Functional Heterogeneity of Embryonic Stem Cells Revealed through Translational Amplification of an Early Endodermal Transcript
Figure 3
Venus positive population may represent an early state in PrEn differentiation.
(A) Venus positive cells express Oct4, but not Nanog. Colonies of HV cells were fixed and immunostained for both Oct4 and Nanog. Primary antibodies specific to Oct4 and Nanog were detected using Alexa 568 conjugated secondary antibodies (red). Images include Venus fluorescence, antibody staining, overlay of Venus and antibody, and bright field for each cell line and the indicated antibodies. (B) Quantitative RT-PCR showing the relative expression of endodermal and pluripotency genes between Venus positive and negative cells obtained from the SSEA-1 positive fractions of two HV clones following flow cytometry. Quantitative PCR analysis was performed to compare transcript levels of Venus with PrEn (Hex, Gata4, Gata6, Dab2, Sox7, Hnf4α, and Pdgfrα), pluripotency (Nanog, Klf4, Rex1, Stella, and Pou5f1) and other lineage (T, Fgf5, Eomes, Flk1, Mixl1, Cdx2, Sox1, Pax6, and Six3) markers in purified cell fractions. Venus positive fractions are represented as green bars and Venus negative black bars. Transcript levels were normalised to the TBP value obtained for each sample. Normalised values are related to the level obtained for the Venus positive fraction in each case.