Spheroid formation and growth in dynamic and static conditions.

<p>(<b>A</b>) Top panels: eGFP-expressing cell aggregates immediately after their separation from EBs cultured in a rotary or in a static condition. Bottom panels: rotary and static cultures of 2-day-old spheroids. (<b>B</b>) Comparative diagrams of spheroid growth in SFs and in Petri dishes. The mean of the spheroid diameter ± standard error of the mean (SEM) are plotted. The sign (***) indicates extremely statistically significant differences (p<0.001) in spheroid size between the dynamic and static cultures.</p

Expression of eGFP and hepatic proteins in spheroid-derived adherent cultures.

<p>(<b>A</b>) Cell colonies outgrowing on collagen Type I matrix. Down-regulation of eGFP fluorescence during the course of the colony growth is evident. (<b>B</b>) Expression of hepatic proteins, using fluorescent immunoassay. For better view of Ecad and lst-1 expression patterns across the cells, the corresponding images are displayed at a magnification ×400. Respective staining of primary hepatocytes (panels <i>a–c</i> and <i>e</i>) or of a liver tissue section (panel <i>d</i>) are shown for comparison. Alexa Fluor® 555-conjugated IgG served as a secondary antibody; cell nuclei were visualized by Hoechst 33342 staining. Merged Alexa Fluor® 555/Hoechst 33342 images are shown. Samples treated with an isotype control antibody instead of the primary antibody stained negatively (data not shown).</p

Glycogen storage and ICG up-take in spheroid-derived adherent colonies.

<p>(<b>A</b>) Glycogen-storing cells in a 12-day-old spheroid-derived adherent culture. (<b>B</b>) Cell cluster in the culture, reversibly up-taking ICG.</p

Engrafted spheroid-derived cells within recipient liver tissue.

<p>(<b>A</b>) eGFP-fluorescent cells and cell clusters observed in liver sections one and two weeks after spheroid transplantation. (<b>B</b>) Expression of Ecad in engrafted cells. “Live” eGFP and merged Alexa Fluor® 555/Hoechst 33342 images are shown.</p

Gene expression profiles of dynamic spheroid cultures, ESCs, and fetal and adult liver tissue.

<p>Due to a big difference between expression values of single genes in particular culture and tissue types, qRT-PCR data are displayed for each gene separately using a logarithmic scale on the y-axes. Mean relative gene expression values ± standard error of the mean (SEM) are plotted. For a better overview, the mean values are additionally listed in tables below the diagrams. Abbreviations ESC, Spher d0, Spher d2, FL, and AL refer to undifferentiated ESCs, 0-day-old and 2-day-old spheroids and to fetal and adult liver, respectively. Statistical significance of the PCR data was evaluated using IBM® SPSS software. Symbols (*), (**) or (***) depict a statistically significant (p<0.05), a highly statistically significant (p<0.01) or an extremely statistically significant (p<0.001) difference in gene expression values, respectively. In control reverse transcription-negative samples and in none-template-containing blank samples, no PCR products have been detected (data not shown).</p