TaqMan Low-Density Array analysis of undifferentiated hES cells cultured on hFFs (hFF) or in suspension (sus).

<p>(A) Significantly lower and higher (p < 0.05) expression of genes in suspension culture relative to hFF culture. The expression values are dCt values relative to the mean expression of <i>ACTB</i>, <i>GAPDH</i> and <i>RAF1</i>, scaled by subtracting the dCt value from the lowest dCt+1 value. The value of dCt+1 was assigned to samples with no expression; thus blue (0) represents no expression, with increasing expression towards red. (B) Cell line-specific differences in gene expression between hFF and suspension culture conditions, with lower expression in suspension versus hFF cultures, and (C) higher expression in suspension versus hFF cultures (p < 0.05 for HS207 and HS360 cells). A list of gene names and abbreviations can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144029#pone.0144029.s011" target="_blank">S6 Table</a>.</p

Percentages of hES cell colonies exhibiting cells positive for DDX4.

<p>Cell colonies were divided into four groups according to their expression profile: no DDX4 expression, <25%, 25%–75% or >75% of DDX4-positive cells present per cell colony. A pool of three experiments (minimum 45 cell colonies) for each cell line and condition was evaluated to define DDX4 expression.</p><p>Percentages of hES cell colonies exhibiting cells positive for DDX4.</p

Expression of mRNA encoding the pluripotency markers <i>NANOG</i> and <i>POU5F1</i>, the germ cell marker <i>DDX4</i> and Sertoli cell markers <i>FSHR</i> and <i>VIM</i> by HS207, HS360 and HS401 cells cultured and differentiated in suspension.

<p>The relative level of mRNA was calculated by the ddCt procedure from the mean of triplicates and statistical analysis performed by way of One-way RM ANOVA. The error bars depict standard deviations. *p <0.05, **p <0.01, ***p <0.001 when comparing undifferentiated with differentiated cells. #p <0.05, ##p <0.01, ###p <0.001 when comparing BMP7-stimulated with unstimulated cells. For an explanation of the abbreviations, see the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144029#pone.0144029.g002" target="_blank">Fig 2</a>.</p

Immunofluorescence detection of WT1 in HS207, HS360 and HS401 cells cultured in suspension.

<p>Nuclear expression of WT1 in cells was observed in all hES cell lines after spontaneous differentiation (A, C, and E) as well as after BMP7-stimulation (B, D and F) in cells mainly located on the edge of the spheres. Nuclear expression of WT1 was observed in Sertoli cells present in adult human testis (G; arrow heads). Negative controls exhibited no specific WT1 staining in either the nucleus or cytoplasm (H and small inserts in a-f). Red: WT1 staining. Blue: DAPI staining marking the nucleus. Scale bars: 100 μm in A to H, and 50 μm in the small inserts.</p

Expression of tight junction proteins and changes in the permeability of the BTB of 8–10 weeks old SPF, GF and CBUT mice.

<p>(<b>A</b>) Representative Western blots for occludin, E-cadherin and β-catenin, and quantification of these blots relative to the levels of GAPDH (means ± SEM., n = 3–6 *<i>P</i><0.05) compared to the values for SPF mice. (<b>B</b>) Immunofluorescent staining for occludin (green) and ZO-2 (red) individually and merged with nuclear staining (DAPI – blue). The scale bar represents 5 µm. (<b>C</b>) Evans blue (EB – red) and nuclear staining (DAPI – blue) showing interstitial cells (arrowheads) and seminiferous tubules (arrows). Scale bar represents 10 µm.</p

Assessment of hormone levels and mRNA levels encoding steroidogenic proteins.

<p>(<b>A</b>) Intratesticular levels of testosterone and serum levels of LH and FSH in 8–10 weeks old SFP, GF and CBUT mice. (a) Levels of intra-testicular testosterone per gram testis weight measured with RIA. Each bar represents the mean ± SEM (n = 6–9). The actual values were 0.315±0.131 pmol/g for SPF mice, 0.025±0.003 pmol/g for the GF group and 0.739±0.517 pmol/g for CBUT mice *P<0.05, **<0.001 as determined by the Mann-Whitney Rank Sum Test for non-parametric independent data. (b) Levels of serum LH as determined by ELISA. Each bar represents the mean ± SEM. The actual values were 179±19 ng/ml for SPF mice, 105±25 ng/ml for the GF group and 167±50 ng/ml for CBUT mice *P<0.05 as assessed with the Student t-Test for independent data. (c) Levels of serum FSH as determined by ELISA. Each bar represents mean ± SEM. The actual values were 169±18 ng/ml for SPF mice, 95±26 ng/ml for the GF group and 123±9 ng/ml for CBUT mice *P<0.05 as assessed by the Student t-Test for independent data. (B) The relative expression of mRNA species encoding steroidogenic and Leydig cell proteins <b>normalized to the housekeeping levels of </b><b><i>18s rRNA</i></b><b> and </b><b><i>Arbpa/Rplp0</i></b><b> mRNA</b> in the testes of 8–10 weeks SPF, GF and CBUT mice. Each bar represents the mean ± SEM for 6 mice from 3 different litters. <i>cyp11a1:</i> cytochrome P450, family 11, subfamily a, polypeptide 1, <i>cyp19a1:</i> cytochrome P450, family 19, subfamily a, polypeptide 1, <i>Hsd3b1:</i> hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1, <i>Hsd17b11</i>: hydroxysteroid (17-beta) dehydrogenase 11 and <i>Insl3:</i> insulin-like 3. <i>Arbpa/Rplp0</i>: ribosomal protein, large, P0.</p

Expression of mRNA encoding the pluripotency markers <i>NANOG</i> and <i>POU5F1</i>, the germ cell markers <i>DDX4</i> and <i>KIT</i> and Sertoli cell markers <i>FSHR</i> and <i>VIM</i> by HS207, HS360 and HS401 cells cultured and differentiated on hFFs.

<p>The relative level of mRNA was calculated by the ddCt procedure from the mean of triplicates and statistical analysis performed by way of One-way RM ANOVA. The error bars depict standard deviations. *p <0.05, **p <0.01, ***p <0.001 when comparing undifferentiated with differentiated cells. #p <0.05, ##p <0.01, ###p <0.001 when comparing BMP7-stimulated with unstimulated cells. Abbreviations: hFFs: human foreskin fibroblasts; hESCs: undifferentiated human embryonic stem cells; diff hESCs: spontaneously differentiated hESCs; BMP7 hESCs: hESCs stimulated to differentiate by bone morphogenetic protein 7 (BMP7). A list of gene names and abbreviations can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144029#pone.0144029.s011" target="_blank">S6 Table</a>.</p

Immunohistochemical detection of POU5F1, DDX4, VIM and AMH in human testis and in HS360 cells cultured with BMP7 in suspension.

<p>Nuclear expression of POU5F1 (arrowheads) was observed in BMP7-stimulated HS360 cells after two weeks in culture (A), as well as in undifferentiated hES cells cultured on hFFs (C). Cytoplasmic expression (arrows) of DDX4 (D), VIMENTIN (G) and AMH (J) was present in BMP7-stimulated HS360 cells and human testicular tissue (F, I and L). Negative controls exhibited no specific staining in either the nucleus or cytoplasm (B, E, H and K; and inserts in C, F, I and L). The organization of Sertoli-like cells among stimulated HS360 cells (red arrow heads in M) and Sertoli cells in the one-year-old human testis (red arrow heads in N) was similar. Scale bars: 100 μm in B, C, E, H, K, M and N and the insert in C; 50 μm in F, I and L and the other inserts; 20 μm in A, D, G and J. Abbreviations: AMH: anti-Mullerian hormone, PAS: periodic acid-Schiff. For an explanation of the other abbreviations, see the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144029#pone.0144029.g002" target="_blank">Fig 2</a>.</p

Immunofluorescence detection of SOX9 in HS207, HS360 and HS401 cells cultured in suspension.

<p>Cytoplasmic expression of SOX9 in cells on the edges of the cell spheres was observed in all hES cell lines after BMP7-stimulation (B, D and F) as well as after spontaneous differentiation (A, C and E). Nuclear expression of SOX9 was observed in Sertoli cells present in adult human testis (G; arrow heads). Negative controls exhibited no specific SOX9 staining in either the nucleus or cytoplasm (H and small inserts in A to F). Green: SOX9 staining. Blue: DAPI staining marking the nucleus. Scale bars: 100 μm in A to H, and 50 μm in the small inserts.</p

Testis weight (A) and sperm count (B) for SPF, GF and CBUT mice at 8 weeks of age.

<p>The values shown are means ± SEM (n = 6) *P<0.05 calculated by the Mann-Whitney Rank Sum Test for non-parametric independent data.</p