qRT-PCR analyses of various genes from ES, MP, SP, and SPTL cells. (A) Expression of stem cell marker gene mRNAs, Oct4, Nanog, and Rex1. Relative expression is shown based on the expression level of ES cells as 1. p < 0.0001 for ES versus MP, SP, or SPTL for all genes. (B) Expression of Gata4 and Gata 6 mRNAs. Relative expression is shown based on the expression level of ES cells as 1. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 for ES vs. MP, SP, or SPTL. (C) Expression of various mRNAs encoded by thyroid differentiation marker genes as indicated. Relative expression is shown based on the expression level of MP cells as 1. p < 0.0001 for MP versus SP or SPTL for all genes. (D) Expression of mRNAs encoded by various thyroid differentiation marker genes in SPTL cells compared with ES cells. Relative expression is based on the expression level of ES cells as 1. p < 0.0001 for ES versus SPTL for all genes. All cells used in these experiments were cultured in the presence of 10% serum. For each mRNA, mean ± SD from representative experiment in triplicate are shown. Note that qRT-PCR was carried out at least twice using samples prepared at different times. Similar results to those shown were obtained. Statistical analysis by unpaired t-test.