Quantitative immunofluorescence of cell cultures at different time points during the DE differentiation protocol on the different ECMP substrates.

<p>(<b>a</b>) Quantification of number of cells, which were attached 24h after seeding on the respective ECMP substrates (day 1). (<b>b</b>) Quantification of the number of cells at the different days during DE differentiation. (<b>c</b>) Quantification of the fraction Sox17 positive cells different days during DE differentiation. (<b>d</b>) Quantification of the fraction Oct3/4 positive cells at different days during DE differentiation. (n = 3, mean ± S.E.M. and * indicate statistical significant differences, P<0.05). ECMP abbreviations are explained in legend in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145389#pone.0145389.g003" target="_blank">Fig 3</a>.</p

(a-d) DE differentiation of hES cells in microtitre plates on selected ECMP combinations.

<p>(<b>a-d</b>) Quantification of Sox17 positive cells and total cell number based on immunofluorescence image analysis of cells differentiated on different ECMP combinations in microtitre plates. n = 3–6, mean ± S.E.M.). * and ** indicate statistical significant differences to Fn, P<0.05 and P<0.005 respectively. Note that not all statistical significant differences are displayed in the graphs. Full data set is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145389#pone.0145389.s007" target="_blank">S3 Table</a>. Abbreviations are explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145389#pone.0145389.s005" target="_blank">S1 Table</a>. <b>(a)</b> Scatter plot of percentage of Sox17 positive cells and the total number of cells for each respective ECMP combination tested (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145389#pone.0145389.s007" target="_blank">S3 Table</a> for descriptions), <b>(b)</b> Scatterplot of percentage Sox17 positive cells and the total yield of Sox17 positive cells (calculated as percentage of Sox17 positive cells * total cell number /100). Linear regression of data excluding six outliers (out of 65) not considered following the linear correlation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145389#sec009" target="_blank">results</a>). r² = >0.95. <b>(c)</b> Percentage of Sox17 positive cells of a selected ECMP combinations. <b>(d)</b> The total cell number of a selected ECMP combinations.</p

The dynamic differentiation of hES cells to DE is influenced by ECMPs.

<p>(<b>a)</b> Schematic overview of the semi-optimal protocol for DE differentiation. hES cells were seeded and kept undifferentiated for 4 days to allow expansion. Subsequently the cells were treated with Wnt3a to direct the cells from the pluripotent stage into the mesendoderm stage, followed by 3 days with Activin A to direct the cells from the mesendoderm stage to the DE stage. <b>(b)</b> Representative immunofluorescence images from three independent experiments of cells cultured on the five different ECMP combinations at different time points during the DE differentiation protocol. The tested ECMP combinations were fibronectin (Fn), collagen II+fibronectin (Col2+Fn), collagen I (Col1), netrin 1+fibronectin (Ne+Fn) and vitronectin (Vn) (scale bar = 200μm). <b>(c)</b> Magnification of cultures on Col1 at day 5 (scale bar = 200μm).</p