Chinese Journal of Tissue Engineering Research ›› 0, Vol. ›› Issue (0): 72-76.doi: 10.3969/j.issn.2095-4344.1870

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Comparison of two passage methods affecting the transfection efficiency of human embryonic stem cells#br#

Sun Li1, 2, Zong Yanyan1, Wei Jianfeng1, 3   

  1. 1Jiangsu Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical University, Xuzhou 221004, Jiangsu Province, China; 2School of Nursing, Xuzhou Medical University, Xuzhou 221004, Jiangsu Province, China; 3Department of Histology and Embryology, School of Basic Medical Sciences, Xuzhou Medical University, Xuzhou 221004, Jiangsu Province, China
  • Received:2019-03-19 Revised:2019-03-27 Accepted:2019-05-31 Online:2019-12-28 Published:2019-12-11
  • Contact: Wei Jianfeng, MD, Associate professor, Jiangsu Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical University, Xuzhou 221004, Jiangsu Province, China; Department of Histology and Embryology, School of Basic Medical Sciences, Xuzhou Medical University, Xuzhou 221004, Jiangsu Province, China
  • About author:Sun Li, Master candidate, Jiangsu Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical University, Xuzhou 221004, Jiangsu Province, China; School of Nursing, Xuzhou Medical University, Xuzhou 221004, Jiangsu Province, China
  • Supported by:
    the Natural Science Foundation of Colleges in Jiangsu Province, No. 14KJB310021; the Foundation of Jiangsu Province Key Laboratory of Encephalopathy Bioinformatics, No. JSBl1403

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Abstract:

BACKGROUND: In the research of human embryonic stem cells, introducing exogenous molecules such as DNA into cells is a common research method, but the transfection efficiency is relatively low. It is crucial to answer the question of how to optimize the existing conditions to improve the transfection efficiency.

OBJECTIVE: To compare the effects of two different passaging methods on H9 transfection efficiency, in order to optimize the conditions required for embryonic stem cell transfection.

METHODS: Human embryonic stem cell lines H9 were cultured for 48 hours after small clone passaging or single-cell passaging. Lipofectamine 3000 was used to transfect pAdTrack-AKT1 fluorescent plasmid into human embryonic stem cells. After 2 days of transfection, the expression of fluorescent plasmids was observed by fluorescence microscope and the transfection efficiency was detected by flow cytometry. RT-qPCR and western blot were used to detect the mRNA and protein expression levels of AKT1 respectively.

RESULTS AND CONCLUSION: Under the fluorescence microscopy, the number of cells expressing fluorescent plasmids in the single-cell passaging group was more than that in the small clone passaging group, and the flow cytometry analysis showed that the transfection efficiency of cells in the single-cell passaging group was (47.18±2.00)%, which was significantly higher than (19.52±0.86)% in the small clone passaging group (P < 0.01). RT-qPCR and western blot analysis showed that the expression levels of AKT1 mRNA and protein in the single-cell passaging group were significantly higher than those in the small clone passaging group (P < 0.01). These findings indicate that single-cell passaging can increase the contact area between cells and transfection reagent liposomes, and improve the transfection efficiency of human embryonic stem cells.

Key words: human embryonic stem cells, small clone passaging, single-cell passaging, liposome transfection, transfection efficiency

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