Characterization of primordial germ cells from EG&K stage X chicken embryos

Primordial germ cells (PGCs) derived from Eyal-Giladi and Kochav (EG&K) stage X embryos (XPGCs) represent the earliest germ cells in poultry. However, little is known about the XPGCs due the difficulty in the isolation and propagation of these cells. In this study, we report a method for the stable isolation and efficient in vitro propagation of XPGCs. We conducted a thorough analysis of the characteristics exhibited by XPGCs. First, we identified an average of 42 PGCs at this stage by using an DAZL-2a-mCherry genetically modified chickens. Three stable cell lines, one male and two female, were established from eight isolates using a filter paper ring and differential adhesion. Germ cell-related protein markers, including DAZL, CVH, and SSEA-1, were detected in the established XPGCs. Re-introduction of the XPGCs into the blastoderms of EG&K stage X embryos or the cardiovascular systems of Hamilton and Hamburger (HH) stage 14 embryos showed that the XPGCs retained the ability to migrate to the gonads after long-term culture. XPGCs demonstrated a lower growth rate in vitro but a higher potential of migrating to the gonads than PGCs derived from the embryonic gonads of HH stage 27-31. The ability to isolate, culture, and characterize XPGCs advances our knowledge of early-stage germ cell development and provides a valuable cell tool for genetic conservation and genome editing in chickens.