Additional Supporting Information may be found in the online version of this article.

sc-12-0751_sm_SupplFigure1.pdf239KSupplementary Figure 1 Cdx2+ cells are proliferative cells EdU incorporation tests assayed for 3 hours (d12) revealed that 45.6±1.51% (n=3) of the ES cell-derived Cdx2+ (green) cells were EdU positive (red). DAPI (blue) indicate nuclei. Scale bars: 100 μm.
sc-12-0751_sm_SupplFigure2.tif2027KSupplementary Figure 2 MEF cells did not express Cdx2. (A) Immunocytochemical analysis of MEF treated with BIO and DAPT. RT-PCR analyses of intestinal markers on day 20. ES cells differentiated on MEF without BIO and DAPT. Scale bars: 100 μm. (B) Immunocytochemical analysis using specific isotype negative control IgGs. Scale bars: 100 μm.
sc-12-0751_sm_SupplFigure3.tif1054KSupplementary Figure 3 There were no undifferentiated ES on day 15 under feeder free condition. Immunocytochemical analysis of undifferentiated ES cells and ES cells-derived cells on day 15 are shown. Nanog+ cells were not observed on day 15. Scale bar: 100 μm.
sc-12-0751_sm_SupplFigure4.tif136KSupplementary Figure 4 FGF inhibitor SU5402 treatment increased initial induction of CDX2. (A) A schematic presentation of the experiment. khES3 cells were cultured on feeders, added with SU5402 from day 10 to 30, and were assayed for CDX2 transcript by real time PCR. (B) Initial induction of CDX2 was potentiated by SU5402, but CDX2 expression in control caught up later. **p < 0.01 versus control (white bars) by Student's t-test (N=3-5).
sc-12-0751_sm_SupplFigure5.tif2100KSupplementary Figure 5 FGF2 inhibited intestinal differentiation in Caco2 cells. (A) A schematic presentation of the experiment. Caco2 cells were cultured to confluency, then differentiated at the presence or absence of FGF2. (B) Bright field views of Caco2 cells under different FGF2 conditions. FGF2 inhibited dome-like morphology of mature intestinal endoderm. (C, D) Flow cytometeric analysis of Caco2 cells treated with graded concentrations of FGF2. Differentiation into CDX2high population was inhibited by 250 ng/ml FGF. (E) RT-PCR analysis of Caco2 cells treated with FGF2. FGF2 inhibited CDX2 expression in Caco2 cells. **p < 0.01 versus control (white bar) by Student's t-test (N=3). Scale bars: 100 μm.
sc-12-0751_sm_SupplTable1.pdf76KSupplementary Table 1 The primer sequences and number of cycles. The primer sequences, cycle numbers for semi-quantitative RT-PCR and real time PCR are shown.
sc-12-0751_sm_SupplTable2.pdf87KSupplementary Table 2 The growth factors tested for potentiating the induction into Cdx2-positive intestinal cells. The growth factors and suppliers used in the experiments are shown.

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