Simplified three-dimensional culture system for long-term expansion of embryonic stem cells

Figure 5 Three-dimensional grown embryonic stem cells were subcultured under two-dimensional culture conditions expressed normal levels of pluripotent markers. A: Light micrographs depicting cell morphology of the initial ESCs used for encapsulation (A1), cultured in three-dimensional (3-D) scaffolds for 2 wk (A2), and subsequently passaged 5 times in 2-D culture (A3); B: Expression of Oct4, Nanog, and Klf4 in ESCs grown in 2-D culture, 3-D grown ESCs, and 3-D grown ESCs, which were subsequently passaged 5 times in 2-D culture as determined by qRT-PCR. Results were expressed as the fold expression ± SE (n = 3) normalized to reference genes Gapdh and β-Actin where a significant (^aP < 0.05 and ^bP < 0.01) increase of marker expression was compared to initial 2-D grown ESCs, using one-way ANOVA and Tukey multiple comparisons test. ESCs: Embryonic stem cells; MEF: Mouse embryonic fibroblasts; qRT-PCR: Quantitative real time polymerase chain reaction; ANOVA: Analysis of variance.