Figure 2.

Characterization of immature syNP4 ES cell derived-CMs. (a) The purity and maturity of ESC-CMs at Day 11 were estimated by costaining with anti-α-SMA (red) and anti-α-actinin (sarcomeric) (green) antibodies. All cells express α-SMA, while 90 ± 2% cells are α-actinin positive. The right figure is a high magnification of the box in the left one. Arrows mark cells with α-SMA positive and α-actinin negative staining. (b) The DNA content of puromycin-selected ESC-CMs generated by mass culture was determined following PI staining. In these experiments (n = 3), the histogram indicates that Day 9 cells contain a higher percentage of G2/M phase cells than Day 14 cells. The number of G0/G1 cells also tends to increase with time of differentiation (P > 0.05). (c) Relative to controls, immature ESC-CMs incubated with 1.5 μM Jc1 had a strong increase in red fluorescence, which based on membrane potentials is indicative of live cells. Moreover, lack of change in membrane potential in either cell population indicates that the data shown from the MTG assay is directly comparable. (d) Typical results from MTG-stained cells assessed by flow cytometry. The green fluorescence signal is directly proportional to the mitochondrial content. As shown in the figure and table, the mitochondrial content increases with time of differentiation. (e) Cell viability of ESC-CMs cultivated under hypoxic and nutrient deprivation conditions. Data are presented as a percentage of control values.