Isolation of stem cells
Murine adipose derived stem cells and bone marrow derived stem cells were obtained from C57Bl/6 mice (Jackson Laboratories, Bar Harbor, ME). They were also obtained from the inbred transgenic strain C57Bl/6-Tg(UBC-GFP)30Scha/J that ubiquitously expresses enhanced green fluorescent protein (Jackson Laboratories). Cells were obtained from 3–4 donors of both sexes from each strain. All donors were 2–4 months old and were individually euthanized by CO2. All procedures performed conform to the requirements of the Animal Welfare Act and protocols approved by the Institutional Animal Care and Use Committee at Tulane University. All mice were maintained under standard housing conditions in a pathogen-free environment with free access to food and water.
mBMSCs and GFPTgBMSCs were obtained from two femurs and two tibiae from each donor which were removed and cleaned of connective tissue. The ends of each tibia and femur were clipped off to expose the marrow. A syringe was inserted into the bone and complete expansion media (CEM) was pushed through the bone to collect the marrow. The marrow was re-suspended in CEM using a pipet and then filtered through a 70 μm nylon mesh filter to remove any particulates. The mixture was then centrifuged at 400 × g for 10 minutes at 4°C, and the pellet was re-suspended in 3 ml CEM. The cells were then plated in an 8.8 cm2 culture plate and washed with media twice over a period of 6 hours to remove hematopoietic cells. CEM consists of Iscove's Modified Dulbecco's Medium (IMDM, Invitrogen, Carlsbad, CA) supplemented with 9% fetal bovine serum (FBS; Atlanta Biologicals, Atlanta, GA), 9% horse serum (HS; Hyclone Laboratories, Logan UT), 100 U/ml penicillin (Invitrogen), 100 μg/ml streptomycin (Invitrogen), 0.25 μg/ml amphotericin B (Invitrogen), and 12 μM L-glutamine (Invitrogen).
Adherent cells were washed after 24 hours, and fresh CEM was added every 3 to 4 days until cells reached approximately 80% confluency. The cells were then washed with PBS and lifted by incubation with 0.5 ml 0.25% trypsin/1 mM ethylenediaminetetraacetic acid (EDTA; Invitrogen) for 2–5 minutes at 37°C. The trypsin was neutralized in 5 ml CEM, and all the cells (passage 1) were replated into a 56.7-cm2 culture dish. The cells were either frozen in liquid nitrogen or expanded further. For freezing, the cells were re-suspended and frozen in 5% dimethylsulfoxide (DMSO), 15% FBS, and15% HS in IMDM by placement in a -20°C freezer for 1 hour, then a -80°C freezer for 1 hour, and finally stored in liquid nitrogen.
mASCs and GFPTgASCs were isolated from inguinal fat pads. The tissue was washed extensively with PBS containing 200 U/ml penicillin (Invitrogen), 200 μg/ml streptomycin (Invitrogen), and 0.50 μg/ml amphotericin B (Invitrogen). The fat was minced with sterile scalpels in 0.075% Collagenase Type I and incubated with the collagenase for 1 hour. The collagenase was then neutralized with CEM and the mixture was re-suspended with vigorous pipetting. The tissue was then washed with PBS and filtered through a 70 μm nylon mesh filter. The suspension was centrifuged at 400 × g for 4 minutes at 4°C, and the pellet was re-suspended in 2 ml CEM. The cells were then plated on a 20.8 cm2 culture dish, and the media was replaced after 24 hours. Adherent mASCs were cultured in the same manner as mBMSCs.
mMSC Differentiation
Cells were plated in 6-well plates at 1000 cells/well and incubated in CEM for 3 days. For osteogenesis, the cultures were then incubated in CEM supplemented with 20 mM glycerol phosphate, 50 ng/mL thyroxine, 1 nM dexamethasone, and 50 μM ascorbate 2-phosphate (all from Sigma, St Louis, MO). The media was changed 2 times per week for 2 weeks. The cells were fixed with 10% formalin for 20 minutes at RT and stained with Alizarin Red, pH 4.1 (Sigma) for 20 minutes at RT.
For the quantitative osteogenesis assay, cells were plated in 96-well plates at 100 cells/well and cultured as aforementioned for 2 weeks. The cells were then stained with Alizarin Red and de-stained with 10% cetylpyridinium chloride for 30 minutes. The amount of Alizarin Red was determined by measuring the optical density (OD) of the solution at 560 nm. The results were then normalized to the protein contents of the samples.
For adipogenesis, the cultures were incubated in CEM supplemented with 5 μg/mL insulin, 50 μM indomethacin, 1 μM dexamethasone, and 0.5 μM 3-isobutyl-1-methylxanthine (IBMX; all from Sigma). The medium was changed 2 times per week for 2 weeks. The cells were fixed with 10% formalin for 20 minutes at RT and stained with 0.5% Oil Red O (Sigma) in methanol (Sigma) for 20 minutes at RT.
For the quantitative adipogenesis assay, cells were plated in 96-well plates at 100 cells/well and cultured as aforementioned for 2 weeks. The cells were then stained with Oil Red O and de-stained with isopropyl alchohol. The amount of Oil Red O was determined by measuring the OD of the solution at 520 nm. The results were then normalized to the protein contents of the samples.
For chondrogenesis, a pellet culture system was used as previously described [24]. Approximately 2 × 105 mMSCs (passage 8 or lower) were placed in a 15-mL polypropylene tube (Falcon, Bedford, MA), and pelleted by centrifugation. The pellet was cultured at 37°C with 5% CO2 in 500 μL chondrogenic media that contained 500 ng/mL bone morphogenic protein-6 (BMP-6; R&D Systems, Minneapolis, MN) in addition to high-glucose DMEM supplemented with 10 ng/mL transforming growth factor-3 (TGF-β3; R&D Systems), 10-7 M dexamethasone (Sigma), 50 μg/mL ascorbate-2-phosphate (Sigma), 40 μg/mL proline (Sigma), 100 μg/mL pyruvate (Sigma), and 50 mg/mL ITS+ Premix (Becton Dickinson, Bedford, MA; 6.25 μg/mL insulin, 6.25 μg/mL transferrin, 6.25 ng/mL selenious acid, 1.25 mg/mL bovine serum albumin, and 5.35 mg/mL linoleic acid). The medium was replaced every 3 to 4 days for 21 days. For microscopy, the pellets were embedded in paraffin, cut into 5 μm thick sections and stained with Toluidine Blue Sodium Borate (Sigma).
FACS analysis
Cells (passage 9 or lower) were trypsinized, collected, washed with PBS, and incubated for 1 hour at 4°C with phycoerythrin (PE)-conjugated antibodies against Sca-1 (Beckton Dickinson [BD]), CD34 (eBiosciences), CD106 (vascular adhesion molecule-1 [VCAM-1]) (Abcam), CD11b (BD), CD45 (BD), IgG2α (BD), IgG2β (BD), or IgG1 (Chemicon). Excess antibody was removed by washing cells with PBS, and cells were fixed in 1% para-formaldehyde. Detection of PE labeling was performed on a FACScalibur cytometer (BD, San Jose, USA), and results were analyzed using FlowJo software.
Examination of Growth Rates
Cells (passage 9 or lower) were plated at 100 cells/cm2 in a 56.7 cm2 culture dish in triplicate in CEM and cultured for 12 days. The media was changed every 3 days, and cells were trypsinized and pelleted every 3 days to analyze fold increase in density with a hemacytometer.
Colony Forming Unit Assay
In order to perform a colony forming unit (CFU) assay, cells (passage 9 or lower) were plated at a density of 100 cells in a 56.7 cm2 culture dish (1.76 cells/cm2). The cells were cultured for 14 days with fresh media added after 7 days. The plates were then washed with PBS and stained with 3% Crystal Violet at room temperature for 30 minutes. All colonies greater than 2 mm in diameter were counted. The CFU assay was performed in triplicate for each donor from three different frozen vials of cells.
