Subcutaneous adipose tissue was harvested during elective abdominoplasty from human adult female patients at Magee Womens Hospital. All samples were waste materials collected as a byproduct of surgery. Deidentified samples were collected under an IRB approved exemption (number 0511186, University of Pittsburgh IRB). Samples were immediately transported to the laboratory and processed on receipt.
Flow Cytometry and Cell Sorting
Fat tissue was thoroughly minced with scissors, digested for 30 min in Hanks' Balanced Salt Solution (HBSS, Invitrogen, Carlsbad, CA) containing 3.5% Bovine Serum Albumin (BSA, Millipore, Charlottesville, VA) and 1 mg/mL collagenase type II (Worthington, Lakewood, NJ) on a shaking water bath at 37°C, and finally disaggregated through successive 425 μm and 180 μm sieves (W.S. Tyler, Mentor, OH). Mature adipocytes were eliminated by centrifugation (400g, ambient temperature, 10 min) and cell pellets were resuspended in NH4Cl-based erythrocyte lysis buffer (Beckman Coulter, Miami, FL, Cat No. IM3630d), incubated for 10 min at room temperature (RT) and washed in Phosphate Buffer Saline (PBS). Viable cell enrichment and debris depletion was achieved on a Ficoll-Hypaque density gradient (Histopaque®-1077, Sigma-Aldrich, St. Louis, MO). Cell suspensions were deposited on slides using a Cytospin-2 cytocentrifuge (Thermo Shandon, Pittsburgh, PA) and Wright-Giemsa stained (Wright Giemsa Stain Hematology Stain Pack, Fisher Diagnostics, Fisher Scientific, Middletown, VA) with an Ames Hema-tek stainer and photographed under bright-field microscopy using a digital camera and software (Spot Insight 2 Meg FW Color Mosaic model 18.2, Diagnostic Instruments) interfaced to a Nikon Labophot microscope.
Freshly isolated cells from the SVF were maintained from then on ice and stained for analytical flow cytometry and cell sorting experiments as previously described (30). Cell suspensions were centrifuged (200g, 7 min) and the cell pellet was preincubated with 5 μL neat mouse serum (Sigma) to minimize nonspecific antibody binding. Cells were simultaneously stained with monoclonal mouse anti-human fluorochrome-conjugated antibodies (2 μL each, CD3-FITC, CD146-PE, CD34-ECD, CD90-PE-Cy5, and CD117-PE-Cy7, all Beckman Coulter, CD31-APC, and CD45-APC-Cy7, BD Biosciences, San Jose, CA).
Analytical samples were fixed with 2% methanol-free formaldehyde (Polysciences, Warrington, PA), permeabilized in PBS with 0.1% saponin (Coulter), 0.5% BSA for 10 min at RT, and incubated with 7.7 μg/mL 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen). Eight-color, 14-parameter data files were acquired on a three-laser CyAn ADP cytometer (Beckman Coulter) at a maximum of 10,000 events per second. Up to 10,000,000 events were acquired per sample. The DAPI signal was acquired independently on both logarithmic and linear (for cell cycle analysis) scales by optimizing voltage and gain settings of two individual photomultiplier tubes (PMT). For compensation purposes, BD Calibrite™ beads (BD Biosciences) and single antibody-stained mouse IgG capture beads (BD Biosciences) were acquired for single fluorochromes (FITC, PE, APC) and tandem-dyes (ECD, PE-Cy5, PE-Cy7, and APC-Cy7), respectively. Initially, regions and gates were set with the aid of “fluorescence minus outcomes” isotype controls. An annotated example is given in the supporting information associated with the on-line version of this article. Offline compensation and analysis were performed using the high throughput parallel processing VenturiOne software (Applied Cytometry, Sheffield, UK). Ten independent samples were analyzed. Summarized results are given as arithmetic means ± SEM, where mean values are compared, and ± SD where sample to sample variability is characterized (31).
Cell sorting was performed using a three-laser MoFlo high speed cell sorter (Beckman Coulter) equipped with a class I biosafety cabinet.
Unfixed eight-color stained samples were suspended in PBS, 2 mM ethylenediaminetetraacetic acid (EDTA, Sigma), 0.5% BSA, supplemented with 2 μg/mL DAPI for exclusion of dead and apoptotic cells. Samples were continuously cooled to 4°C and four-way sorting was performed at 10,000-to-20,000 events per second. Samples were collected into sterile polypropylene tubes containing 500 μL fetal calf serum (FCS, Atlanta Biologicals, Lawrenceville, GA) and plated in uncoated BD Falcon™ plates/flasks (BD Biosciences) at a density of 10,000 to 25,000 cells per cm2. Adherent sorted and unsorted cells were expanded in equal volumes of Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) + DMEM (Invitrogen) and supplemented with 10% FCS, 0.1 μM dexamethasone (Sigma), 100 U/mL penicillin (Sigma), 50 μg/mL gentamycin sulfate (Sigma).
Small (∼1 cm3) pieces of freshly harvested adipose tissue were washed in PBS, 15% sucrose, immersed in 7.5% gelatin (Sigma) in PBS, 15% sucrose (Sigma), and held for at least 2 h at 4°C, before being frozen by repetitive immersion in liquid nitrogen-cooled 2-methylbutane (Fisher Scientific, Fair Lawn, NJ). Sections (6 to 8 μm) were cut on a Microm cryostat, fixed for 5 min in ice cold acetone (Fisher Scientific), and then stored at −80°C. Before staining, sections were dried at RT and postfixed for 5 min in ice cold acetone. Tissue rehydratation and all subsequent washes were performed by two 5-min incubations in Dako Wash Buffer (Dako, Carpinteria, CA). All incubations were completed at ambient temperature.
For immunohistochemistry experiments, endogenous peroxidase activity was neutralized with Dual Endogenous Enzyme Block solution (Dako) for 10 min. Specimens were washed and nonspecific secondary antibody binding was prevented by a 1 h incubation with PBS, 5% goat serum (Sigma), 0.05% Tween20 (Dako). Sections were incubated with prediluted uncoupled mouse anti-human antibodies for 1 h. Dako Universal Mouse Negative Control (Dako) was substituted for primary antibodies for a negative control. Specimens were washed and incubated for 30 min with horseradish-peroxidase (HRP)-labeled polymers (Dako Envision+ Dual Link System-HRP, Dako). Staining was completed by incubating the rinsed specimens with 3,3′-diaminobenzidine (DAB+) substrate-chromogen (Dako) for 1 to 3 min. Finally, tissue sections were washed in Dako Wash Buffer, stained for 3 min with Hematoxylin (Dako), and rinsed in deionized water. Dehydrated specimens were mounted in nonaqueous medium (Cytoseal™280, Richard-Allan Scientific, Kalamazoo, MI) and photographed under bright-field microscopy using a Spot Insight 2Meg FW Color Mosaic digital camera (Diagnostic Instruments).
For fluorescent immunostaining, rehydrated tissue sections were pretreated with goat serum blocking solution and incubated with primary unconjugated antibodies for 1 h. Washed sections were sequentially incubated for 1 h with biotinylated secondary goat anti-mouse antibody (Dako) and for 30 min with streptavidin-Cy3 (Sigma). Nuclear staining was attained through 5-min incubation with (300 nM) DAPI (Invitrogen). Slides were mounted in 1:1 PBS/glycerol (Sigma) or Prolong Gold anti-fade reagent (Invitrogen) and observed under an epi-fluorescence microscope (Nikon Eclipse TE 2000-U).
Primary antibodies used in these studies were anti-CD31 (ready-to-use, Dako, Cat No. N1596 and 1:50, SantaCruz Biotechnology, Santa Cruz, CA, Cat No. SC13537 for immunohistochemistry and immunofluorescence experiments, respectively), anti-CD34 (1:50, BD Biosciences, Cat No. 347660), anti-CD90 (1:100, BD Biosciences, Cat No. 550402), anti-CD146-FITC (1:20, AbD Serotec, Raleigh, NC, Cat No. MCA2141F), and anti-α-SMA-FITC (1:200, Sigma, Cat No. F3777). All antibodies and streptavidin-Cy3 were diluted in PBS, 5% goat serum, 0.05% Tween20.
Adipogenic Differentiation and Oil Red O Staining
For adipogenic differentiation, expanded adherent cells were trypsinized (5 min incubation with 0.25% Trypsin/EDTA, Cellgro, Mediatech, Manassas, VA) and transferred into replicate (4, 5) 24-well plates (50,000 cells/well) and allowed to adhere overnight. Culture medium was supplemented with 1 μM dexamethasone, 0.5 μM isobutylmethylxantine, 60 μM indomethacin, and 10 μg/mL insulin (all Sigma). After 21 days, cells were fixed in 2% formaldehyde (Polysciences, Inc.) in PBS for 15 min at room temperature. Fixed cells were washed in 60% isopropanol and stained for 10 min with Oil Red O (Sigma) at RT. After five consecutive washes in deionized water, the presence of red stained lipid was documented using bright-field microscopy. The number of cells per low power field (10× objective, five–eight fields) with at least five large Oil Red O+ vesicles was recorded for each culture. For cells derived from each sorted population, Student's paired two tailed t-test was performed comparing the log10 of Oil Red O+ cells derived from each fraction to the log10 of Oil Red O+ cells in unsorted cells from the same experiment. Since the difference of logs is equivalent to a ratio in the arithmetic domain, this statistic provided a convenient assessment of adipogenic potential relative to unsorted cells.