Isolation and culture of adipose-derived stem cells
The Twitcher mice and their littermates were obtained from breeding pairs carrying a natural mutation in the GALC gene. Breeding pairs were originally purchased from the Jackson Laboratory (Bar Harbor, ME). All the protocols and experimental procedures were approved by the Institutional Animal Care and Use Committee at Tulane University. Genotyping of Twitcher mice was performed on post-natal day 14 by real time PCR modified as previous described . The subcutaneous fat pads were isolated from the post-natal day 34 moribund Twitcher mice and their wild type littermates, rinsed with Hank’s balanced salt solution (HBSS) (Life Technologies, Grand Island, NY) to remove blood and hair contamination, and digested with 0.1% collagenase type 1 solution (Life Technologies) for approximately 4 hours at 37°C under vigorous agitation. The digested adipose tissue samples were then filtered through a 70-μm nylon mesh cell strainer (BD Biosciences, Bedford, MA), and centrifuged at 500×g for 10 minutes at room temperature (RT). The pellets were re-suspended and cultured in the growth medium DMEM: F12 (Life Technologies) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals, Atlanta, GA), 2 mM L-glutamine (Life Technologies), and 1% antibiotic/antimycotic (Penicillin/streptomycin/amphotericin, Life Technologies).
The following antibodies were used to define the surface markers expressed by the ASCs: CD29-FITC (fluorescein isothiocyanate), CD34-FITC, CD106-PE (vascular cell adhesion molecule-1 (VCAM-1)), CD31-PE (phycoerythrin), CD45-PE, CD11-PE, and Sca1-PE (stem cell antigen-1). All of the antibodies were purchased from BD Biosciences. The ASCs were cultured to 70% confluence, trypsinized, pelleted, and re-suspended in 500 μl phosphate buffered saline (PBS) (Life Technologies). The cells were incubated with the antibodies for 30 minutes at RT, then washed with PBS, and analyzed by Cytomics FC500 (Beckman Coulter, Brea, CA). The results were analyzed with CXP analysis software (Beckman Coulter).
Colony forming unit assay
ASCs at passage 3 were seeded onto 56.7 cm2 Nunc cell culture plates (Nalge Nunc International, Rochester, NY) in 5 replicates at a total of 100 cells per plate. Growth media was changed every 3–4 days. After 14 days, the cells were washed with PBS, stained with 3% crystal violet in 100% methanol for 30 minutes at RT, and then washed with deionized (DI) water at least 3 times to remove excess dye. All colonies greater than 2 mm in diameter were counted.
ASCs at passage 3 were seeded with a density of 6×104 cells/well on 6-well Nunc plates (Nalge Nunc International) and cultured to approximately 90% confluence before adipogenic and osteogenic differentiation media were added. Adipogenic differentiation medium was ASC growth medium supplemented with 5 μg/ml insulin, 50 μM indomethacin, 1 μM dexamethasone and 0.5 μM 3-isobutyl-1-methylxanthine (all media supplements were purchased from Sigma, St Louis, MO). Osteogenic differentiation medium was ASC growth medium supplemented with 1nM dexamethasone, 20 mM β-glycerolphosphate, 50 μM L-ascorbic acid 2-phosphate sesquimagnesium salt, and 50 ng/ml L-thyroxine sodium pentahydrate. Media were changed twice per week for 3 weeks. For adipogenic differentiation, the cells were washed with PBS, fixed with 10% formalin (Sigma) for 20 minutes at RT, washed again with PBS, stained with Oil Red-O (Sigma) for 20 minutes at RT, washed again with PBS until wash was clear. For the detection of osteogenesis, the cells were washed with PBS, fixed with 10% formalin for 20 minutes at RT, washed again with DI water, stained with Alizarin Red (Sigma) for 20 minutes at RT, washed again with DI water until wash was clear. Images were acquired at 10× for adipogenic differentiation and 4× for osteogenic differentiation on Nikon Eclipse TE200 (Melville, NY) with Nikon Digital Camera DXM1200F using the Nikon ACT-1 software version 2.7.
The *levels of adipogenic and osteogenic differentiation were also quantitated. For the quantitation of adipogenic differentiation, the accumulated lipids were eluted with isopropanol after images were captured. The amount of Oil Red O was measured by recording the optical density (OD) of the solution at 584 nm. The results were normalized to the protein content of the samples with the BCA assay (Thermo Scientific, Rockford, IL). For the quantitative osteogenesis assay, the cells were de-stained, after images were taken, with 10% cetylpyridinium chloride (Sigma) for 30 minutes at RT. The amount of Alizarin Red was determined by measuring the OD of the solution at 584 nm. The results were normalized to the protein content of the samples.
Real-time PCR analysis of osteogenic markers in WtASC and TwiASC
Total RNA was extracted from differentiated WtASC and TwiASC on day 7, day 14, and day 21 using RNeasy Mini Kit (Qiagen, Valencia, CA). RNA concentration and purity were assessed by Nanodrop 2000C spectrophotometer (Thermo Scientific). First strand cDNA syntheses were performed using iScript cDNA Synthesis kit (Biorad, Hercules, CA) after RNA was first treated with DNase (Life Technologies). Primers for mouse alkaline phosphatase (ALP), Runx-2 (RUNX), and osteocalcin (OCN) were commercially synthesized (IDTDNA, Coralville, Iowa), and the sequence of the primers was as previously described . The housekeeping gene β-actin (UniGene: Mm328431) was used as internal reference. The PCR reactions were performed using CFX96 Real Time System (Biorad) in a total final volume of 25 μl containing 12.5 μl SYBR green supermix (Biorad), 1 μl forward primer and 1 μl reverse primer (400 nM), and 1 μl template (500 ng). Reaction mixtures were incubated at 95°C for 3 minutes, and reactions were allowed to proceed via 45 cycles of melting at 95°C for 15 seconds, annealing and extension at 57°C for 30 seconds. Quantification was calculated using ΔΔCt method .
ASCs at passage 3 were seeded onto 56.7 cm2 Nunc cell culture plates in triplicate with a seeding density of 500 cells/cm2. Growth medium was changed every 3–4 days. Every 2 days for a total of 10 days, the cells were trypsinized, pelleted, re-suspended in 500 μl PBS, and counted using a Countess® Automated Cell Counter (Life Technologies) to analyze the fold increase in cell number.
Macrophage and ASCs co-culture
Primary macrophages were obtained through thioglycolate-elicited peritoneal isolation using a slightly modified published method . Briefly, 1 ml 3% (w/v) sterile thioglycolate (Sigma) in DI water was injected intraperitoneally into C57Bl6 mice (Jackson Lab) to elicit peritoneal exudate cells. Four days after thioglycolate injection, cells were harvested by peritoneal lavage using 10 ml PBS, treated with red blood cell lysing buffer (Sigma) to remove the red blood cells, assessed for macrophage purity by flow cytometry using FITC-labeled IgG anti-CD11b (BD Biosciences), and finally plated onto 24-well Nunc cell culture plates (Nalge Nunc International) with 200,000 cells per well in RPMI medium (Life Technologies) supplemented with 10% heat inactivated FBS and 1% penicillin/streptomycin. After 6-hour incubation, the non-adherent cells were removed by vigorous washing with PBS. Then 0.4 μm pore size 24 well transwell inserts (Corning, Lowell, MA) were placed into the 24-well plate, and TwiASCs or WtASCs were plated in each transwell insert with 40,000 cells per well (ASC: macrophage ratio=1:5). Cells were cultured overnight, incubated another 24 hour after the addition of 30 ng/ml lipopolysaccharide (LPS, Sigma), and then total RNA was extracted from the macrophages using RNeasy Mini Kit. RNA concentration and purity were assessed by Nanodrop 2000C spectrophotometer.
Real-time quantitative PCR
RNA was first treated with DNase, and first strand cDNA syntheses were performed using iScript cDNA Synthesis kit. Real-time PCR assay was performed to analyze the levels of mouse inflammatory cytokines IL-1α, IL-1β, TNFα, IL-6 and IL-10 expressed by macrophages transwell co-cultured with TwiASCs and WtASCs. PCR analyses were performed using CFX96 Real Time System in a total volume of 20 μl containing 10 μl Taqman mastermix (Applied Biosystems, Foster City, CA), 1 μl primer and probe mix (Applied Biosystems), and 2 μl template. Reaction mixtures were incubated at 50°C for 2 minutes and 95°C for 10 minutes, and reactions were allowed to proceed via 40 cycles of melting at 95°C for 15 seconds, annealing and extension at 60°C for 1 minute. The housekeeping gene β-actin (UniGene: Mm328431) was used as internal reference. Quantification was calculated using ΔΔCt method . Controls were RNA from macrophages cultured alone with and without LPS.
Groups of data were analyzed by one-way analysis of variance (ANOVA). For pair-wise comparisons, the F-test was used to determine whether a given pair of population variances was equal (α<0.05). This information was then used in designating the appropriate t-tests (typically heteroscedastic) to perform for comparing the means of population pair, with significance defined as P<0.05. All values were reported as mean± SD.