Molecular, genomic, and functional characterization of co-cultured AEC.

<p><b>A</b>) The top images are two representative examples of migration testes perfomed in cultured and co-cultured AEC during the first week of incubation. As indicated by the+symbol, only the AEC co-cultured with fetal tendon explants show a migration activity. <b>B</b>) Representative images of immunocytochemistry showing the distribution of cytokeratin 8 and α-SMA in AEC co-cultured with fetal primary tenocytes after 14 or 28 days of incubation. All images show cell nuclei in blue (DAPI) and both the proteins in red (Cy3). At day 14, the co-cultured AEC organized in monolayer display high levels of cytokeratin 8, and undetectable levels of α-SMA. A similar molecular phenotype is displayed by monolayered AEC cultured alone (small insets) or co-cultured with fetal tenocytes (arrows in large figures) for 28 days. By contrast, an opposite behaviour is observed in co-cultured AEC that organized cell-aggregates. Scale bar for all images = 100 µm. <b>C</b>) Q-FISH detection of telomere length in freshly isolated AEC (AEC), in cultured AEC (AEC 28 days), in AEC co-cultured with fetal explants (AEC+fetal explants 28 days), and fetal tenocytes. The top figure is a representative image showing several hybridized red telomeres (Cy3) within two interphase nuclei stained in blue with DAPI. Scale bar = 15 µm. The three box plots indicate the Telomere area (TEA), the feret maximum (TEF), and the mean densitometric value (MEAND) parameters. The horizontal lines express the 5th, 25th, 50th, 75th, and 95th percentile of the distribution. The box stretches from the 25th to the 75th percentile, and therefore contains the middle half of the scores in the distribution. The median is shown as a line across the box, meanwhile the mean value as a black square within the box. ^* indicates data of TEA, TEFmax, and MEAND that resulted significantly different from AEC for <i>p</i><0.01 after One Way ANOVA test followed by <i>post-hoc</i> Tukey test. <b>D</b>) Three representative normal karyotypes obtained by freshly isolated AEC (day 0), cultured AEC and cells co-cultured in the presence of fetal explants. <b>E</b>) Flow cytometry for the major histocompatibily (MHC) class I and II molecules performed on ovine peripheral blood mononuclear cells (PBMC; top image) to test the ovine reactivity of both the antibodies and on freshly isolated AEC (bottom images) to demonstrate the presence of the MHC class I and the absence of MHC class II antigens.