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STEM_95_sm_suppinfofigure1.tif2124KSupporting Information Figure 1. Wnt3a induces of hESC mesendoderm formation, cell viability, and cardiomyogenesis in cell line-independent manner. H1 (A-C) or H7 (D-F) ESC were treated with control or Wnt3a CM for 48 hours and analyzed for mesendoderm formation (A & D), EB formation (B & E), and cardiomyogenesis (C & F).
STEM_95_sm_suppinfofigure2.tif1771KSupporting Information Figure 2. A. Human ESC were treated with vehicle, rWnt1 (300 ng/ml), rWnt2 (300 ng/ml), rWnt3a (100 ng/ml), rWnt7a (300 ng/ml), or BIO (300 nM) for 48 hours, harvested and subjected to RT-PCR analysis detecting Brachyury gene expression. B. Cells were treated with vehicle, IGF1 (100 ng/ml), PMA (200 nM), ISO (5 mM), or rWnt3a (30 ng/ml) for 48 hours, harvested and subjected to RT-PCR analysis detecting Brachyury gene expression. C. Cells were treated as in 2A-B and cultivated as described in Materials and Methods. On day 6, cells were visualized under the microscope.
STEM_95_sm_suppinfofigure3.tif2026KSupporting Information Figure 3. Effects of β-catenin/canonical signaling pathway on hESC EB formation, mesendoderm formation, and cardiomyogenesis. A. Human ESC were treated with control or Wnt3a CM for 48 hours, fixed, and immunostained for Brachyury (red) and β-catenin (green). B-C. Cells were treated as described in 3A, harvested, and subjected to immunoblot analysis probing for total or active β-catenin. Fold changes of normalized signals of total and active β-catenin are shown as means of two distinct experiments. D. Cells were treated with vehicle, IGF1 (100 ng/ml), PMA (200 nM), ISO (5 mM), or rWnt3a (30 ng/ml) for 48 hours, harvested, and subjected to immunoblot analysis probing for total β-catenin.
STEM_95_sm_suppinfofigure4.tif1949KSupporting Information Figure 4. Suppression of stem cell genes. Signals were derived from microarray analysis data. Fold changes in detection signals of day-23 beating clusters against undifferentiated hESC are shown for genes solely expressed in stem cells. Data are shown as mean±SD, n=4, #p<0.01.
STEM_95_sm_suppinfofigure5.tif1351KSupporting Information Figure 5. Wnt3a enhanced expression of activating but not inhibiting TGFβ family related genes. Signals for these genes were obtained from microarray analysis. Fold changes in detection signals of day-2, control- or Wnt3a-treated against undifferentiated hESC are shown for genes implicated in activating or inhibiting TGFβ signaling pathway. Data are shown as mean±SD; n=4; *p<0.05.
STEM_95_sm_suppinfofigure6.tif2047KSupporting Information Figure 6. Beating clusters exhibited little or no increase in none-cardiac related genes. Signals shown represent data derived from microarray analysis. Fold changes in detection signals of day-23 differentiated against undifferentiated hESC are shown for genes specific to liver (A), pancreatic (B), lung (C), blood (D), or neural cells (E). Data are shown as mean±SD, n=4. Broken line represents no change (1-fold change).
STEM_95_sm_suppinfofigure7.tif1919KSupporting Information Figure 7. Beating clusters exhibited an increase in genes characteristic of other cells. A-C. Signals shown represent data derived from microarray analysis. Fold changes in detection signals of day-23 differentiated against undifferentiated hESC are shown for genes specific to endothelial (A), epithelial (B), or fibroblast (C) cells. Data are shown as mean±SD, n=4. D. Human ESC were treated with Wnt3a CM for 48 hours and cultivated as described in Materials and Methods. Beating clusters (day 16-30) were microdissected, dissociated into single cells and cultivated on 0.1% gelatin-coated slide for another 3 days. Cells were fixed and immunostained with antibodies against Nkx2.5 and Vimentin or smooth muscle Actin (SMA). Images were taken using fluorescent microscope with 20X objective lens.
STEM_95_sm_suppinfotable1.tif605KSupporting Information Table 1. Primers for Taqman real time PCR analysis.
STEM_95_sm_suppinfotable2.tif1618KSupporting Information Table 2. Primers for SYBR Green real time PCR analysis.

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