Fig. 1
Characterization of canine adipose-derived mesenchymal stem cells (cAD-MSCs) used in this study. A Morphology of cAD-MSCs were observed under a phase-contrast microscope at 40X and 100X magnifications. B Flow cytometer was used for characterization the presence of MSC markers (CD29, CD44, and CD90), and the leukocyte common antigen (CD45) on the cell surface. C Expression of the stemness-related genes (Oct4 and Rex1) and the proliferation gene (Ki-67) were revealed by RT-qPCR with Gapdh normalization. D Population doubling time of cAD-MSCs in different serial passage numbers was estimated by counting cAD-MSCs after trypsinization. The linear regression curve was visualized along with a grey region of 95% confidence interval. The equation indicates the senescence rate as a slope with an annotation of the adjusted coefficient of determination (Radj2). E Adipogenic differentiation potential at day 28 post-induction was affirmed by Oil Red O staining and adipogenic related-mRNA expression (Lep and Lpl). F Osteogenic differentiation potential after 14-day induction was revealed by Alizarin Red S staining and the expression of osteogenic mRNA markers (Ocn and Runx2). G Chondrogenic differentiation potential at day 21 post-induction was assessed by Alcian Blue staining and chondrogenic mRNA markers (Col2a1 and Sox9). The mRNA expressions for multi-lineage differentiation were normalized with the reference gene (Gapdh) and the undifferentiation control. The scale bars represent 200 μm. In boxplots, mean values of each group were annotated at the bottom, and global statistics were stated at the top. The bars indicate pairwise comparisons (ns; no significance or p-value > 0.05, *; p-value ≤ 0.05). Abbreviation: AU; arbitrary unit