Culture And Differentiation Of Chicken Primordial Germ Cells

This paper was focus on isolating primordial germ cells (PGCs) from gonads at stage 19 and stage 28 by Ficoll density-gradient centrifugation. After cultured and subcultured , PGCs were induced at different cultured time, differentiation media and suspension culture, in order to discuss the condition of directional induction and the ability of undifferentiation maintenance. The results showed:1. The purified PGCs were cultured in DMEM media, supplemented with 10% fetal bovine serum, 2% chicken serum, 2 mmol/L glutamine, 1mmol/L sodium pyruvate, 5.5 X 10~-5mol/L β-mercaptoethanol, 5ng/mL hSCF, 10UI/mL mLIF, 10ng/mL bFGF, 0.04ng/mL hIL-11, 10ng/mL IGF, 100U/mL gentamycin sulfate. The PGCs can adhere to the cell layer, form cell colonies and proliferate. When subcultured, PGCs still could form cell colonies and proliferate, it is also positive to AKP. These indicated that under mentioned culture system, second passage PGCs are also undifferentiation.2. The second passage PGCs were induced at retinoic acid, the differentiation rate of PGCs showed no difference (P>0.05) among 3 kinds of culture time, and at the same induction medium, the harm to PGCs along with β-ME concentration.3. Under the same culture time, the different induction media showed: induction medium and were the best way to induce PGCs to differentiate into neuron-like cells, and there is no significant difference between them, but very significant difference (P<0.01) compared to other induction media. The effect of induction medium D are not good, and showed very significant difference (P<0.01) to others. There is no significant difference (P>0.05) between induction medium and , but they showed very significant difference (P<0.01) compared to D. Special Nissl body was found byhistochemistry after induction.4. The second passage PGCs at stage 19 and 28 were suspension cultured in DMEM media, supplemented with 10% fetal bovine serum, 2% chicken serum, 2 mmol/L glutamine, lmmol/L sodium pyruvate, 5.5 X 10~-5mol/L P-mercaptoethanol, PGCs can differentiate into vascular cells in presence of a few feeder cells, or they can form simple embryoid bodies under suspension culture.