Characterization of stromal vascular fraction and adipose stem cells from subcutaneous, preperitoneal and visceral morbidly obese human adipose tissue depots

Fig 1. Perivascular and hematopoietic populations classified for analytical flow cytometry. SVF of subcutaneous, preperitoneal and visceral adipose tissues from morbidly obese patients was analyzed by flow cytometry. First, viable cells were identified by 7AAD exclusion (A). Second, viable cells were distributed in a Forward versus Side Scatter plot (B, C, D) and further analyzed according to CD45 expression (E, I, M). The gates were set on non-hematopoietic (CD45neg—NHe) and hematopoietic (CD45pos—He) cells. Third, CD45neg cells (red) were analyzed for CD34 and CD146 expression (F, J, N) or CD34 and CD31 expression (G, K, O). A blue gate was set on the CD146posCD34neg cells to identify the pericytes. A subset of supra-adventitial cells (SA), which were CD34posCD31neg, was identified (green gate) whereas the endothelial progenitor cells (EP) were identified as CD34posCD31pos (purple gate). Among the He cells, two monocyte-macrophage populations were identified (H, L, P): adipose tissue resident macrophages were identified as CD14posCD206pos cells (upper right quadrant) and a population of CD14posCD206neg cells (lower right quadrant). (Q-S) Compensation controls of fluorescence…

Fig 2. SVF contents of subcutaneous, preperitoneal and visceral adipose tissues from morbidly obese patients. The quantitative data of the flow cytometry analysis shows the distribution of the non-hematopoietic (A, B, C) and hematopoietic (D, E, F) cell subpopulations. The percentage of each subpopulation analyzed from each patient is expressed. Values obtained from different cell samples harvested from the same patient are connected with lines. p values under graphs resulted from statistical tests. Asterisk represents the p value from the post-test of statistical analyses: (*) p <0.05. n = 9 to 11, according to the population. SVF: stromal vascular fraction; SC: Subcutaneous; PP: preperitoneal; VC: visceral; AT: adipose tissue.

Fig 3. Morpho-phenotypical characterization of ASC from the subcutaneous, preperitoneal and visceral adipose tissues of morbid obese patients. ASC from the subcutaneous (A), preperitoneal (B) and visceral (C) showed fibroblastic cells. (A, B, C) phase contrast. Bar size: 100 micrometers. The histograms show the ASC surface expression of CD34 (D-F), CD146 (G-I); CD105 (J-L); CD73 (M-O) and CD90 (P-R). The histograms of the ASC from the subcutaneous (D, G, J, M, P), preperitoneal (E, H, K, N, Q) and visceral (F, I, L, O, R) depots are representative of independent experiments (n = 3). Gray histogram—negative control; blue histogram—expression of the indicated CD. ASC: adipose stem cell; SC: Subcutaneous; PP: preperitoneal; VC: visceral; CD: cluster of differentiation.

Fig 4. Differential cytokine secretion of ASC derived from distinct abdominal white adipose tissue depots. The concentration of cytokine secreted in the cell culture supernatant quantified using a multiplex assay is expressed as the mean ± SEM (A-E). p values under graphs resulted from statistical tests. The asterisks represent the p values from the statistical post-tests: (*) p <0.05. Assays were performed in triplicates for each of the 3 patients evaluated. SC: Subcutaneous; PP: preperitoneal; VC: visceral; IL: interleukin; MCP-1: Monocyte Chemoattractant Protein 1; G-CSF: Granulocyte colony-stimulating factor; PAI-1: Plasminogen activator inhibitor-1.

Fig 5. Differential lipid accumulation capacity and cytokine secretion of induced ASC derived from subcutaneous, preperitoneal and visceral depots. The ASC were cultured under lipid accumulation stimulus for up to 3 weeks. Lipid accumulation was analyzed by Oil Red O staining (A-C). The total Oil Red O dye captured was measured in a spectrophotometer. The Graph (D) shows the quantitative results of the Oil Red O staining. ANOVA tests comparing the absorbance were performed. Cytokine secretion in the supernatant of the induced cells is expressed in graphs (E) IL-6, (F) IL-8, (G) MCP-1, (H) adiponectin, (I) G-CSF, (J) PAI-1. p values under graphs resulted from statistical tests. The asterisks represent the p values from the statistical post-tests: (*) p <0.05. (A-C) Optical microscopy—bar size: 50 micrometers. Assays were performed in triplicates for each of the 3 patients evaluated. ASC: adipose stem cell; SC: subcutaneous; PP: preperitoneal; VC: visceral; IL: interleukin; MCP-1: Monocyte Chemoattractant Protein 1; G-CSF: Granulocyte colony-stimulating factor; PAI-1: Plasminogen activator inhibitor-1.