Figure 6.

Exogenous NO arrests the differentiation induced by bFGF withdrawal in hESCs cultured in a feeder-free system. (a) Upper panel: Representative phase contrast images showing the morphology of hESCs cultured for five passages in the presence (left) or the absence (middle) of bFGF and in the absence of bFGF plus 2 μM DETA-NO (right). Images are representative of 10 experiments. Lower panel: immunofluorescence images of cells positive for Oct4 in hESCs cultured for five passages in the presence (left) or absence (middle) of bFGF or in the absence of bFGF plus 2 μM DETA-NO (right). Images are representative of three experiments. Scale bars are 20 μm. (b) qRT-PCR analyses of Oct4, Sox-2 and Nanog in H181 hESCs cultured for 10 days under the indicated conditions. β-actin was used as the endogenous control and Ct values were normalized with respect to the Ct of cells cultured in the presence of bFGF. Data are the mean ± S.E.M. of three experiments. ^*P≤0.005 versus cells cultured in the absence of bFGF. ^**P≤0.005 versus cells cultured in the presence of LIF. (c) Western blot analysis of Oct4, Sox-2 and Nanog in H181 hESCs cultured for 10 days under the indicated conditions. Representative images and densitometry quantifications are from five independent experiments. (d) qRT-PCR analysis of Brachyury and Gata4 expression in H181 hESCs . β-actin was used as the endogenous control and Ct values were normalized with respect to the Ct of cells cultured in the absence of bFGF. Data are the mean ± S.E.M. of three experiments. ^*P≤0.005 versus cells cultured in the absence of bFGF. (e) Confocal images of cells positive for SSEA-4 in hESCs cultured for five passages in the presence (left panel) or absence (middle panel) of bFGF or in the absence of bFGF plus 2 μM DETA-NO (right panel). Nuclei were counter-stained with DAPI. Images are representative of three experiments. Scale bars are 20 μm