Miura et al. 10.1073/pnas.0409397102. |
Supporting Materials and Methods
Mice.
The CD18-null (CD18–/–) mice and their sex-matched C57BL/6J littermates were obtained by breeding heterozygotes of the CD18–/– mice (1). The CD18–/– mice have been backcrossed >10 generations into the C57BL/6J background. Immunocompromised bg-nu/nu-xid nude mice were purchased from Harlan–Sprague–Dawley. Mice were maintained in sterile microisolator cages and all animal experiments were performed with the approval of the committee of the American Red Cross (protocols no. 407 and 447) and the National Institute of Dental and Craniofacial Research (protocol no. 04-317).Analysis of Bone Phenotypes
. The CD18-deficient mice and their sex-matched C57BL/6J littermates at the ages of 5-15 weeks were used to analyze bone phenotypes. Radiographs of left femurs were taken by Faxitron (Wheeling, IL). Quantitative analysis for bone mineral density (BMD) on left femurs was based on dual energy x-ray absorptiometry (DXA) by the use of a Piximus (GE Lunar, Madison, MI). Distal femoral metaphyses were analyzed by microcomputed tomography (mCT-20; Scanco Medical, Bassersdorf, Switzerland) as described in ref. 2. The scanning regions were confined to secondary spongiosa and were »0.30 mm in thickness. Using 2D images, a region of interest was manually drawn near the endocortical surface. Cancellous bone morphometric indices were assessed using 3D image reconstructions, including bone volume/total volume (BV/TV) percentage, trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp). Peripheral quantitative computed tomography (pQCT) analysis of the distal femora was performed using an XCT Research M scanner (Stratech, London; Norland Co., Trumbull, CT) as described in ref. 2. Briefly, scans were obtained at 2.25 and 2.75 mm from the distal condyles and cancellous BMD. Machine cancellous BMD precision (based on the manufacturer’s data) was ±3 mg/cm3, while the coefficient of variation in our laboratory based on repeat scans was 2.26%. For histological analysis, left femurs were harvested from mice, fixed with 2% paraformaldehyde, decalcified with 10% EDTA (pH 8.0), and embedded in paraffin. Sections were deparaffinized, hydrated, and stained with hematoxylin/eosin. To analyze the whole skeleton, 1-week-old mice were dissected to remove skin, muscle, and fat and kept in acetone to remove further fat for 3 days. They then were stained with 0.09% alizarin red S and 0.05% Alcian blue in a solution containing ethanol, glacial acetic acid, and water (67:5:28) for 48 h at 37°C. After staining, the mice were transferred to 1% potassium hydroxide until the skeleton was clearly visible. The mice were preserved in 100% glycerol with gradual increase in concentration.Mouse BMSSC Culture.
Preparation and expansion of the mouse BMSSCs were performed based on a method described in ref. 2. Mouse bone marrow (BM) cells (1.5 × 107) harvested from long bones were seeded into 10-cm culture dishes (Corning, Corning NY), incubated for 3 h at 37°C to allow attachment of adherent cells, and washed twice with PBS to remove nonadherent cells. BM cells (1.5 × 107) from long bones of guinea pigs then were added as feeder cells. To prevent proliferation in culture, the feeder cells were g-irradiated (Caesium-137) with 6,000 cGy by a Gammacell-1000 Irradiator (Atomic Energy of Canada Ltd., Ontario) before seeding. The mouse culture medium consisted of a-MEM (GIBCO/BRL; Invitrogen), 20% FBS (Equitech-Bio, Kerrvile, TX), 2 mM L-glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin (Biofluids Inc., Rockville, MD), 10 nM dexamethasone (Sigma–Aldrich), and 55 mM 2-mercaptoethanol (GIBCO/BRL; Invitrogen). BMSSCs formed adherent colonies after 7–16 days of culture, which was designated as passage 0. Primary BMSSCs were lifted to disperse the colony-forming cells and seeded on freshly prepared culture dishes. These cells were passaged when they reached confluence and used for further experiments.Human BMSSCs Culture.
Human BM aspirates from healthy adult volunteers were purchased from AllCells, LLC (Berkeley, CA). To identify putative BMSSCs, single-cell suspension of 1 × 106 of bone marrow mononuclear cells (BMNCs) were seeded into 15-cm culture dishes (Falcon; BD Biosciences, San Jose, CA), and nonadherent cells were removed after 3 h of incubation at 37°C. The adherent cells were cultured with a-MEM supplemented with 15% FBS, 100 mM L-ascorbic acid 2-phosphate (Wako Pure Chemical, Osaka), 2 mM L-glutamine, and a combination of 100 units/ml penicillin and 100 mg/ml streptomycin. The human culture medium was changed on days 7 and 14, if the cells were not passaged by day 14. Subsequent passages were performed when the cells were approaching confluent. BMSSCs of the second to the fifth passages were used for further experiments unless specifically mentioned.Dual-Color Cell Sorting of Fresh Human Bone Marrow by FACS
. Procedures for sorting of the human BMSSC population are described in refs. 3 and 4. Approximately 1-3 × 108 BMNCs were sequentially incubated with STRO-1 supernatant (IgM), anti-IgM-biotin, streptavidin microbeads, and finally streptavidin-FITC (Caltag, Burlingame, CA) before being separated on a Mini MACS magnetic column (Miltenyi Biotec, Auburn, CA). The FITC-labeled STRO-1 positive BMNCs isolated by MACS were colabeled with a mouse anti-human CD18 mAb (clone 6.7, Pharmingen/BD Biosciences) for 30 min on ice, washed, and incubated with PE-conjugated goat anti-mouse IgG (Pharmingen/BD Biosciences) for an additional 20 min on ice. After washing, cells were sorted for both CD18 and STRO-1 using a FACStarPLUS flow cytometer (Becton Dickinson). The sorted human BMSSCs, either STRO-1bright/CD18+ or STRO-1bright/CD18–, were used for further experiments.Flow Cytometric Analysis of BMSSCs.
The sorted human STRO-1bright/CD18+ BMSSCs were incubated with either of the primary Abs or their corresponding isotype-matched control Abs at a concentration of 10 mg/ml for 1 h on ice. Primary Abs were as follows: mouse IgG1 anti-human CD14, CD34, CD45 (DAKO Cytomation, Carpinteria, CA); mouse IgG1 anti-human CD44 (H9H11) and IgG2a anti-human CD146 (CC9); mouse IgG1 anti-human CD90, CD105, CD166 (Pharmingen; BD Biosciences); mouse IgG1 anti-human CD106 (6G10) (kindly provided by B. Masinovsky, ICOS Corp., Bothell, WA). Isotype-matched control mouse mAbs were 1B5 (IgG1) and 1A6.11 (IgG2b) (kindly provided by L. K. Ashman, University of Newcastle, New South Wales, Australia). After washing, the cells were incubated with the secondary detection reagents, goat anti-mouse IgG1- or IgG2b-FITC conjugated Abs (1/50) (Southern Biotechnology Associates) for 45 min on ice. After washing, the samples were analyzed using an Epics-XL-MCL flow cytometer (Beckman Coulter). For dual-color FACS analysis of mouse BMSSCs, single-cell suspension of P0 BMSSCs (1 × 106) were incubated with a pair of FITC- and PE-conjugated Abs or their corresponding isotype-matched controls (each Ab at 10 mg/ml) for 45 min on ice. After washing, the samples were analyzed using a FACScan flow cytometer (Becton Dickinson). The percentage of cell population in each quadrant was calculated using the FACSCAN program. All Ab conjugates were purchased from Pharmingen/BD Biosciences unless specifically mentioned, including PE-conjugated rat anti-mouse CD18 (C71/16, IgG2a), FITC- and PE-conjugated Sca-1 (E13-161.7, IgG2a), FITC-conjugated rat anti-mouse CD14 (rmC5-3, IgG1), FITC-conjugated anti-CD34 (49E81, IgG2a), and their corresponding FITC- and PE-conjugated IgG controls.In Vitro
Differentiation Potentials of the Sorted Human BMSSCs. Osteogenic differentiation of the sorted human STRO-1bright/CD18+ BMSSCs was induced in the presence of 100 mM L-ascorbate-2-phosphate, 3 mM inorganic phosphate, and 10 nM dexamethasone. Calcium deposits were identified by Alizarin Red S staining after 3 weeks of cultivation (5). Adipogenesis was induced in a-MEM supplemented with 15% FBS, 100 mM L-ascorbate-2-phosphate, 0.5 mM isobutyl-methylxanthine, 0.5 mM hydrocortisone, 60 mM indomethacin, and 10 mg/ml recombinant human insulin. Oil Red O staining was used to identify lipid-laden fat cells after 2 weeks of cultivation, as described in ref. 6. Chondrogenic differentiation was assessed by histochemical staining with Alcian blue (pH 1) in aggregate cultures treated with 100 mM L-ascorbate-2-phosphate, 2 mM sodium pyruvate, 1% insulin/transferring/selenous acid mixture (ITS; BD Biosciences), 100 nM dexamethasone, and 10 ng/ml TGF-b as described in ref. 7.In Vitro
Mineralization Induction of Mouse BMSSCs. For the mineralization induction of mouse BMSSCs in vitro, 2 mM b-glycerophosphate (Sigma–Aldrich) and 100 mM L-ascorbic acid 2-phosphate (Wako Pure Chemical) were added to the mouse culture medium. After 6 weeks of cultivation, the cultures were stained with 1% Alizarin red to examine calcium accumulation in the cells. The images were captured with a scanner (Epson), and mineralized areas were quantified using TOTALAB software (Nonlinear Dynamics, Durham, NC). Mineral deposits were expressed as a ratio relative to WT mouse BMSSC-mediated mineralization.BMSSC-Mediated Bone Formation in Vivo.
Approximately 4.0 × 106 mouse BMSSCs were mixed with 40 mg of hydroxyapatite/tricalcium phosphate (HA/TCP) ceramic powder (Zimmer, Warsaw, IN), and the mixture was implanted s.c. into the dorsal surface of 8- to 10-week-old immunocompromised bg-nu/nu-xid nude mice as described in ref. 8. The transplants were recovered 7 weeks after implantation and fixed with 2% paraformaldehyde, decalcified with 10% EDTA (pH 8.0), and embedded in paraffin. For quantification of bone formation in the transplants, sections were deparaffinized, hydrated, and stained with hematoxylin/eosin. Five to seven representative fields at ´50 magnification were selected for each BMSSC transplant. The total bone area within each field was calculated using the program NIH IMAGE as described in ref. 9 and expressed as a percentage of bone formation by WT BMSSCs.Colony-Forming Unit Fibroblast (CFU-F) Assay.
The CFU-F assay was performed as described in ref. 10. Mouse BM cells (1-5 × 106) harvested from long bones were seeded into T-25 culture flasks (Nalge Nunc, Rochester, NY), incubated for 3 h at 37°C to allow attachment of adherent cells, and then rinsed twice with PBS to remove nonadherent cells. BM cells (1.5 × 107) from long bones of guinea pigs then were added as feeder cells. To prevent proliferation in culture, the feeder cells were g-irradiated (Caesium-137) with 6,000 cGy by a Gammacell-1000 Irradiator (Atomic Energy of Canada) before seeding. Culture medium consisted of a-MEM, 20% FBS, 2 mM L-glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin, and 55 mM 2-mercaptoethanol. Adherent colonies were fixed with methanol between days 7 and day and stained with an aqueous solution of saturated methyl violet (Sigma–Adlrich). The sorted human BMSSCs, either STRO-1bright/CD18+ or STRO-1bright/CD18–, were plated on the culture dishes. Colony-forming efficiency assays were performed at day 14 of culture after staining of the cultures with 0.1% (wt/vol) toluidine blue in 1% paraformaldehyde. Colonies containing ³50 cells were counted as colonies under a dissecting microscope.Cell Proliferation Assays.
The proliferation of BMSSCs was assessed by BrdUrd incorporation of the cells. Mouse BMSSCs were seeded at 5 × 105 cells on two-well chamber slides (Nalge Nunc), incubated with BrdUrd solution (Zymed) for 20 h. The number of BrdUrd positive cells were detected using a BrdUrd staining kit (Zymed) according to the manufacturer’s instruction with hematoxylin counterstaining. For quantification of BrdUrd positive cells, 10 representative images captured at ´200 magnification were used to calculate BrdUrd positive cell number. Cell proliferation was shown as a percentage of BrdUrd-positive cells over total nucleated cells.Osteoclast Activity.
In vivo osteoclastic activity was determined by measuring the serum concentrations of C-terminal telopeptides of type 1 collagen, obtained from the peripheral blood of 8-week-old mice, using the Ratlap ELISA kit (Osteometer BioTech A/S, Herlev, Denmark) according to the manufacturer’s instructions. The number of mature osteoclasts was determined by the tartrate-resistant acid phosphate (TRAP) staining, as described in ref. 11. Briefly, the left femurs were harvested from mice, fixed with 2% paraformaldehyde, decalcified with 10% EDTA (pH 8.0), and embedded in paraffin. Sections were deparaffinized, hydrated, and stained for TRAP. The TRAP solution was a mixture of the following two solutions: 9.6 mg of naphthol AS-BI phosphate substrate (Sigma–Aldrich) dissolved in 0.6 ml of N,N-dimethylformamide and 84 mg of fast red-violet LB diazonium salt (Sigma–Aldrich), 58.2 mg of tartaric acid (Sigma–Aldrich), 240 ml of 10% MgCl2, and 4 ml of 3 M sodium acetate buffer (pH 5.0) dissolved in 56 ml of distilled water. The mixture was passed through a 0.22-mm filter before use. The sections were incubated for 30 min in the TRAP solution at 37°C in the dark and then washed with distilled water for 10 min, followed by a counterstaining with hematoxylin. For quantification of TRAP positive cells in the bones, four representative images were captured under microscope with a ´200 magnification and then analyzed using the program NIH IMAGE. The results were expressed as the number of TRAP positive cells per total bone area.Cell Adhesion Assays.
The same numbers of WT and CD18–/– BMSSCs were seeded on treated plastic chamber slides (Nalge Nunc) in the culture medium. After 3 h of incubation, the chamber slides were gently washed with PBS twice to remove nonadherent cells, and the number of adherent cells was determined by manual counting of five representative fields at ´100 magnifications.Retroviral-Mediated CD18 Expression in BMSSCs.
Murine CD18 cDNA was subcloned into the retroviral expression vector MGIN (12), using the EcoRI and NotI restriction sites. To achieve better expression, a Kozak sequence (ACCATGG) was inserted before the initiation codon of the CD18 protein. After confirming the correctness of the inserted CD18 sequence by DNA sequencing, the retroviral expression vector MGIN-CD18 was transfected into the packaging cell line GP+E-86 (13), using Lipofectamine (Invitrogen). After G418 (600 mg/ml) selection of the transfected cells, individual clones were established by picking up single colonies, and the titers of their viral supernatants were determined by infecting NIH 3T3 cells and counting the colonies formed upon G418 selection. The clones that produced the highest retroviral titer [»1 × 106 colony-forming units (CFU) per ml] were used to prepare retroviral supernatants, which were subsequently concentrated to 6 × 106 CFU/ml and used to infect the CD18-deficient BMSSCs in the presence of 8 mg/ml polybrene. Three days later, the medium was replaced with freshly prepared viral supernatants, and the infected BMSSCs were used 1 day later for in vivo bone formation experiments. To evaluate the infection efficiency, a portion of the infected cells were kept for additional 3 days and then analyzed by FACS using a PE-conjugate of rat anti-mouse CD18 (mAb C71/16) and by immunoblot using a rabbit anti-CD18 cytoplasmic domain Ab (14).Western Blot Analysis.
Cells were lysed in M-PER extraction reagent (Pierce), and protein concentrations were measured using Bio-Rad Protein Assay. To examine CD18 expression in mouse BMSSCs, total cell lysates from the same number of WT, CD18-/-, and CD18-reconstituted (with either full-length or cytoplasmic tail-truncated CD18) BMSSCs were applied onto NuPAGE gel (Invitrogen) at »3 × 104 cells per lane. The separated proteins were transferred onto poly(vinylidene fluoride) membranes (Millipore) and blocked with a solution containing 10 mM Tris•HCl (pH 7.5), 154 mM NaCl, 4% BSA, 1% milk, and 0.05% Tween 20 for 60 min at room temperature (RT). After washing, the membrane was incubated with rabbit anti-CD18 cytoplasmic tail Abs (1:1,000 dilution) in incubation buffer [10 mM Tris•HCl (pH 7.5), 154 mM NaCl, 0.5% BSA, 0.05% Tween 20] for 60 min at RT. The membranes then were washed and incubated with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology) at 1:5,000 dilutions in incubation buffer for 30 min at RT. After washing with T-TBS, the membranes were reacted with HRP substrate (Pierce) to visualize positive bands on x-ray films (Eastman Kodak). To examine CD18 expression in human BMSSCs, total cell lysates from human BMSSCs at different passages were prepared. In each lane, 12 mg of protein were loaded and analyzed as described above. To examine Cbfa1 expression, 2.2 mg of total protein lysate from WT or CD18-/- BMSSCs were applied to NuPAGE and detected by immunoblot using rabbit anti-Cbfa1 (Oncogene Research Products, Cambridge, MA) Ab at 1:500 dilution. To examine the effect of TGF-b treatments, mouse BMSSCs were serum-starved with a-MEM supplemented with 2% FBS for 16 h and then treated with 2 ng/ml TGF-b (R&D Systems) for the indicated periods. Phosphorylation of Smad2 was examined by loading equal amount of cell lysate (12 mg) in each lane and detected by immunoblot using rabbit anti-phospho-Smad2 Ab (Cell Signaling Technology, Beverly, MA) at 1:500 dilution. Each membrane also was stripped using the stripping buffer (Pierce) and reprobed with mouse anti-a-actinin mAb (Upstate Biotechnology, Lake Placid, NY) or anti-b-actin (Sigma–Aldrich) to quantify the amount of proteins loaded.Statistical Analysis.
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