Fig. 2. Effect of Activin and FGF on the pluripotent status of hESCs. (A) Efficiency of FGF and different members of the TGFß family in maintaining pluripotency marker expression of hESCs in feeder-free conditions. hESCs were grown in CDM for 7 days in the presence of different growth factors and then maintenance-of-pluripotency-marker expression was established by determining the number of colonies expressing Oct-4 using immunofluorescence. Colonies were allocated to three arbitrary categories corresponding to different levels of pluripotency marker expression: colonies containing a preponderance of cells expressing Oct-4 (100% Oct-4, yellow column and green fluorescence, upper right panel), colonies consisting of a relatively even mixture of cells positive and negative for Oct-4 expression (50% Oct-4, grey column and green fluorescence, middle right panel) and colonies containing few or no Oct-4 expressing cells and thus totally differentiated (0% Oct-4, blue column and bottom panel). Nuclei are stained with Hoechst 33258 dye (blue fluorescence). Bar, 100 µM. Values represent the mean and standard deviation of three separate experiments. (B) Expression of pluripotency and differentiation markers during differentiation of hESCs in the absence or presence of Activin (10 ng/ml), recombinant Nodal (recNodal) (50 ng/ml) and TGFß1 (1 ng/ml). RNAs were extracted every four days for 16 days (D4-D16), then reverse-transcriptase-PCR analysis was performed to detect the expression of the genes denoted. (C) Long term expression of the pluripotency markers Tra-1-60, SSEA-3, and SSEA-4 in hESCs grown either on feeder or in CDM supplemented with Activin (10 ng/ml) and FGF2 (12 ng/ml). hESCs were grown for 10 passages ( 40 days) in adherent conditions and then the fraction of pluripotent cells was established using FACS to detect expression of Tra-1-60 (upper panels), SSEA-3 (middle panels), and SSEA-4 (bottom panels). hESCs grown on feeder layers were used as positive controls (left panels). (D) Effect of the Activin/Nodal/TGFß receptor inhibitor SB431542 (SB) and the FGF receptor inhibitor SU5402 (SU) on hESCs grown in feeder-free conditions in CDM. Wild-type hESCs and Nodal-hESCs were grown for 7 days in CDM in the presence or absence of Activin and FGF, and in the presence or absence of the SB and SU inhibitors. FACS was used to determine the fraction of Tra-1-60 expressing cells. Values represent the mean and standard deviation of three separate experiments. (E) Effect of SB431542 (SB) and SU5402 (SU) inhibitors on Oct-4 expression of hESCs grown on Matrigel in feeder cell-conditioned medium. H9 cells were grown for 10 days in the absence (upper row) or in the presence of 20 µm SB (middle row) or 10 µm SU (lower row) inhibitors. The level of differentiation was established by immunofluorescence to determine the expression of Oct-4 (green fluorescence, right panels). Nuclei are shown by Hoechst staining (blue fluorescence). Bar, 100 µM.