Oct4 and Pax6 expression in hESCs, TR−/S4+ cells and NPCs.

<p>(A) Western blotting image and quantitative histogram showing Oct4 and Nanog protein expression in the three cell types. (B) Flow cytometry analysis of Oct4 and Pax6 expression in the three cell types. Percentages of total Oct4 positive and high Oct4 expressing cells are indicated. (C) Immunostaining of Oct4 and Pax6 proteins in H7 hESCs, day 6 neural differentiation (ND-d6) and rosette-forming neuroepithelial cells. Scale bar = 20 µm. Arrows indicate cells with high Oct4 and low Pax6 expression; arrowheads indicate cells with high Pax6 and low Oct4. (D) Bisulphite DNA sequencing of the <i>Oct4</i> promoter region in hESCs, TR−/S4+ cells and NPCs. The transcription starting site and the corresponding location of CpG are indicated. Open and closed circles indicate unmethylated and methylated CpG, respectively.</p

Differentiation of H1 hESCs to definitive endoderm.

<p>H1 hESCs were treated with high levels of Activin A and LY294002 for 3 days as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037129#s4" target="_blank">Materials and Methods</a>. The cells were stained with Tra-1-81 and SSEA4 antibodies and analysed for their gene expression by q-RT-PCR at the indicated time points during the differentiation. (A) Expression of marker genes were analysed by qRT-PCR at the indicated time points during the differentiation. (B) Phase-contrast images and histogram of flow cytometry analysis on Tra-1-81 and SSEA4 antibody staining at the indicated time points. Scale bar = 100 µm.</p

Sequential loss of Tra-1-81 and SSEA4 expression during neural differentiation of hESCs.

<p>(A) & (B) Expression of Tra-1-81, SSEA4 and SSEA1 during neural differentiation of H1 (A) & H7 (B) hESCs respectively on the indicated days of differentiation. Phase-contrast images are shown. Scale bar = 50 µm. (C) Immunostaining of Tra-1-81 and SSEA4 in H7 hESCs and at day 6 of their neural differentiation. Scale bar = 20 µm. (D) Expression of Tra-1-81, SSEA4 and SSEA1 in hESC-derived neural progenitor cells. Days of the differentiation are indicated.</p

Gene expression profile in undifferentiated hESCs, TR−/S4+ cells and neural progenitor cells (NPCs).

<p>(A) Flow cytometry analysis of cells co-stained with Tra-1-81 and SSEA4 antibodies in the three stages of neural differentiation and in purified TR−/S4+ cells. (B) qRT-PCR analysis of marker gene expressions in TR−/S4+ cells, hESCs and NPCs. Standard deviations were calculated from at least three independent experiments. (C) RT-PCR analysis of LIFR in undifferentiated H1 hESCs, sorted TR−/S4+ cells, unsorted day 9 differentiated cells (9 d diff) and NPCs. Each lane represents an independent experiment.</p

Neural differentiation of TR−/S4+ cells.

<p>TR−/S4+ cells were further differentiated for in N2B27 medium supplemented with noggin for 1–2 weeks, then without noggin. (A) Phase-contrast images of further culture of TR−/S4+ in neural differentiation media for 3–4 weeks. (B–F) Immunostaining with the indicated antibodies at different time points: nestin and Sox1 (3–4 weeks), MAP2 and β-tublin III (6 weeks) and GFAP (15 weeks). Scale bar represent 100 µm.</p

Top 15 functions of the genes up- or down- regulated in TR−/S4+ and early EB cells.

*<p>Number in the bracket represents the position of gene ontology ranking in EB cells.</p

Differentiation of TR−/S4+ cells by cell aggregate formation.

<p>(A) Images of cell aggregates at day 3 and 7 of differentiation, respectively. EB-like structures are visible by day 7 (arrows). Scale bar = 100 µm. (B) Images of cell aggregates cultured for a further 7 days after disassociation. Scale bar = 100 µm. (C) qRT-PCR analysis of markers in hESCs, TR−/S4+ and their differentiated progeny (TR−/S4+ EB) two weeks after initiation of differentiation (1 week in suspension and 1 week after dissociation onto adherent dish). (D) Immunostaining with indicated antibodies on differentiated progeny of TR−/S4+ cells 1 week after dissociation onto coverslip. Scale bar = 50 µm.</p