Effects of DMSO on the Pluripotency of Cultured Mouse Embryonic Stem Cells (mESCs)

Figure 3

Exposure to small percentages of DMSO for 48 hours does not negatively affect pluripotency marker expression or the differentiation potential of mESC. (a) Representative immunofluorescence images of plated colonies presenting the pluripotency marker Nanog (red) and counterstained with Hoechst 33342 (blue) (600x magnification) after a 48 h incubation with/without DMSO. (b) RT-PCR analysis for Oct4, Nanog, Rexo1, and Essrb mRNA gene expression normalized for endogenous beta-actin (Actb) at the 48 h time point. At least four independent experiments were performed, and the results are presented as . (c, d) Relative protein expression of the pluripotency factors Oct4 and Nanog evaluated by Western blot analysis and normalized by β-actin expression at the 48 h time point. At least three independent experiments were performed, and the results are presented as . (e–g) Embryoid bodies (EBs) were generated from mESCs cultured for 48 h in the presence/absence of DMSO. After the differentiation protocol, every condition was able to generate fully differentiated cultures as shown in (e) by phase-contrast microscopy (100x magnification). (f, g) Western blot analysis and quantification of the protein levels of the three embryonic leaflet markers α-FP, SMA, and β-3-TUB normalized by the expression of the loading control β-actin. At least four independent experiments were performed, and the results are presented as . Statistical significance was considered when .

(a)

(b)

(c)

(d)

(e)

(f)

(g)