Enhanced cardiomyogenic differentiation of P19 embryonal carcinoma stem cells.
OBJECTIVE
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.


METHODS
P19 cells were plated at a density of 1x10(6) 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 microM 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, alpha-actin, alpha-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 microM 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.


CONCLUSIONS
Taken together, these results demonstrated that cardiomyogenic differentiation of P19 cells was enhanced by a combination of different experimental factors.
Yang
Jihyun
J
Department of Cardiology, College of Medicine, Korea University, Seoul, Korea.
Ko
Seok-Jin
SJ
Kim
Beom-Suk
BS
Kim
Hyun-Seo
HS
Park
Sangheon
S
Hong
Doran
D
Hong
Soon-Woong
SW
Choi
Ji-Hyun
JH
Park
Chi-Yeon
CY
Choi
Seung-Cheol
SC
Hong
Sun-Jun
SJ
Lim
Do-Sun
DS