Department of Cardiology, College of Medicine, Korea University, Seoul, Korea.
Korean Circ J. 2009 May;39(5):198-204. doi: 10.4070/kcj.2009.39.5.198. Epub 2009 May 28.
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.
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.
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.
Taken together, these results demonstrated that cardiomyogenic differentiation of P19 cells was enhanced by a combination of different experimental factors.
我们研究了不同浓度血清、5-氮杂胞苷和培养时间对 P19 胚胎癌细胞向心肌细胞分化的影响，旨在开发一种有效的心肌细胞谱系生成方案。
将 P19 细胞以 1x10(6)细胞/10cm 细菌培养皿的密度铺板，在 1%二甲基亚砜存在的条件下培养 96 小时形成胚体。将胚体在含 2%或 10%胎牛血清的培养基中培养 10 或 15 天，同时加入 0、1 或 3μM 5-氮杂胞苷。
实时定量聚合酶链反应（PCR）分析显示，心肌特异性基因如 GATA4、α-肌动蛋白、α-肌球蛋白重链和心肌肌钙蛋白 T 的信使核糖核酸（mRNA）表达在 15 天培养组中明显高于 10 天培养组。此外，无论培养时间如何，高血清组的心肌特异性基因表达均高于低血清组。1μM 5-氮杂胞苷对 P19 细胞的心肌分化作用最有效，与血清浓度无关。此外，5-氮杂胞苷对心肌分化的刺激作用在低血清培养条件下比高血清培养条件下更为显著。P19 细胞的心肌分化进一步通过心肌特异性抗体的免疫染色得到证实。
综上所述，这些结果表明 P19 细胞的心肌分化是由不同实验因素的组合增强的。
