Fig 1.
Generation of omentum-derived adult mouse mesothelial cell lines.
(A) Following 7 days of culture, primary MCs had migrated out of an omentum explant (outlined by stippled line). MCs in these cultures had a typical cobblestone-type appearance (arrowheads). (B-D) Passaged MCs overall retained their epithelial phenotype even though some cells adopted a slightly elongated shape (arrows) (B, P1). This appearance was un-affected by passage number (C, P5; D, P24). Scale bar represents 50 μm (A, B, D; C).
Fig 2.
Expression of mesothelial markers over passages.
(A) Immunofluorescence analysis of Wt1 and mesothelin (Msln) in P4 to P24 MCs showed that expression of both proteins was present throughout passages. Scale bars represent 25 μm (Wt1/DAPI) and 50 μm (Msln/DAPI). (B) qPCR analysis of mRNA expression for Wt1 showed significant downregulation in cultured P5, P10 and P25 MCs relative to OMC. (C) mRNA expression of Msln (C) was upregulated in the cultured cells compared to OMC. Significant differences compared to OMC were determined using a one way ANOVA with Sidak’s multiple comparison test where; ****p < 0.0001, ***p < 0.001 and **p < 0.01.
Fig 3.
Cultured mesothelial cells expressed both epithelial and mesenchymal characteristics by immunofluorescence.
(A) OMC cells showed strong ZO1 tight junctional bands at the cell-cell contacts, while ZO1 expression had a punctate appearance (arrowheads) at the cell perimeter In P4 and P24 MCs (B-C). (D) Cytokeratin intermediate filaments localised across the cytoplasm in OMC cells, while in P4 and P24 MCs the expression was reduced to the perinuclear regions (arrowheads; E-F). Expression of Vim (G-I) and αSMA (J-L) was similar between OMC, P4 and 24 MCs. Scale bar 50μM (A-L).
Fig 4.
Long-term culture influenced epithelial gene expression.
Analysis of mRNA expression levels for the epithelial genes Cdh1 (A) and Krt8 (B) showed significant down regulation in P5, P10 and P25 MCs relative to OMC. By contrast, expression of mesenchymal markers Vim (C) and αSMA (D) showed no significant change in mesothelial cell cultures of increasing passage. Significant differences compared to OMC were determined using a one way ANOVA with Sidak’s multiple comparison test where; ***p < 0.001, **p < 0.01, and *p<0.05.
Fig 5.
Clonogenic potential of mesothelial cells.
(A) Single cell from which one of the clones was started by dilution cloning. (B) MC clone at 2 weeks after cloning. (C) MC clone at confluence. (D) MC clone cells after passaging. Immunofluorescence staining for the mesothelial marker Wt1 (E-F), epithelial markers Cytokeratin (CK) (G) and ZO1 (H), and the mesenchymal markers Vimentin (Vim) (I) and α smooth muscle actin (αSMA) (J) in cells of mesothelial cell-derived clones revealed expression patterns similar to uncloned cells. The stem cell marker Bmi1 was detected in clonal MCs (K-L). Scale bars are 50 μm (A, B, C, D, E-J, K-L).
Fig 6.
Mesothelial cells expressed stem/progenitor cell markers.
Bmi1 and Sox9 nuclear localisation was detected through immunofluorescence in OMC (A, D; G, J), P4 (B, E; H, K) and P24 cells (C, F; I, L), respectively. Relative expression of stem cell markers in passaged MCs was maintained for Sox2 (N), while Sox9 (M) and CD34 (O) were downregulated in the passaged cells, respectively. Significant differences compared to OMC were determined using a one way ANOVA with Sidak’s multiple comparison test where; **p < 0.01, and *p<0.05. Scale bars are 25 μm (A-F) and 50 μm (G-L).
Fig 7.
Analysis of osteogenic and adipogenic potential.
Using Alizarin S red staining, Calcium deposits could be detected in P13 MCs (B) and P21 MSCs (D), indicating osteogenic differentiation, while cells under control conditions failed to exhibit the deposits (A, C). Fat droplet accumulation could be detected in P5 MCs (F) and, slightly more pronounced in MSCs (H). Control conditions showed no generation of fat droplets (E, G). Expression analysis by qPCR revealed that the osteogenic marker Sparc was up-regulated in the earlier passages (I), with a significant 6-fold change in P13 MCs. A significant 3.7-fold increase in expression was observed in P5 MCs for the adipogenic gene PPARγ (K). A Student’s t-test was used to calculate significance. Scale bars are 30 μM.
Fig 8.
Mesothelial cells are found within the developing kidney rudiment.
MCGFP+ were seen situated around Six2-expressing cells in the cap mesenchyme (A-C), and close to Pax2- (D-F) and Wt1- (G-I) expressing nascent nephron structures. In very few cases, Wt1- and Pax2-expression seemed to colocalise with the GFP fluorescence of the MCGFP+ cells (arrowheads, D-I). An S-shaped body is outlined in (I). GFP-positive MCs were found closely attached to Laminin of the basement membrane of Megalin-expressing proximal tubules (arrows, J-L). Scale bars are 50 μm (A-C, D-L).
Fig 9.
Mesothelial cells responded to the nephrogenic environment.
After isolation from the chimeric rudiments (KRA), MCGFP+ cells were analysed by qPCR in comparison to non-treated MCs of the same passage (Control). Relative expression analysis revealed that the epithelial marker ZO1, the mesenchymal marker Vim and the EMT regulators Snai1, Zeb1 and Twist1 were significantly up-regulated, while Wt1, SDF1 and Snai2 were not significantly changed. A Student’s t-test was performed to determine statistical significance.