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First published online September 22, 2005
doi: 10.1242/10.1242/jcs.02553

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Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells

Department of Surgery and Cambridge Institute for Medical Research, Addenbrooke's Hospital, University of Cambridge, Hills Road, Cambridge, CB2 2XY, UK

View larger version (41K): [in a new window]   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.

View larger version (153K): [in a new window]   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.

View larger version (33K): [in a new window]   Fig. 3. Interactions between FGF and Activin signalling pathways in hESCs. (A) Real-time quantitative PCR (QPCR) analysis of Oct-4 and Cripto expression in hESCs grown for four days in CDM supplemented with Activin (10 ng/ml) or FGF (40 ng/ml). Tra-1-60 positive and negative cells were sorted using FACS to separate undifferentiated and differentiated cells. Normalised Oct-4/PBGD (left panel) and Cripto/PBGD (right panel) mRNA levels were measured by QPCR. hESCs grown on feeders and differentiated cells from EBs were used respectively as positive and negative controls. Similar results were obtained in three independent experiments. (B) Effect of Cripto on hESCs grown in feeder-free conditions in CDM. Wild-type hESCs were grown for 7 days in CDM in the presence or absence of Cripto, 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. (C). Western blot analysis of Smad2/3 phosphorylation in hESCs grown in feeder-free conditions. hESCs were grown for 6 days in CDM supplemented with 10 ng/ml Activin and 12 ng/ml FGF2 without feeders. Then hESCs were grown in different culture conditions for 2 hours. HepG2 cells were used as a positive control. Nuclear proteins were extracted and the expression of the phosphorylated form of the Smad2 protein was analysed using western blot (upper panel). Alternatively, total cellular extracts were used to confirm that expression of the Smad2 protein was the same in all the conditions (lower panel). Similar results were obtained with hESCs grown on feeders (data not shown). (D) Western blot analysis of Smad1/5/8 phosphorylation in hESCs grown in feeder-free conditions in the presence or absence of BMP4 (100 ng/ml). hESCs were grown for 6 days in CDM supplemented with 10 ng/ml Activin and 12 ng/ml FGF2 without feeders. Then hESCs were grown in different culture conditions for 2 hours. Nuclear proteins were extracted and the expression of the phosphorylated form of the Smad1/5/8 protein was analysed using western blot (upper panel). Alternatively total cellular extracts were used to confirm that expression of the Smad2 protein was the same in all the conditions (lower panel). (E) Recombinant BMP4 activates the Tlx2-lux reporter in feeder-free conditions. H9 cells were transiently transfected with the Tlx2-lux vector. After transfection, cells were incubated 48 hours in the absence (Neg) or in the presence of the SU inhibitor (10 µM) or BMP4 (100 ng/ml) alone or combined with FGF2 (40 ng/ml) or SU inhibitor (10 µM). Normalised luciferase activity is expressed as the mean ± SD from three informative experiments.

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