Female C3H/HeJ, C57BL/6J, and C3MRL-Faslpr/J mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Female immunocompromised mice (Beige nude/nude XIDIII) were purchased from Harlan (Indianapolis, IN, USA). All animal experiments were performed under the institutionally approved protocols for the use of animal research (USC #10874 and 10941).
Anti Oct4, SSEA4, Runx2, OCN, active β catenin and β catenin were purchased from Millipore (Billerica, MA, USA). Anti alkaline phosphatase (ALP) antibody was purchased from Abcam (Cambridge, MA, USA). Anti Sca-1-PE, CD34-PE, CD34-FITC, CD45-PE, CD73-PE, CD4-PerCP, CD8-FITC, CD25-APC, CD3ε and CD28 antibodies were purchased from BD Bioscience (San Jose, CA, USA). Anti Foxp3-PE, IL17-PE, and IFNγ-APC antibodies were purchased from eBioscience (San Diego, CA, USA). Unconjugated anti CD34, CD73, and CD105, NOS2 were purchased from Santa Cruz Biosciences (Santa Cruz, CA, USA). Anti β actin antibody was purchased from Sigma (St. Louis, MO, USA).
Isolation of mouse bone marrow mesenchymal stem cells (BMMSCs)
The single suspension of bone marrow derived all nucleated cells (ANCs) from femurs and tibias were seeded at a density of 15 × 106 into 100 mm culture dishes (Corning, NY, USA) at 37°C and 5% CO2. Non-adherent cells were removed after two days and attached cells were maintained for 16 days in alpha minimum essential medium (α-MEM, Invitrogen, Grand Island, NY, USA) supplemented with 20% fetal bovine serum (FBS, Equitech-bio, Kerrville, TX, USA), 2 mM L-glutamine, 55 μM 2-mercaptoethanol, 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). Colony-forming attached cells were passed once for further experimental use.
Preparation of Extracellular Matrix (ECM) coated dishes
ECM coated dishes were prepared as described previously . Briefly, 100% confluence of BMMSCs was cultured in medium with 100 nM L-ascorbic acid phosphate (Wako Pure Chemical, Richmond, VA, USA). After two weeks, cultures were washed with PBS and incubated with 0.005% Triton X-100 (Sigma) for 15 minutes at room temperature to remove cells. The ECM was treated with DNase I (100 units/ml; Sigma) for 1 hour at 37°C. The ECM was washed with PBS three times and stored in 2 ml of PBS containing 100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml fungizone (Invitrogen) at 4°C.
Isolation of BMMSCs in culture suspension (S-BMMSCs)
Bone marrow-derived ANCs (15 × 106) were seeded into 100 mm culture dishes and cultured for two days. The culture supernatant with floating cells was collected and centrifuged to obtain putative non-attached BMMSCs. The cells were re-seeded at indicated numbers on ECM-coated dishes. After 2 days, the floating cells in the cultures were removed with PBS and the attached cells on ECM were maintained for an additional 14 days. Colony-forming attached cells were passed once and sub-cultured on regular plastic culture dishes for further experiments. For some stem cell characterization analyses, we collected SSEA4 positive S-BMMSCs using the MACS magnetic separation system (Milteny Biotech, Auburn, CA, USA) and expanded in the cultures.
Colony forming unit-fibroblastic (CFU-F) assay
One million cells of ANCs from bone marrow were seeded on a T-25 cell culture flask (Nunc, Rochester, NY, USA). After 16 days, the cultures were washed with PBS and stained with 1% toluidine blue solution in 2% paraformaldehyde (PFA). A cell cluster that had more than 50 cells was counted as a colony under microscopy. The colony number was counted in five independent samples per each experimental group.
Cell proliferation assay
The proliferation of BMMSCs and S-BMMSCs was performed using the bromodeoxyuridine (BrdU) incorporation assay. Each cell population (1 × 104 cells/well) was seeded on two-well chamber slides (Nunc) and cultured for two to three days. The cultures were incubated with BrdU solution (1:100) (Invitrogen) for 20 hours, and stained with a BrdU staining kit (Invitrogen). BrdU-positive and total cell numbers were counted in ten images per subject. The BrdU assay was repeated in five independent samples for each experimental group.
Population doubling assay
A total of 0.5 × 106 cells of BMMSCs and S-BMMSCs was seeded on 60 mm culture dishes at the first passage. Upon reaching confluence, the cells were passaged at the same cell density. The population doubling was calculated at every passage according to the equation: log2 (number of harvested cells/number of seeded cells). The finite population doublings were determined by cumulative addition of total numbers generated from each passage until the cells ceased dividing.
Flow cytometric analysis of mesenchymal stem cell surface molecules
BMMSCs or S-BMMSCs (0.2 × 106 cells) were incubated with 1 µg of R-Phycoerythrin (PE). (PE)-conjugated antibodies or isotype-matched control immunoglobulin Gs (IgGs) (Southern Biotech, Birmingham, AL, USA) at 4°C for 45 minutes. Samples were analyzed by a fluorescence-activated cell sorting (FACS)Calibur flow cytometer (BD Bioscience). For dual color analysis, the cells were treated with PE-conjugated and fluorescein isothiocyanate (FITC)-conjugated antibodies or isotype-matched control IgGs (1 µg each). The cells were analyzed on FACSCalibur (BD Bioscience).
The cells subcultured on eight-well chamber slides (Nunc) (2 × 103/well) were fixed with 4% PFA. The samples were incubated with the specific or isotype-matched mouse antibodies (1:200) overnight at 4°C, and treated with Rhodamine-conjugated secondary antibodies (1:400, Jackson ImmunoResearch, West Grove, PA, USA; Southern Biotechnology, Birmingham, AL, USA). Finally, chamber slides were mounted using Vectashield mounting medium containing 4', 6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA).
In vivo bone formation assay
A total of 4.0 × 106 cells was mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) ceramic powders (40 mg, Zimmer Inc., Warsaw, IN, USA) and subcutaneously transplanted into eight-week-old immunocompromised mice. After eight weeks, the transplants were harvested, fixed in 4% PFA and then decalcified with 5% ethylenediaminetetraacetic acid (EDTA; pH 7.4), followed by paraffin embedding. The paraffin sections were stained with H & E and analyzed by an NIH Image-J. The newly-formed mineralized tissue area from five fields was calculated and shown as a percentage to total tissue area.
In vitro osteogenic differentiation assay
BMMSCs and S-BMMSCs were cultured under osteogenic culture conditions containing 2 mM β-glycerophosphate (Sigma), 100 μM L-ascorbic acid 2-phosphate and 10 nM dexamethasone (Sigma). After induction, the cultures were stained with alizarin red or alkaline phosphatase.
In vitro adipogenic differentiation assay
For adipogenic induction, 500 nM isobutylmethylxanthine, 60 μM indomethacin, 500 nM hydrocortisone, 10 μg/ml insulin (Sigma), 100 nM L-ascorbic acid phosphate were added to the culture medium. After 10 days, the cultured cells were stained with Oil Red-O and positive cells were quantified by using an NIH Image-J. Total RNA was also isolated from cultures after 10 days induction for further experiments.
In vitro chondrogenic differentiation assay
For chondrogenic induction, 1 × 106 cell pellets were cultured under chondrogenic medium containing 15% FBS, 1% ITS (BD), 100 nM dexamethasone, 2 mM pyruvate (SIGMA), and 10 ng/ml transforming growth factor beta 1 (TGFβ1) in (D)MEM (Invitrogen) for threeweeks. Cell pellets were harvested at three weeks post induction, fixed overnight with 4% PFA and then, sections were prepared for staining.
Reverse transcriptase polymerase chain reaction (RT-PCR) analysis
Extraction of total RNA and RT-PCR were performed according to standard procedures. Primer information is described in Additional materials and methods [see Additional file 1].
Western blotting analysis
A total of 20 µg of protein was used and SDS-PAGE and western blotting were performed according to standard procedures. Detailed procedures are described in Additional materials and methods [see Additional file 1]. β-actin on the same membrane served as the loading control.
Hematopoietic differentiation of BMMSCs and S-BMMSCs
BMMSCs and S-BMMSCs were cultured onto 35 mm low attach culture dishes (2 × 104/dish, STEMCELL Technologies, Vancouver, BC, V5Z 1B3, Canada) under hematopoietic differentiation medium (STEMCELL Technologies) with or without erythropoietin (EPO; 3 U/mL) for seven days. Whole bone marrow cells and linage negative bone marrow cells (Linage-cells) were used as positive controls. The results are representative of five independent experiments.
S-BMMSCs and BMMSCs were treated with 1 mM L-NG-monomethyl-arginine (L-NMMA) (Cayman Chemical, Ann Arbor, MI, USA) or 0.2 mM 1400 W (Cayman Chemical) to inhibit total nitric oxide synthase (NOS) or inducible nitric oxide synthase (iNOS), respectively.
Measurement of nitric oxide production
BMMSCs (0.2 × 106/well) were cultured on 24-well plates with or without cytokines (IFNγ, 25 ng/ml; IL-1β, 5 ng/ml, R&D Systems, Minneapolis, MN, USA) and chemicals (L-NMMA, 1 mM; 1400 W, 0.2 mM) at the indicated concentration and days. The supernatant from each culture was collected and nitric oxide concentration measured using a Total Nitric Oxide and Nitrate/Nitrite Parameter Assay kit (R&D Systems) according to the manufacturer's instruction.
Cell apoptosis and cell survival assay
The transwell system (Corning) was used for co-culture experiments. A total of 0.2 × 106 of S-BMMSCs or BMMSCs was seeded on each lower chamber. Activated spleen cells (1 × 106/chamber), which were pre-stimulated with plate-bound anti CD3ε antibody (3 µg/ml) and soluble anti CD28 antibody (2 µg/ml) for two days, were loaded in the upper chambers. Both chambers were filled with a complete medium containing (D)MEM (Lonza, CH-4002 Basel, Switzerland) with 10% heat-inactivated FBS, 50 µM 2-mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate (Sigma), 1% non-essential amino acid (Cambrex, East Rutherford, NJ, USA), 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin. To measure the spleen cells viability, cell counting kit-8 (Dojindo Molecular Technologies, Rockville, MD, USA) was used. For apoptosis of spleen cells analyses, Annexin V-PE apoptosis detection kits I (BD Bioscience) were used and analyzed on FACSCalibur (BD Bioscience).
In vitro CD4+CD25+Foxp3+Tregs and Th17 induction
CD4+CD25- T-lymphocytes (1 × 106/well), collected using a CD4+CD25+ Treg isolation kit (Miltenyi Biotec), were pre-stimulated with plate-bound anti CD3ε antibody (3 µg/ml) and soluble anti CD28 antibody (2 µg/ml) for two days. These activated T-lymphocytes were loaded on 0.2 × 106 BMMSCs or S-BMMSCs cultures with recombinant human TFGβ1 (2 ng/ml) (R&D Systems) and recombinant mouse IL2 (2 ng/ml) (R&D Systems). For Th17 induction, recombinant human TFGβ1 (2 ng/ml) and recombinant mouse IL6 (50 ng/ml) (Biolegend, San Diego, CA, USA) were added. After three days, cells in suspension were collected and stained with anti CD4-PerCP, anti CD8a-FITC, anti CD25-APC antibodies (each 1 µg) for 45 minutes on ice under dark conditions. The cells were then stained with anti Foxp3-PE antibody (1 µg) using a Foxp3 staining buffer kit (eBioscience) for cell fixation and permeabilization. For Th17, cells in suspension were stained with anti CD4-FITC (1µg, Biolegend) for 45 minutes on ice under dark conditions followed by intercellular staining with anti-IL 17 antibody (1µg, Biolegend) using a Foxp3 staining buffer kit. The cells were analyzed on FACSCalibur.
Allogenic mouse S-BMMSC transplantation into MRL/lpr mice
Under general anesthesia, C3H/HeJ-derived BMMSCs, S-BMMSCs, L-NMMA pre-treated BMMSCs (1 mM for five days), or CD34+/CD73+ double sorted cells (0.1 × 106 cells/10 g body weight) were infused into MRL/lpr mice via the tail vein at 10 weeks of age (n = 6 each group). In the control group, MRL/lpr mice received PBS (n = 5). All mice were sacrificed at two weeks post transplantation for further analysis. The protein concentration in urine was measured using a Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA).
Measurement of autoantibodies, albumin, soluble runt-related NF-κB ligand (sRANKL) and C-terminal telopeptides of type I collagen (CTX)
Peripheral blood serum samples were collected from mice. Autoantibodies, sRANKL and CTX were analyzed by ELISA using commercially available kits (anti-dsDNA antibodies and ANA; alpha diagnostics, albumin and sRANKL; R&D Systems, CTX; Nordic Bioscience Diagnostics, Herlev, Rigion Hovedstaden, Denmark) according to their manufactures' instructions. The results were averaged in each group. The intra-group differences were calculated between the mean values.
Flow cytometric analysis of Tregs and Th17 cells
To detect Tregs, peripheral blood mononuclear cells (PBMNCs) (1 × 106) were treated with PerCP-conjugated anti-CD4, FITC-conjugated anti-CD8a, APC-conjugated anti-CD25 antibodies, and stained with R-PE-conjugated anti-Foxp3 antibody using a Foxp3 staining buffer kit (eBioscience). To measure Th17 cells, PBMNCs (1 × 106) were incubated with PerCP-conjugated anti-CD4, FITC-conjugated anti-CD8a, followed by treatment with R-PE-conjugated anti-IL-17 and APC-conjugated anti-IFNγ antibodies using a Foxp3 staining buffer kit. The cells were then analyzed on FACSCalibur .
Student's t-test was used to analyze statistical difference. P values less than 0.05 were considered significant.