Enhanced Cardiomyogenic Differentiation of P19 Embryonal Carcinoma Stem Cells

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 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. CONCLUSION:Taken together, these results demonstrated that cardiomyogenic differentiation of P19 cells was enhanced by a combination of different experimental factors.

Cardiac side population cells exhibit endothelial differentiation potential

Recent studies have shown that side population (SP) cells, isolated from adult myocardium, represent a distinct cardiac progenitor cell population that exhibits functional cardiomyogenic differentiation. However, information on the intrinsic characteristics and endothelial potential, of cardiac SP cells, is limited. The present study was designed to investigate whether cardiac SP cells exhibit endothelial differentiation potential. The cardiac SP cells more highly expressed the early cardiac transcription factors as well as endothelial cell markers compared to the bone marrow-SP cells. After treatment with VEGF, for 28 days, cardiac SP cells were able to differentiate into endothelial cells expressing von Willebrand factor as determined by immunocytochemistry. Furthermore, expression of endothelial cell markers increased several-fold in VEGF-treated cardiac SP cells compared to the control group when assessed by real-time PCR. We also confirmed that cardiac SP cells provided a significantly augmented ratio of ischemic/normal blood flow, in the cardiac SP cell-transplanted group compared with saline-treated controls on postoperative days 7, 14, 21 and 28, in a murine model. These results show that cardiac SP cells may contribute to regeneration of injured heart tissues partly by transdifferentiation into angiogenic lineages.