spherical chondrocyte-like aggregates revealed intense Alcian Blue staining of glycoproteoglycans (Figure 2D). In contrast to the high osteogenic and chondrogenic differentiation of canine BMMSC, we found a low degree of adipogenesis, with limited vacuole formation and Oil Red O staining (data not included). Int. J. Mol. Sci. 2020, 21, 5129 A C B Ꭰ 4 of 21 Figure 2. Micrographs documenting multipotent characteristics of canine BMMSC. Note differentiation into osteocytes (B, Alizarin Red) compared to control (A) and chondrocytes (D), Alcian Blue) compared to control (C). Scale bars A, B, C, D = 50 μm. 2.2. Multilineage Potential Three-lineage potential was confirmed using the commercial StemPro® Differentiation Kit and corresponding protocol for induction towards chondrogenic, osteogenic and adipogenic lineages. After 14 days of incubation in cell-type corresponding differentiation medium, we confirmed that canine BMMSC show a high degree of biomineralizing osteogenesis with visual staining of calcium deposits expressing Alizarin Red positivity (Figure 2B). Furthermore, chondrocytes migrating from spherical chondrocyte-like aggregates revealed intense Alcian Blue staining of glycoproteoglycans (Figure 2D). In contrast to the high osteogenic and chondrogenic differentiation of canine BMMSC, we found a low degree of adipogenesis, with limited vacuole formation and Oil Red O staining (data not included). Int. J. Mol. Sci. 2020, 21, 5129 A C B Ꭰ 4 of 21