Cultivation and passage of chicken primordial germ cells in vitro

摘要

This paper investigates proper conditions and factors for the cultivation and cryopreservation of chicken primordial germ cells (PGCs). PGCs were isolated from germinal ridges at the 19th chick embryo stage with 0.25% trypsin-0.04% EDTA at room temperature cultured in the DMEM with high concentrations of glucose supplemented with 10% fetal bovine serum, 2% chicken serum, 1 mmol/L glutamine, 5.5 ×105 β-mercaptoethanol, 100 U/ml gentamycin and 1 mmol/L sodium pyruvate, with or without hSCF (5 ng/mL), mLIF (10 U/ml), bFGF (10 ng/ml), hIL-11 (0.04 ng/ml), IGF (10 ng/ml). After three passage, chicken embryonic fibroblast cells were used as a feeder layer. Cell morphology, colony formation, proliferation capability, karyotype, alkaline phosphatase activity and cell differentiation were characterized. Results showed that the Medium II which wassupplemented with 5 factors was better than the Medium I which was without the factors. PCGs cultured in the Meium Ⅱ could be subcultured five times and still maintained an undifferentiated status with normal chromosome karyotype, the cytoplasm of PCGs exhibited positive reaction for PAS and alkaline phosphatase. Chicken primordial germ cells could be induced into neuron type cells by vitamin A. It seems that the cultured chicken PGCs maintained characters of pluripotent stem cells[Acta Zoologica Sinica 51(4): 723–731, 2005].