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

Figure 3 Expression of pluripotent markers in three-dimensional grown embryonic stem cells. ESCs in scaffolds prepared with Dex-SH with 33% thiol substitution were cultured and analyzed for the expression of selected markers, Oct4, Nanog, Klf4, and Sox2 using qRT-PCR at week 0, 1, 2, 3, and 6. Results of experiments 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 pluripotent marker expression was compared to initial 2-D grown ESCs (day 0), using one-way ANOVA with the non-equal variance hypothesis and Dunnett’s multiple comparisons test. Results depicted showed upregulation of Oct4, Nanog, and Klf4, but Sox2 was maintained at levels similar to the control. Expression of Oct4 and Nanog was successively and significantly higher at week 1, 2 and 3 until the onset of degradation (week 6), at which point their expression was decreased to initial levels. ESCs: Embryonic stem cells; Dex-SH: Thiol funtionalized dextran; qRT-PCR: Quantitative real time polymerase chain reaction; 2-D: Two-dimensional.