In vitro generation and characterization of chicken long-term germ cells from different embryonic origins

Primordial germ cells (PGCs) are the precursors of differentiated germ cells. Located in the epiblast of a stage X (EG&K) embryo, the PGCs translocate anteriorly to the germinal crescent and migrate, within 48 to 56 hours of development, through the blood vascular system to the germinal ridges where they become the gonadal germ cells (GGCs). We aim to generate, compare, and determine the basic characters of the in vitro long-term cultured PGCs derived from (1) the chicken blastodermal cells (at stages IX-XII); (2) the chicken blood of a 2-day old embryo (stages 14-17 Hamburger Hamilton [HH]); and (3) the long-term cultured gonocytes taken from male gonads of a 5- to 6-day-old embryo (stages 29-30 HH). In presence of fibroblast growth factor, chicken blastodermal cells are able to long-term proliferate and generate small, round, alkaline phosphatase-positive cell clusters. Molecular characterization shows that these selected and amplified clusters show a PGC-like cell profile, as they express cPOUV (a pluripotent-associated marker), NR6A1/GCNF and DDX4/CVH (germ cell-specific genes). Both chicken PGCs and GGCs, obtained from embryonic blood and gonads, at 14 to 17 HH and 29 to 30 HH, respectively, generate long-term germ cell cultures and positively react in vitro to periodic acid-Schiff. Immunochemical analyses reveal that these cell lines are specifically recognized by anti-SSEA-1, anti-EMA-1, anti-CVH, anti-β1-integrin, and anti-CEACAM antibodies. The presence of surrounding cells may suggest a stronger dependency toward the niche process for the GGCs. The reactivity of chicken embryonic germ cells obtained from the two different sources to the specific markers used in this study was not altered through the culture. In conclusion, the morphologic analysis specific for chicken PGCs and GGCs will further contribute to quick and reliable characterization of long-term cultured in vitro chicken germ cells.