Human adipose tissue collection and stromal vascular fraction isolation
Human adipose tissue was obtained from three healthy female donors after informed consent under approval of the Stanford University Institutional Review Board (Protocol no. 2188). Both abdominoplasty and suction-assisted lipoaspiration specimens were collected from each patient. Patients were female, 36–54 years of age, and had no known comorbidities. SAL was performed at a negative pressure of 760 mmHg using a 5 mm rounded, blunt cannula.
Lipoaspirate was processed to obtain the stromal vascular fraction as described previously . Briefly, lipoaspirate was washed with sterile phosphate-buffered saline, followed by removal of the oil and blood/saline layers. The remaining fat layer was digested with Type II collagenase (Sigma-Aldrich; St. Louis, MO) in Medium 199 (Cellgro; Manassas, VA, USA) in a 37 °C water bath at 180 rpm for 30 min. The mixture was centrifuged at 1500g for 20 min at 4 °C and the supernatant was discarded. The cellular pellet was re-suspended in Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen; Carlsbad, CA, USA) with 10 % fetal bovine serum (FBS), filtered through a 100 µm pore size cell strainer (Corning; Corning, NY, USA), centrifuged at 300g for 15 min, and the supernatant was discarded once again. The cell pellet was then re-suspended in red cell lysis buffer and centrifuged once more time before re-suspending the stromal vascular fraction (SVF) in complete medium. Excised abdominoplasty specimens were de-epithelialized, mechanically minced into small pieces, and then digested and processed in the same manner as the lipoaspirate samples.
Fluorescence-assisted cell sorting analysis
Our group recently demonstrated significant differences in the transcriptional profiles of primary ASCs when compared to cultured cells stressing the importance of using primary or very early passage cells in in all translational studies . Therefore we utilized freshly isolated SVF and stained it for immediate fluorescence-activated cell sorting (FACS) to identify the ASC fraction. ASCs were defined by the established surface marker profile CD45-/CD31-/CD34+ [16, 19, 20]. Mouse anti-human monoclonal antibodies CD31-PE, CD45-PeCy7, and CD34-APC (BD Biosciences; San Jose, CA, USA) were used and propidium iodide staining was employed to exclude dead cells. Analysis was performed using a BD FACSAria machine (BD Biosciences).
In vitro viability assay
Freshly extracted ASCs from SAL and excisional fat were seeded into a 96-well plate for determination of viability by MTT assay (Vybrant MTT Cell Proliferation Assay Kit, Invitrogen; Carlsbad, CA, USA).
In vitro osteogenic differentiation
ASCs derived from SAL lipoaspirates and excised adipose tissue at passage two were cultured in osteogenic differentiation media (ODM), containing 10 % FBS, 1 % penicillin/streptomycin, 100 μg/mL ascorbic acid, and 10 mM β-glycerol 2-phosphate . An alkaline phosphatase assay (Sigma-Aldrich) was performed after 7 days in culture with ODM, and mineralization was assessed using Alizarin Red staining at day 14. Alizarin Red staining was extracted with 20 % methanol and 10 % acetic acid in distilled water, and quantified using a spectrophotometer at 450 nm.
Total RNA was harvested immediately prior to beginning osteogenic stimulation with ODM at day 0, then again at day 7 and day 14 in osteogenic culture, and processed using the RNeasy Mini Kit (Qiagen; Hilden, Germany). Reverse transcription was performed using TaqMan Reverse Transcription Reagents (Invitrogen). An ABI Prism 7900HT Sequence Detection System (Applied Biosystems; Foster City, CA, USA) was used to perform quantitative real-time polymerase chain reaction (qRT-PCR) with Power SYBR Green PCR Master Mix (Applied Biosystems) as the reporter. qRT-PCR analysis was conducted to detect gene expression levels of the early osteogenic marker Runt-related transcription factor-2 (RUNX-2) as well as the late osteogenic marker osteocalcin (OCN). Expression levels of RUNX-2 and OCN were normalized to beta-actin expression values.
In vitro adipogenic differentiation
Cells from both groups were passaged twice and seeded in standard 6-well plates in triplicate at equal density. After reaching 70 % confluence, ASCs were cultured in adipogenic differentiation medium (ADM), consisting of DMEM, 10 % FBS, 1 % penicillin/streptomycin, 10 μg/mL insulin, 1 μM dexamethasone, 0.5 mM methylxanthine, and 200 μM indomethacin. Lipid accumulation was determined using Oil Red O (ORO) staining after 7 days in culture with ADM. Staining was imaged using a Leica DC300 camera and Leica DM IL inverted contrasting microscope at 10× magnification, then extracted with isopropanol, and quantified by absorbance spectrophotometry at 520 nm.
Total RNA was harvested at day 0 and day 7 of adipogenic induction culture. Expression levels of the adipogenic differentiation markers peroxisome proliferator-activated receptor γ (PPAR-γ), fatty acid binding protein 4 (FABP4/AP2), and lipoprotein lipase (LPL) were determined at two time points during adipogenic differentiation. Gene expression values were normalized to beta-actin.
All mice were housed in the Stanford University Veterinary Service Center in accordance with NIH and institution-approved animal care guidelines. All procedures were approved by the Stanford Administrative Panel on Laboratory Animal Care.
In vivo excisional wound model
Nude male Crl:CD-1-Foxn1
nu mice (Charles River Laboratories, Wilmington, MA, USA http://www.criver.com) between 8 and 12 weeks of age were randomized to three treatment groups: unseeded hydrogel control or hydrogel seeded with human ASC isolated from SAL lipoaspirates or resected adipose tissue. Pullulan-collagen hydrogel was produced as and seeded as described previously [5, 22]. Briefly, 2.5 × 105 human ASCs suspended in 15 μL of PBS solution were pipetted onto hydrophobic wax paper and the hydrogel absorbed the cells actively by capillary, hydrophobic and entropic forces . As previously described , two 6 mm full thickness wounds were created at the dorsum of each mouse. Each wound was held open by donut shaped silicone rings sutured on with 6-0 nylon sutures to prevent wound contraction and allow for healing by granulation. Wounds were covered with an occlusive dressing (Tegaderm, 3 M, St. Paul, MN, http://www.3m.com). Photographs were taken on days 0, 3, 5, 7, 9, 11, 13 and 15 and wound area was measured using ImageJ software (National Institute of Health, Bethesda, MD, http://www.nih.gov) (n = 8 wounds/group).
Assessment of wound vascularity
To evaluate wound vascularity, immunohistochemical staining for the endothelial cell marker CD31 was performed as described previously (n = 8 wounds/condition) . Briefly, wounds were harvested upon closure and processed for paraffin sectioning. Seven micron thick paraffin sections were stained with primary antibody (1:100 Rb α CD31, Ab28364, Abcam, Cambridge, UK, http://www.abcam.com) overnight at 4 °C, followed by secondary antibody staining (1:400 AF547 Gt α Rb, Life Technologies, Grand Island, NY, USA http://www.lifetechnologies.com). Cell nuclei were visualized with the nuclear stain DAPI. ImageJ (National Institute of Health, Bethesda, MD, USA http://www.nih.gov) was used to binarize immunofluorescent images taken with the same gain, exposure, and excitation settings as previously described . Intensity threshold values were set automatically and quantification of CD31 staining was determined by pixel-positive area per high power field.
Data are shown as mean ± SEM. Statistical analyses were performed using GraphPad Prism software (GraphPad Software, Inc.). Statistical comparisons were made using Student’s t-tests and ANOVAs, with Bonferroni corrections for multiple comparisons where appropriate. A *p value of < 0.05 was considered statistically significant.