Files in this Data Supplement:
Table S1. RT-PCR primer sequences and conditions.
Fig. S1. Chemical Structure of 1m.
Fig. S2. Treatment of hESCs with compound 1m induces differentiation and does not inhibit proliferation and viability. (A) Histogram plots of Shef-3 hESCs analysed by flow cytometry following immunostaining with antibodies towards Tra-1-60 and SSEA4. Cells were treated with BIO or 1m or left untreated as controls and cultured for 7 days on either MEFs or on Matrigel in mTeSR®1 media. Control histograms are shown in the inserts and percent positive cells are indicated. (B) XTT cell proliferation and viability assay. Shef-3 hESCs were cultured on MatrigelTM-coated 24 well trays in mTeSR®1 media containing serial dilutions of compound 1m. Cells were cultured for 6 days, with media and compound being refreshed every other day. Metabolic activity was measured by adding to each well, 250 µl of a solution containing 1 mg/ml XTT (2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) and 25 µM phenazine methosulphate. The plates were incubated for 4 hours at 37°C and the soluble formazan product generated by the metabolically active cells was measured at 450nm on a BioTek plate reader. For each experiment, the average reading from 4 replicates were determined and expressed as a fold increase over the DMSO alone-treated (0) samples. The graph indicates the mean and s.e.m. obtained from 3 independent experiments.
Fig. S3. Treatment of hESCs with compound 1m induces differentiation. Shef-1 hESCs were cultured on MEFs and treated with either BIO or 1m for 7 days. (A) Images show typical colonies formed. Scale bar represents 1 mm. (B) Histogram plots of hESCs analysed by flow cytometry following immunostaining with antibodies towards Tra-1-60 and SSEA4. (C) RNA was extracted and RT-PCR analyses performed using primers specific for OCT4 and NANOG. (D) Protein extracts were immunoblotted using an antibody specific to OCT4. Blots were stripped and reporobed with anti-SHP-2 antibodies to assess equal loading.
Fig. S4. Compound 1o induces differentiation and activates Wnt/β-catenin signalling. (A−D) Shef-3 hESCs were treated with the indicated concentration of 1o and cultured for 7 days on either MEFs or on MatrigelTM in mTeSR®1 media. (A) Images show typical colonies formed following the indicated treatments. (B) Histogram plots of hESCs analysed by flow cytometry following immunostaining with antibodies against the pluripotency markers Tra-1-60 or SSEA4. While the percentage of SSEA4 positive cells recorded for the 1o/MEF conditions remains high it is clear from the histogram plot that the peak fluorescence intensity has decreased compared to control. One possible explanation is that factors produced by the feeders slow loss of SSEA expression. (C) RNA was extracted from the cells and RT-PCR analyses performed using primers specific to the pluripotency genes OCT4 and NANOG and to the house-keeping gene β-ACTIN. (D) Immunoblotting was performed using an antibody specific to OCT4. Blots were stripped and reprobed with anti-GAPDH antibodies to assess equal loading. (E) Shef-1 hESCs were cultured either on MEFs or on MatrigelTM in mTeSR®1 media and treated with 1o for 30 min. Protein extracts were prepared and immunoblotting performed using antibodies detecting phosphorylated forms of β-CATENIN and ERK1/2. The same immunoblot in each case was reprobed for total β-CATENIN and ERK1 to assess loading. (F) TOPFlash luciferase reporter assay of Shef-1 hESCs treated for 24 hr with 1o at the indicated concentrations. Luciferase activity is expressed as a fold increase in activation compared with the normalised TOPFlash luciferase activity in untreated (Con) cells. A control (FOPFlash), containing mutant TCF binding sites, was also run in parallel. Data represent the mean +/- SEM of 3 independent experiments.
Fig. S5. Differentiation towards the DE induced by 1m and BIO. (A) Shef-1 hESCs were treated with 2 µM 1m for the times indicated and immunostained with antibodies recognizing the endodermal markers FOXA2 and SOX17. (B) Shef-3 hESCs were treated with 2 µM BIO for the times indicated and immunostained with antibodies recognizing FOXA2, SOX17 and HNF4α.
Fig. S6. Differentiation of BIO-derived DE cells to hepatocyte-like cells. Shef-3 hESCs were treated with 2 µM BIO for 5 days to induce differentiation to the DE. Cells were then cultured under hepatic induction and maturation conditions as outlined in Materials and Methods and Fig. 5A. (A) Expression of the early hepatic markers HNF4α, AFP and TTR in hESCs at stage II and stage III of differentiation were analysed by immunofluorescence. Scale bar represents 50 µm. (B) RT-PCR analyses of RNA prepared from cells at the indicated stages of differentiation, using primers specific to the genes indicated.
