A practical approach for the stable isolation and cultivation of chicken gonadal primordial germ cells with mitotically inactivated STO feeder cells.

OBJECTIVE: Establishing chicken primordial germ cells (PGCs) in vitro is critical for producing genetically modified (GM) chickens. Efficient and reliable isolation and cultivation of PGCs remain significant challenges in advancing avian genetic modifications. To address these challenges, we employed a streamlined and practical approach for the efficient isolation and stable cultivation of chicken gonadal PGCs. METHODS: Chicken gonadal PGCs were isolated from embryonic gonads, surgically removed and dissociated using trypsin. The PGCs were isolated by exploiting differential adhesion properties, allowing fibroblasts to attach while PGCs remained suspended. Cultivation was performed with mitotically inactivated SIM mouse embryo-derived thioguanineresistant (STO) feeder cells under optimized culture conditions. RESULTS: PGCs proliferated robustly, reaching over 105 cells within one month, which is comparable to previously reported methods. Characterization assays confirmed the expression of PGC-specific markers, including SSEA-1 and DAZL, along with pluripotencyrelated genes such as OCT4 and NANOG. Additionally, injected PGCs successfully migrated to recipient embryonic gonads, where their presence was confirmed by fluorescence analysis and PCR. CONCLUSION: This study highlights the effectiveness of the STO feeder-based culture system in avian germ cell research, contributing to progress in the production of germline chimeric and GM chickens.