Chinese Journal of Tissue Engineering Research ›› 2014, Vol. 18 ›› Issue (19): 3069-3074.doi: 10.3969/j.issn.2095-4344.2014.19.019

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Sodium-calcium exchanger promoter promotes differentiation of mouse induced pluripotent stem cells into cardiomyocytes in vitro

Dai Bo1, Li Hong-tao1, Liu Ze1, Xiao Ding-zhang2, Yu Xi-yong2, Chen Bin1, He Jian-xin1, Xiang Ding-cheng1, Qiu Jian1, Wang Yi-gang3   

  1. 1Guangzhou General Hospital of Guangzhou Military Region, Guangzhou 510010, Guangdong Province, China
    2Guangdong Academy of Medical Sciences, Guangdong General Hospital, Guangdong Cardiovascular Institute, Guangzhou 510080, Guangdong Province, China
    3Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, Cincinnati, Ohio 45267, USA
  • Revised:2014-03-10 Online:2014-05-07 Published:2014-05-07
  • About author:Dai Bo, M.D., Associate chief physician, Guangzhou General Hospital of Guangzhou Military Region, Guangzhou 510010, Guangdong Province, China
  • Supported by:

    the National Natural Science Foundation of China, No. 81270189; the Natural Science Foundation of Guangdong Province, No. S2011040000852

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Abstract:

BACKGROUND: Inducing pluripotent stem cells has been considered as a promising treatment for ischemic heart disease. However, an ideal inducing method has not been found yet.
OBJECTIVE: To investigate the role of sodium-calcium exchanger (NCX1) promoter in the differentiation of mouse induced pluriptent stem cells (miPS) into cardiomyocytes.
METHODS: The pLVX-IRES-ZsGreen1 vectors which contain NCX1 promoter constructed by recombinant DNA technology were co-transfected to 293FT cells with ViraPowerTM Lentiviral Packaging Mix. The recombinant lentiviruses infected with miPS were selected and purified by puromycin. miPS were recovered and passaged to form embryoid bodies. The embryoid bodies were induced by differentiation medium containing various concentrations of the virus titer. The number of beating embryoid bodies were calculated. The expression profiles of the myocardial intra-markers were tested to determine the differentiation efficiency of iPSC by RT-PCR and immunofluorescence analysis.
RESULTS AND CONCLUSION: pLVX-IRES-ZsGreen1 vectors which contain NCX1 promoter were constructed and confirmed by PCR. Virus could be packaged, purified and concentrated successfully. The recombinant lentivirus to transduce miPS was sorted by flow cytometry. In contrast to NCX1-/GFP- cells, NCX1+/GFP+ cells were differentiated and developed prominent beating areas with sustained contractile activity for additional 4 days, and demonstrated positive expression of gap communication marker CX43 and cardiac troponin. The expressions of GATA4, MEF2c and Nkx2.5 in the NCX1+ cells were 4.2, 7.5, and 2.5 times those in NCX1- cells. Results showed the NCX1 promoter can promote the cardiac differentiation of miPS.


中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程


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Key words: stem cells, multipotent stem cells, mice, sodium-calcium exchanger, lentivirus

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