significantly increased the speed and efficacy of hPGCLC induction. RNA sequencing analysis revealed that this improvement was due to an increased cellular capacity to cope with hypoxic stress and avoid apoptosis. The N2B27 medium promoted enhanced mitochondrial activity, enabling cells to better manage hypoxic stress while reducing the production of reactive oxygen species. Moreover, through gradient concentration experiments, we demonstrated that the addition of the common antioxidant N-acetyl-L-cysteine at an optimized concentration further enhanced the efficiency of PGCLC induction under GK15 conditions. In summary, we have established an optimized induction system that enhances the efficiency of hPGCLC differentiation by improving cellular resilience to hypoxic stress and apoptosis.

Keywords

human primordial germ cell-like cell / N2B27 / hypoxia / N-acetyl-L-cysteine

Fundings

This research was funded by the National Key R&D Program (Grant Nos. 2022YFC2702800 and 2021YFC2700302 to Y.Y.), as well as the National Natural Science Foundation of China (Grant Nos. 82122025 to Y.Y., 82221005 to J.S., and 82201763 to L.L.).

Acknowledgments

We thank the Jiang Min research group from Westlake University for providing the mitochondrial dye.

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