Establishment and optimization of the two-step induction system for generating primordial germ cell-like cells from chicken embryonic stem cells.

Primordial germ cells (PGCs) are the progenitor cells of sperm and eggs. Xenotransplantation of chicken PGCs can achieve germline transmission. However, there are still challenges in obtaining many PGCs from endangered birds in vitro. In this study, at first, by incorporating 2i factors, the embryonic stem cells (ESCs) culture conditions were optimized, successfully yielding and validating pluripotent ESCs clones. Then, during induction ESCs, bFGF, activin A, and 1% KSR were added to Epiblast-like cells (EpiLCs). Quantitative real-time polymerase chain reaction (qRT-PCR) showed Pax6, Eomes, and Vimentin expression patterns similar to primary epiblast, indicating successful EpiLCs induction. During EpiLCs to Primordial germ cell-like cells (PGCLCs) transformation, we evaluated BMP4, BMP8b, EGF, LIF, and SCF combinations' impact on induction efficiency. Flow cytometry, qRT-PCR, and immunofluorescence showed high expression of Cvh, C-kit, Dazl, CVH, and DAZL in PGCLCs, suggesting successful EpiLCs differentiation. Induced PGCLCs injected into 2.5-day chick embryos migrated to gonads by day 7-7.5, demonstrating migration and colonization. This study optimized a two-step protocol for in vitro differentiation of chicken ESCs into PGCLCs. This research's results not only provide a reference for obtaining many PGCLCs in vitro but also open up a new approach for the development and application of genetic resource preservation technology in domestic chickens.