Safety of Allogeneic Canine Adipose Tissue-Derived Mesenchymal Stem Cell Intraspinal Transplantation in Dogs with Chronic Spinal Cord Injury

Figure 3

Characterization of canine AT-MSCs. (a) Phase-contrast micrograph showing AT-MSCs adhered to a plastic culture flask at passage 3. (b) Immunophenotypic profile. Flow cytometry analyses were carried out for each marker separately. The percentages of positive cells are shown in the graph. (c, d) Adipogenic differentiation. Cells were grown in regular (c) or adipogenic (d) medium for 21 days, fixed, and stained with oil red O to evaluate the presence of red-colored lipid drops in the cytoplasm. The insets correspond to higher magnifications of areas boxed in (d). (e–h) Osteogenic differentiation. Alkaline phosphatase (ALP) activity was measured after 7 or 14 days of culture in osteogenic medium (right bars) or regular medium (left bars), as indicated in (e). Bars labeled with the same letters are significantly different (). Von Kossa staining revealed more mineralization foci at day 7 in induced cells (f). (g, h) show the absence or presence of mineralization deposits in osteogenic and regular medium, respectively. (i–l) Chondrogenic differentiation. (i) Scheme representing the micromass assay. (j) Micromass generated after 21 days in chondrogenic medium after being processed, cut, and stained with alcian blue and counterstained with neutral red to evidence cell nuclei. (k) Selected area in (j), showing the typical structure of cartilage tissue with nuclei eccentrically placed in lacunae and an alcian blue-stained matrix revealing the presence of glycosaminoglycans. (l) No micromasses were observed after 21 days in control medium. Conversely, the droplets were disassembled giving rise to a homogeneous cell monolayer.

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