Table 1.
Mean Fluorescence Intensity (MFI) of skin and lung MPCs surface markers.
Table 2.
Primer sequences for RT-PCR.
Figure 1.
Mesenchymal precursors isolated from adult mouse tissue.
A. Representative phase contrast images of one explant out of three independent explant assays performed with three different animal tissues are shown. To note outgrowth of mesenchymal precursor cells (MPCs) already at day 2 from skin and lung tissue explants surrounded by a ring of Matrigel. Images were taken 1, 2, 7 and 8 days after explant attachment. Scale bars, 100 µm (explants) and 30 µm (cultures). B. Growth and morphology of MPC cultures. Skin and lung MPCs obtained from explants were maintained in culture, and representative phase contrast images taken 1, 2 and 3 days after plating. Scale bars, 100 µm (upper panel) and 30 µm (lower panel).
Figure 2.
Characterization of skin and lung MPCs.
A. Representative growth curves for skin (red) and lung (green) MPCs in complete medium. One out of nine wells of representative cultures in a Real Time Cell Analyzer is shown. Cell Index represents the relative number of proliferative cells. Differences between skin and lung were statistically significant by ANOVA (*p<0.03). B. Flow cytometry analysis of surface molecule expression on skin and lung mesenchymal precursors. One group of representative histograms out of nine independent experiments is shown. Table 1 shows the media of the mean intensity fluorescence and the variance for all nine experiments. C. RT-PCR analysis of gene expression profile in skin and lung MPCs. The panel shows one representative agarose gel analysis of amplified products out of three independent experiments for each explant. Primer sequences are shown in Table 2 and summary of the results is shown in Table 3.
Table 3.
Gene expression profile of skin and lung MPCs.
Figure 3.
Cytoskeletal protein expression and localization in MPCs.
A. Western blot showing constitutive expression of cytoskeletal components in mesenchymal precursors from skin and lung. One out of three independent experiments is shown. B. Representative fluorescence images of skin and lung MPCs, showing the distribution of α-actin, β-tubulin and β1-integrin rabbit anti-mouse antibody). Scale bar, 1 µM.
Figure 4.
Skin and lung MPCs were grown in complete medium or in epithelial differentiation medium (MIX medium) over 7 days. Images were taken along the seven days of differentiation. To note that cells grown in MIX medium acquired epithelial morphology, consistent with gene expression changes (Table 4). Three independent experiments were performed. Scale Bar, 100 µm.
Table 4.
Changes on lineage marker expression of MPCs cultured for 7 days in epithelial differentiation medium (MIX medium) compared with growth complete medium.
Figure 5.
Mesenchymal precursor migration.
A. Migration of undifferentiated skin (red) and lung (green) MPCs analyzed by a well insert assay in the Real Time Cell Analyzer. One representative experiment out of 9 independent wells of MPCs cultures is shown. Migrated Cell index is the relative number of migrated cells. Differences between skin and lung at 12 h were statistically significant by ANOVA (*p<0.04). B. Migration of skin and lung MPCs in response to cytokines. The indicated stimuli were placed in the lower chambers in the migration assay. One representative experiment out of 9 independent wells of lung and skin MPCs cultures in presence of cytokines is shown. Data were analyzed as in A. Differences between control and stimulated cells were statistically significant by ANOVA (*p<0.05; **p<0.04). Table with the mean of migrated cell index for the nine control or stimulated MPCs clones is shown. CTRL, un-stimulated control cultures.