Figure 1.
Molecular characterization of freshly isolated AEC.
A) Levels of surface and intracellular stemness markers analyzed by flow cytometry and expressed as Mean Fluorescence Intensity (MFI) ratio. B) The mRNA content of tendon-related genes analyzed by RT-PCR. The bars show the standard error calculated on 3 independent experiments. C) Two representative images of cytokeratin 8 (epithelial marker) and α-SMA (mesenchymal marker) proteins detected in AEC by using an immunocytochemistry approach. The images show the blue nuclei counterstained with DAPI, and both proteins in red (Cy3). Scale bar for all images = 50 µm. D) The in vitro differentiation of AEC into endoderm (liver: bottom image) and ectoderm (neural cells: top image) cell lineages are documented by the immunocytochemistry detection of nestin and albumin, respectively. Nuclei were counterstained with DAPI. The mesodermal osteogenic in vitro differentiation (central image) was documented by the Alizarin Red staining. Scale bar for all images = 50 µm.
Table 1.
Proliferation activity recorded in AEC maintained in culture for 28 days alone or in co-incubation with tenocytes and tendon explants derived from fetal or adult calcaneal tendons.
Figure 2.
Morphology of differentiated AEC toward tenocyte lineage after co-culture.
The four top images reproduce the more representative phenotypes acquired by AEC (monolayer, circular aggregates, elongated, and tendon-like structures) during the 4 weeks of co-incubation. The bigger images were obtained with the aid of a contrast microscope, while the small ones, inserted in the corner, represent a low magnification recorded under a stereomicroscope. In the lower part of the Table, the incubation intervals (expressed in weeks) required to obtain these different phenotypes are indicated. Scale bar for all images = 50 µm.
Figure 3.
Molecular, genomic, and functional characterization of co-cultured AEC.
A) 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) 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. C) 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 p<0.01 after One Way ANOVA test followed by post-hoc Tukey test. D) Three representative normal karyotypes obtained by freshly isolated AEC (day 0), cultured AEC and cells co-cultured in the presence of fetal explants. E) 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.
Figure 4.
COL1 distribution in AEC aggregates developed in co-culture.
The images exemplify the COL1 protein distribution recorded by immunohistochemistry in the more representative typologies of cell aggregates obtained during AEC co-cultures. The images show in blue the nuclei counterstained with DAPI and in green the COL1 protein (Alexa Fluor 488). The COL1 protein is undetectable when the AEC are organized in monolayer independently from the cultural conditions adopted (co-cultured AEC, monolayer; AEC cultured alone, small inset). COL1 starts to appear in AEC forming cell aggregates and reaches its highest and widespread distribution within the tendon-like structures. In the early phase, COL1 is localized within the fusiform cells that start to be oriented along the longitudinal axis of the tendon-like structures. Later, the protein was either localized into the AEC or deposited within the extracellular matrix (tendon-like: late phase). Scale bar for all images = 100 µm.
Figure 5.
OCN, Cx32 and Cx43 proteins distribution in AEC co-cultured with fetal tenocytes or tendon explants.
Representative images showing osteocalcin (OCN: lower panel) and connexins (Cx32 or Cx43: upper panel) immunolocalization in AEC cultured alone (AEC) and in AEC co-cultured fetal derived cell/tissues that developed 3D structures. The pictures show the cell nuclei in blue (DAPI), OCN or Cx32 proteins in green (Alexa Fluor 488) and Cx43 in red (Cy3). The co-expression of Cx proteins on AEC cell aggregates were analyzed with a double immunostaining. OCN and both the Cx proteins were undetectable in AEC cultured alone (AEC) and in AEC organized within elongated aggregates. By contrast, AEC that differentiated 3-D tendon-like structures co-expressed Cx32 and Cx43. The Cx43 protein shows higher levels in the early tendon-like structures with a clear membrane localization, while Cx32, more abundant in late structures, is localized either on the membrane or into the cytoplasm. OCN appears as a cytoplasmatic protein within the fusiform shaped cells forming the tendon-like structures (early phase). Its intracellular levels progressively increased during the process of in vitro tendon differentiation (late phase). As indicated in the corner box showing a representative image of an ALP assay, tendon-like structures did not display any osteogenic foci. Scale bar for all images = 50 µm.
Figure 6.
Expression profile of tendon/ligament-related genes in co-cultured AEC.
The mRNA content of SCXB, COL1, COL3, TNMD, THSB4, OCN was analyzed in cultured AEC or in AEC co-cultured with fetal or adult tenocytes/tendons explants by using RT-PCR. The semi quantitative analyses of mRNA levels were normalized for GAPDH gene and expressed as mean of 3 different replicates ± SD. The data were compared by One Way ANOVA test followed by post-hoc Tukey test. * Values significantly different from AEC group for p<0.05; a values significantly different from fetal tendon group for p<0.05; a′ values significantly different from adult tendon group for p<0.05.
Table 2.
Details of primary antibodies used in flow cytometry analysis.
Table 3.
Details of primary and secondary antibodies used for immunohistochemistry.
Table 4.
Primer sequences used in RT-PCR analyses.