Identification and Culture of Primordial Germ Cells and Somatic Cells from Undifferentiated Embryonic Chick Gonads and Formation of Presumptive Embryoid Body Precursors

Primordial germ cells (PGCs) are pluripotent stem cells and exist as embryonic precursors to germ cells in vivo. PGCs have been shown to differentiate into embryonic germ (EG) cells which can aggregate to form embryoid bodies (EBs) in vitro. EGs within EBs are capable of differentiating into a variety of cell types; thus EBs can be useful for generating large numbers of stem cells for regenerative medicine. The objectives of this study were to harvest and culture primordial germ cells from indifferent gonads of the embryonic domestic chick and assess factors affecting survival, proliferation, and development of gonadal cells (somatic and germ) in vitro. PGCs were identified based on size and staining with pluripotent stem cell markers. Formation of cell aggregates, presumptive EB precursors, was observed throughout all gonadal cultures in the presence of PGCs. Gonadal cultures treated with busulfan to eliminate PGCs had only limited aggregate formation. Cultures supplemented with fibroblast growth factor showed increased aggregate formation. Co-culture with embryonic testes or ovaries seemed to influence somatic cells surrounding aggregates. PGC-derived EGs were identified immunocytochemically within aggregates using pluripotent stem cell and germ cell markers (anti-SSEA1, anti-SSEA4, and anti-EMA1). In vitro culture conditions for dissociated embryonic gonad cells in this study may provide a basis for expanding embryonic germ cells and directing their development for germ stem cell development, in vitro gamete production, and other stem cell application