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

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

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

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

Histology of mouse limb skeletal muscle at embryonic day E14.5.

<p>Cross sections of the lower hind limb of a WT fetus (A-C), a RYR1^+/- fetus (D-F), a RYR1^-/- fetus (G-I), a Ca_v1.1^+/- fetus (J-L), and a Ca_v1.1^-/- fetus (M-O), respectively. At E14.5, the skeletal muscle of the hind limb of a WT fetus (A, B) as well as of a RYR1^+/- fetus (D, E) already harbor muscle fascicles (surrounded by dotted line) consisting of numerous muscle fibers while myoblasts were virtually absent. In contrast, the skeletal muscle of the hind limb of a RYR1^-/- (G, H), a Ca_v1.1^+/- (J, K), and a Ca_v1.1^-/- (M, N) fetus, respectively, exhibits disorganization (asterisks) or complete absence of muscle fascicles and numerous myoblasts. Immunohistochemistry with anti-activated caspase-3 reveals prominent apoptosis only in nuclei of the myotubes of a Ca_v1.1^-/- fetus at E14.5 (O, arrows). H&E staining (A, B, D, E, G, H, J, K, M, and N); original magnification x100 (A, D, G, J, M) and x200 (B, E, H, K, N). Immunohistochemistry with rabbit anti-mouse activated caspase-3 (clone C92-605; BD Biosciences) and slight counterstaining with hemalum; original magnification x400. Scale bars correspond to 100 μm in all microphotographs.</p

Gross fetal morphology at E14.5 and E18.5.

<p>Photographs of whole fetuses were obtained at E14.5 and E18.5 for RYR1^+/+ (WT), RYR1^+/- and RYR1^-/- littermates (A), as well as for Ca_v1.1^+/+ (WT), Ca_v1.1^+/- and Ca_v1.1^-/- littermates (B).</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

DEGs specific for the E14.5 to E18.5 development of WT, RYR1^-/- or Ca_v1.1^-/- skeletal muscle.

<p>(A) A Venn diagram, showing the number of DEGs identified in the MA analyses at E18.5 compared to E14.5 in WT, RYR1^-/- and Ca_v1.1^-/- limb skeletal muscle. Numbers in the overlapping and non-overlapping areas represent the amount of shared and not shared DEGs between genotypes, respectively. Wiki Pathways (B, C, D), GO BP (E, G, I) and GO CC (F, H, J) enrichment analyses of the DEGs found exclusively in WT (483 DEGs, blue charts), RYR1^-/- (91 DEGs, yellow charts) and Ca_v1.1^-/- (171 DEGs, red charts) from E14.5 (control) to E18.5, respectively. The ten most significantly enriched categories for each analysis are shown. Enrichment analyses (B–J) were performed via the Enrichr online tool [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194428#pone.0194428.ref031" target="_blank">31</a>], length of the bars is proportional to the significance (p-value). Gray bars in (J) indicate a p-value ≥ 0.05.</p

Analysis of all DEGs found in skeletal muscle development from E14.5 to E18.5.

<p>GO BP (A, C and E) and Wiki Pathways (B, D, F) enrichment analyses of all DEGs found in WT (A, B), RYR1^-/- (C, D) and Ca_v1.1^-/- (E,F) from E14.5 (control) to E18.5. The ten most significantly enriched categories for each analysis are shown. Enrichment analyses (A–F) were performed via the Enrichr online tool [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194428#pone.0194428.ref031" target="_blank">31</a>], length of the bars is proportional to the significance (p-value).</p

Biological processes affected by the RYR1^-/- and Ca_v1.1^-/- mutations at E14.5.

<p>GO BP enrichment analyses were performed for the DEGs identified in the RYR1^-/- (A) and Ca_v1.1^-/- (B) samples when compared to WT littermates samples at E14.5. The ten most significantly enriched categories for each analysis are shown. Arrows indicate categories presented as heat maps in (C) and (D). The enrichment analyses was performed via the Enrichr online tool [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194428#pone.0194428.ref031" target="_blank">31</a>], length of the bars represents the significance (p-value). Heatmaps were generated for the DEGs enriched in the “Regulation of neuron differentiation” biological process in RYR1^-/- samples (C) and for the DEGs enriched in “Muscle contraction” biological process in Ca_v1.1^-/- samples (D). The heatmaps were generated from the MAs intensity levels of each gene via ClustVis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194428#pone.0194428.ref032" target="_blank">32</a>]. Hierarchical average linkage clustering using the Euclidean distance was performed for all rows and columns.</p