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KMID : 0368120090390050198
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Korean Circulation Journal
2009 Volume.39 No. 5 p.198 ~ p.204
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Enhanced Cardiomyogenic Differentiation of P19 Embryonal Carcinoma Stem Cells
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Yang Ji-Hyun
Lim Do-Sun
Park Chi-Yeon
Choi Ji-Hyun
Ko Seok-Jin
Kim Beom-Suk
Kim Hyun-Seo
Park Sang-Heon
Hong Do-Ran
Hong Soon-Woong
Seung-Cheol Choi
Hong Sun-Jun
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Abstract
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Background and Objectives : We investigated the effects of different concentrations of serum, 5-azacytidine, and culture time on the cardiomyogenic differentiation of P19 embryonal carcinoma stem cells in the course of developing an efficient protocol for generating the cardiomyogenic lineage.
Materials and Methods : P19 cells were plated at a density of 1×106 cells on 10-cm bacterial dishes for 96 hours in the presence of 1% dimethyl sulfoxide to form embryoid bodies. The embryoid bodies were cultured in medium with 2% or 10% fetal bovine serum for an additional 10 or 15 consecutive days in the presence of 0, 1, or 3 μM 5-azacytidine.
Results: Quantitative real-time polymerase chain reaction (PCR) analysis showed that the messenger ribonucleic acid (mRNA) expression of cardiac muscle-specific genes, such as GATA4, α-actin, α-myosin heavy chain, and cardiac troponin T, were significantly higher in the 15-day culture groups than in the 10-day culture groups. Furthermore, the cardiac muscle-specific genes were expressed more in the high-serum groups compared to the low-serum groups regardless of the culture time. Cardiomyogenic differentiation of the P19 cells was most effective in 1 μM 5-azacytidine regardless of the serum concentrations. In addition, the stimulation effects of 5-azacytidine on cardiomyogenic differentiation were more significant under low-serum culture conditions compared to high-serum culture conditions. Cardiomyogenic differentiation of P19 cells was further confirmed by immunostaining with cardiac muscle-specific antibodies.
Conclusion: Taken together, these results demonstrated that cardiomyogenic differentiation of P19 cells was enhanced by a combination of different experimental factors.
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KEYWORD
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Cell differentiation, Embryonal carcinoma stem cells, Myocytes, cardiac, Serum
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