Fig. 6. Generation of mesoderm and the anterior PS from genetically unmanipulated hES cell lines, KhES-3 and HES-3 cells. (A) Phase-contrast microscopy of KhES-3 cells, treated with BIO at different concentrations for 3 days. Scale bar: 100 µm. (B,C) The qPCR analysis on RNA isolated after 3 days from untreated KhES-3 cells (vehicle, v) or BIO-treated cells (BIO, 5 µM) with or without Noggin (Nog; 250 ng/ml) for 3 days was performed using specific primers for the genes indicated. (D) Expression of N-cadherin and FOXA2 in BIO-treated KhES-3 cells. The hES cells were cultured with BIO (5 µM) in the presence or absence Noggin for 3 days, and stained with specific antibodies as indicated. Scale bar: 100 µm. (E) Phase-contrast microscopy and localization of β-catenin in BIO-Acetoxime-treated HES-3 cells. Cells were cultured for 3 days with BIO-Acetoxime that exhibits greater selectivity for GSK3 than for BIO (left panels, phase-contrast images), and subjected to immunostaining with anti-β-catenin antibody (right panels). Scale bars: 100 µm. (F) qPCR analysis of RNA isolated from HES-3 cells, with or without BIO-Acetoxime (0-10 µM) for 3 days, was performed as described in B. (G) Expression of N-cadherin and FOXA2 in BIO-Acetoxime-treated HES-3 cells. The HES-3 cells were cultured with BIO-Acetoxime (10 µM) in the presence or absence Noggin for 3 days, and subjected to immunostaining with anti-N-cadherin and anti-FOXA2 antibodies. Scale bar: 100 µm.