Sun Changhua, Wang Yingjie, Jin Kai, Song Jiuzhou, Zuo Qisheng, Zhang Yani, Chen Guohong, Li Bichun
Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, China.
Department of Food technology, College of Biochemical Engineering, Yangzhou Polytechnic College, Yangzhou, China.
J Cell Biochem. 2019 Feb;120(2):1376-1385. doi: 10.1002/jcb.27173. Epub 2018 Nov 16.
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.
近年来，鸡胚胎干细胞（ESCs）研究因在基因治疗和再生医学中的广泛应用而备受关注。然而，所采用的方法仍不成熟。在本研究中，我们发现边界清晰的鸡胚胎干细胞克隆能够稳定表达碱性磷酸酶以及SSEA-1、SOX2和OCT4等标记蛋白。此外，含有10 μmol/L维生素C（VC）的培养基可显著促进胚胎干细胞的增殖。而且，转染了p:增强绿色荧光蛋白（pEGFP）-hTERT的胚胎干细胞在含有外源因子（mLIF + bFGF + hSCF）和VC的培养基中能够传代培养超过十代，并且这些胚胎干细胞克隆在冷冻保存后仍可再生成活。定量实时聚合酶链反应结果表明，胚胎干细胞永生化过程中SSEA-1、SOX2和OCT4的表达无显著差异，且第十代胚胎干细胞仍能够表达标记蛋白SSEA-1、SOX2和OCT4。我们的结果表明建立了一种胚胎干细胞固定化体系，并且胚胎干细胞在体外培养时保持了其多能性。
