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

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HEP_23506_sm_Supportingfig1.TIF8070KSupplementary Figure 1: Effect of diverse signaling pathway inhibitors on endoderm differentiation. H9 cells were grown for 3 days in culture conditions inductive for endoderm differentiation (CDM + Activin + FGF2 + BMP4 +Ly) (Day1, Day2 and Day3). Alternatively hESCs were grown in culture conditions inductive for endoderm differentiation for 2 days and then for an additional day in the absence of Activin/Nodal signaling (SB431542 10 μM + FGF2 + BMP4 + Ly) (SB), in the absence of BMP signaling (Noggin 200 ng/ml + Activin + FGF2 + Ly)(Noggin), in the absence of FGF signaling (SU5402 10 μM + Activin + BMP4 + Ly)(SU5402) and in the absence of PI3Kinase inhibition (Activin + FGF2 + BMP4) (AFB). RNAs were extracted at the indicated time point, and then Q-PCR analyses were performed to detect the genes denoted. Data represent the mean of three independent experiments and error bars indicate standard deviation.
HEP_23506_sm_Supportingfig2.TIF6448KSupplementary Figure 2: Immunostaining analyses showing the absence of Oct-4 expression in hESCs grown for 3 days in culture conditions inductive for endoderm. HESCs grown in the presence of usual dose of Activin (10 ng/ml) or in the presence of high dose of Activin were used as positive controls.
HEP_23506_sm_Supportingfig3.TIF2701KSupplementary Figure 3: Endoderm cells generated from hESCs express Wnt3. H9 cells were grown for 3 days in culture conditions inductive for endoderm differentiation (CDM + Activin + FGF2 + BMP4 +Ly). RNAs were then extracted and Q-PCR analyses were performed to detect Wnt3 transcripts. Data represent the mean of three independent experiments and error bars indicate standard deviation.
HEP_23506_sm_Supportingfig4.TIF4966KSupplementary Figure 4: Urea secretion from Hepatic cells generated from hESCs. Urea was measured in the media after 30 min (light grey column) and 60 min (dark grey column) of incubation in presence of ammonium. Human Fetal Hepatocytes were used as positive control.
HEP_23506_sm_Supportingtext.doc43KSupplementary Material and Methods.

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