Fig. 1. Activity of the Activin/Nodal/TGFß signalling pathway and consequences of Activin/Nodal signalling inhibition on hESC pluripotency. (A) Nuclear co-localisation of Smad2 (upper panel, green fluorescence) and Smad3 (lower panel, green fluorescence) with Oct-4 (red fluorescence) in hESCs grown on feeders. Nuclei stained with Hoechst 33258 dye (blue fluorescence). Combined Smad and Oct-4 staining (right panels). Bar, 50 µM. (B) Number of hESCs colonies generated after stable transfection of pTP6 expression vectors for the human recombinant Green Fluorescent Protein (hrGFP), Lefty2 (Lefty) and Cerberus-Short. hESCs were transfected using Lipofectamine 2000 (see Materials and Methods) and the numbers of colonies generated were determined after 10 days of puromycin selection. In the pTP6 vector, the expression of the transgene is linked to the expression of the puromycin resistance gene though an IRES (internal ribosome entry site). Therefore, all the hESC colonies resistant to puromycin expressed the relevant transgene. (C) Expression of Activin, Nodal and TGFß1 in feeder cells, grown in the presence (+) or absence (-) of FGF2, and in hESCs and EBs that had differentiated for 14 days in CDM, using reverse-transcriptase PCR. ß2 microglobulin (ß2M) was used as a loading control. (D) Effect of the Activin inhibitor follistatin on hESC pluripotency. Wild-type-, Lefty-, Cerberus-Short- and Nodal-hESCs were grown for 10 days (1 passage) in the presence of 0, 100 or 200 ng/ml follistatin. FACS was used to determine the fraction of Tra-1-60-expressing cells. Values represent the mean and standard deviation of three separate experiments.