Table 1.
Specific induction media compositions.
Table 2.
Semi-quantitative scoring system used in the evaluation of adipogenic differentiation.
Table 3.
Sequences of primers used for RT-PCR analysis.
Fig 1.
Cell growth of canine AT- and UC-MSCs.
Doubling times of frozen-thawed AT-MSCs (□) and UC-MSCs (■) over four passages of in vitro culture. Data represent the mean ± SD. Different symbols indicates significant differences: * and ** for AT-MSCs, ° and °° for UC-MSCs (P <0.05).
Fig 2.
Spheroid formation and scratch assays of canine AT- and UC-MSCs.
A-B) Volume reconstruction of an AT-MSC (A) and UC-MSCs (B) spheroid, obtained after 24 h of hanging drop culture. C-D) Scratch assay on AT-MSCs (C) and UC-MSCs (D) at T0 and after 24 h (T1) of cell growth (Magnification 4X).
Fig 3.
Differentiation potential of AT- and UC-MSCs.
Canine AT- and UC-MSCs cultured toward adipogenic, chondrogenic and osteogenic differentiation. Magnification: 20X for all pictures except for 4X of osteogenic differentiated cells (bars = 100 μm).
Fig 4.
RT-PCR analysis of gene expression in canine AT- and UC-MSCs.
Products from AT- and UC-MSCs samples are visualized over time, from passage 0 (P0) to passage 4 (P4) of in vitro culture.
Fig 5.
Real-Time ATP Production Rate Assay of basal OCR (blue spheres) and ECAR (blue squares) rates of AT-MSCs (A) and OCR (orange spheres) and ECAR (orange squares) rates of UC-MSCs (B). After detection of OCR and ECAR rates, oligomycin (1.5 μM) and rotenone plus antimycin A (0.5 μM) were serially injected at fixed times in order to allow to detect the mitochondrial and glycolytic ATP production rates. C) AT- and UC-MSCs quantification of ATP production by mitochondrial oxidative phosphorylation (dashed rectangle) or by the glycolytic pathway (glucose conversion to lactate) (filled rectangle). D) Energy map of AT-MSCs and UC-MSCs with OCR vs ECAR of AT- (blue spheres) and UC-MSCs (orange spheres) are plotted. E) The plot shows the ratio between the mitochondrial ATP production rate and the glycolytic ATP production rate (logarithmic scale). Data expressed as points (A, B, D, E plots) and column chart (C plot) represent the mean ± SD (vertical bars) from three experiments carried out on different cell preparations. * indicates significant differences (P<0.05).
Fig 6.
Metabolic measurements in MSCs.
A) Mitochondrial respiration profile (OCR) of AT-and UC-MSCs under basal conditions and after the addition of 1.5 μM oligomycin, 2.0 μM and 0.5 μM FCCP and a mixture of rotenone plus antimycin A (0.5 μM) injections where indicated by the dotted lines. B) Mitochondrial parameters (basal respiration, ATP production, proton leak, maximal respiration, spare respiratory capacity) in AT- and UC-MSCs. C) Basal ECAR and ECAR changes after addition of the mitochondrial inhibitors: 1.5 μM oligomycin, 2.0 μM and 0.5 μM FCCP and a mixture of rotenone plus antimycin A (0.5 μM) addition on AT- and UC-MSCs. Data expressed as points (A, C) and column chart (B) represent the mean ± SD (vertical bars) from three experiments carried out on different cell preparations. * indicates significant differences (P<0.05).