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Enhanced Cardiomyogenic Differentiation of P19 Embryonal Carcinoma Stem Cells
양지현,김범석,김현세,박상현,홍도란,홍순웅,최지현,박치연,최승철,홍순준,임도선 대한심장학회 2009 Korean Circulation Journal Vol.39 No.5
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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