Fig. 1. Characterization of canine adipose tissue-derived mesenchymal stem cells (cAD-MSCs). (A) The cAD-MSCs obtained from canine adipose tissue were able to attach to the culture plates and expand in vitro. 40× (left), 100× (right). (B) Flow cytometry histograms show the expression of surface markers (CD34, CD44 and CD90) by cAD-MSCs populations compared with controls.
Fig. 3. Effects of TN and 3D-TN culture systems on cell phenotype in cAD-MSCs. (A) Schematic diagram of TN and 3D-TN culture systems. (B) Cellular morphology of TN and 3D-TN-cultured cells. 40×. mRNA expressions of (C) cell surface markers (CD29, CD34, CD44, CD45 and CD90), (D) Ki-67 and PCNA were confirmed by qRT-PCR and normalized to the GAPDH level. GAPDH was used as a housekeeping control gene. Each data from three independent experiments was evaluated and expressed as the mean ± SD (***p < 0.0001).
Fig. 2. Effects of 2-dimentional (2D) and transwell (TN) culture systems on cell phenotype in cAD-MSCs. (A) Schematic diagram of 2D culture and TN culture system. (B) mRNA expression of proliferation genes (Ki-67 and PCNA) were confirmed by qRT-PCR and normalized to the GAPDH level. GAPDH was used as a housekeeping control gene. Each data from three independent experiments was evaluated and expressed as the mean ± SD (*p < 0.05, ***p < 0.0001).
Fig. 4. Differentiation potentials of TN and 3D-TN culture systems in cAD-MSCs. (A) Using the specific staining kit, chondrocytes (Alcian Blue) and osteocytes (Alizarin Red) were positively stained in two groups. 100×. (B) The mRNA expressions of chondrocyte (COL2A1 and SOX9) and osteocyte (OC and TGF-β1) related genes were detected by qRT-PCR and normalized to the GAPDH level. GAPDH was used as a housekeeping control gene. Each data from three independent experiments was evaluated and expressed as the mean ± SD (**p < 0.001, ***p < 0.0001).
Table 1. List of primers used for quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR)
Table 2. Composition of differentiation media
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