In recent years, considerable attention has been paid to chicken embryonic stem cells (ESCs) studies in relation to extensive applications in gene therapy and regenerative medicine. However, the approaches used are still immature. In this study, we showed that the chicken ESCs clones with a clear border can express alkaline phosphatase and marker proteins such as SSEA-1, SOX2, and OCT4 stably. In addition, culture medium containing 10 μmol/L of vitamin C (VC) could significantly promote the proliferation of ESCs cells. Moreover, ESCs transfected with p:enhanced green fluorescent protein (pEGFP)-hTERT could be subcultured more than tenth generations in culture medium containing exogenous factors (mLIF + bFGF + hSCF) and VC, and these ESCs clone could still be regenerated following cryopreservation. Quantitative real-time polymerase chain reaction results showed that there was no significant difference between SSEA-1, SOX2, and OCT4 expression during ESCs immortalization and that the tenth generation of ESCs was still able to express marker proteins SSEA-1, SOX2, and OCT4. Our results showed that an immobilized system for ESCs was established, and the ESCs were cultured in vitro maintaining their pluripotency.
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