Bone marrow preparation
Our study was approved by the local institutional review board (ethical committee). Bone marrow (BM) was taken under sterile conditions from randomly chosen donors (neither metabolic nor neoplastic diseases) with informed consent during orthopaedic operations: From each donor 5 ml of whole BM was collected in a sterile heparinized syringe.
MSC isolation and cell culture
To isolate MSCs from whole BM we used the density gradient technique as described previously [37]. Briefly, 5 ml bone marrow was resuspended in 10 ml PBS (Cambrex Bio Science, Verviers, Belgium) and laid over 15 ml Lymphoflot (sodium diatrizoate 9.1% [w/v], ficoll 5.7% [w/v]; Biotest AG, Dreieich, Germany). After centrifugation (20 minutes at 1000 × g without braking) the mononuclear cells were harvested, washed twice with PBS, transferred to a 75 cm2 culture flask (Corning Inc., Schiphol-Rijk, Netherlands) and incubated (37°C, 5% humidified CO2) with normal medium containing desoxyribonucleotides, ribonucleotides, ultra glutamine 1 (α-MEM, Cambrex Bio Science), 100 I.U./ml Penicillin (Cambrex Bio Science), 100 μg/ml Streptomycin (Cambrex Bio Science) and 10% heat inactivated Fetal Calf Serum (FCS) (Cambrex Bio Science). After 24 hours the non-adherent cells were removed and the adherent cells were cultured and characterized.
Characterization of human MSCs by in vitro differentiation and FACS analysis
MSCs are functionally characterized by in vitro differentiation assays [38]. We evaluated the differentiation potential into three mesenchymal lineages: Adipogenic, osteogenic and chondrogenic differentiation. The MSCs were treated for 21 days with adipogenic medium, osteogenic medium or normal medium (control) as described previously [39, 40] (modification: No addition of Amphotericin B): Adipogenic medium contained DMEM + 20% FCS with 1.0 μM dexamethasone (Sigma, Deisenhofen, Germany), 0.5 mM isobutylmethylxanthine (Sigma), 0.2 mM indomethacine (Sigma) and 0.01 mg/ml insulin (Sigma). Osteogenic medium contained normal medium with 10-8 M dexamethasone (Sigma), 0.2 mM ascorbic acid (Sigma) and 10 mM β-glycerolphosphate (Sigma). After 21 days the cell culture was stained with oil red O for in vitro adipogenesis. Briefly, after removal of the medium and washing twice with PBS, 2 ml of 10% formalin were added followed by an incubation time of 30 minutes. After removing the formalin and washing the cell layer with sterile water 2 ml of isopropanol (60%, Bio Whittaker, Verviers, Belgium) were added for 2 minutes. The isopropanol was removed and 2 ml of a filtered working solution of oil red O (3 parts oil red O stock solution [300 mg oil red O powder (Sigma) + 100 ml 99% isopropanol (Sigma)] + 2 parts deionized water) were pipetted onto cells and left there for five minutes. Thereafter the plate was rinsed with tap water and the cells were counterstained with 2 ml haematoxylin (Sigma) for 1 minute. The osteogenic differentiated cells as well as control cells were cytochemically stained for alkaline phosphatase using a commercial staining kit according to the manufacturer's (Cambrex Bio Science) recommendations: after removal of the medium and washing twice with PBS, 2 ml citrate fixative (12.5 ml citrate solution + 32.5 ml acetone + 4 ml 37% formaldehyde) were added for 1 minute, followed by staining with 2 ml alkaline-dye (0.5 ml sodium nitrite + 0.5 ml FRV-alkaline solution + 22.5 ml deionized water + 0.5 ml naphtole AS-BI alkaline solution) for 30 minutes. The cell layer was washed twice and counterstained with 2 ml haematoxylin (Sigma) for 1 minute. Chondrogenic differentiation was performed using a commercially available mesenchymal functional differentiation kit (Cat. Nr. SC006, R&D Systems, Wiesbaden, Germany). The MSC of each population were treated with the chondrogenic differentiation procedure: 250 × 103 cells were transferred into a 15 ml tube. After centrifugation (200 × g), 1.0 ml basal medium (D-MEM/F-12, Bio Whittaker) and 0.5 ml chondrogenic differentiation medium were added and replaced every 2-3 days. The cell suspension was cultured in the tube forming a solid pellet. After 14 days the chondrocyte pellet was removed, squeezed on a glass slide and stained with 1% Alcian Blue 8GX (Serva, Heidelberg, Germany) in 3% acetic acid (pH 2.5).
FACS analysis was performed with FACScan (BD Biosciences, San Jose, CA, USA) using BD CellQuest Pro software. At subconfluency (1 × 106 cells) the cells were detached with Accutase™ (PAA Laboratories, Cölbe, Germany)) and washed (PBS + AccuMax™ [PAA Laboratories]). Each probe contained a cell suspension with 5 × 105 cells in FACS-buffer (PBS + 1% bovine serum albumine [Sigma] + 0.1% FCS [Cambrex Bio Science]). The PE-conjugated antibody (anti-human-CD14, -CD29, -CD34, -CD44, -CD45, -CD59, -CD71, -CD73, -CD90, -CD105, -CD106, -CD146, -CD166, -CD271, HLA class I, HLA class II) was added. After an incubation time of 20 minutes and 2 washing steps the probe was ready for analysis. All antibodies, except for anti-human-CD271 (Miltenyi Biotech, Bergisch Gladbach, Germany), were from BD Biosciences.
Contrast Media and cell labeling
Resovist® (SHU 555A, Bayer Schering AG, Berlin, Germany), an MRI contrast agent already approved for clinical use in Europe, Japan and Australia, is organ-specific and used for liver imaging [41–43]. It consists of superparamagnetic iron oxide (SPIO) nanoparticles (4-6 nm) [44] coated with carboxydextran (mean hydrodynamic diameter 60 nm), which is accumulated by phagocytosis in von Kupffer cells. For an enhanced iron loading of the cells, the transfection reagent (TA) jetPEI™, a linear polyethylenimine (PolyPlus Transfection, Illkirch, France) was used. Cell labeling was performed for 4 h with 60 μg/ml Resovist® in combination with jetPEI™ in 6-well-plates seven days after seeding of 5 × 104 cells as described previously [19]. First, 10 μl of the TA in a total volume of 50 μl PBS was carefully added to 50 μl of the Resovist® solution, mixed and preincubated for 30 min. The following incubation was carried out in the incubator at standard conditions (37°C, 5% humidified CO2). After the labeling, cells were washed three times with PBS, harvested and processed for further plating with or without magnets.
Magnets
Magnets (magnetic material: NeoSint N33H (alloy of neodymium, dysprosium, boron, cobalt and iron), remanence: > 1.140 mT, magnetic field force on the surface: 600 mT, coercive force, BHC = > 851 kA/m, JHC = > 1.353 kA/m, max. residual energy > 247 kJ/m3, magnetisation direction: axial, coating: nickel, diameter: 15 mm, thickness: 3 mm (ppm materials GmbH, 70794 Filderstadt, Germany)) were fixed on plastic dishes and the 75 cm2 culture flasks and 6-well plates were firmly positioned with direct contact above the magnets (Figure 2A-C). The 75 cm2 culture flasks were used for the proliferation and viability assays, the transmission electron microscopy, the quantification of the total iron load and the adipogenic and osteogenic differentiation analyses, the 6-well plates were used for the colony formation analyses and the microarray experiments. The following conditions were investigated: unlabeled MSCs without exposition to magnetic fields (MSC - m), unlabeled MSCs with exposition to magnetic fields 24 hours after seeding into the 75 cm2 culture flasks and 6-well plates (MSC + m(24 h)), unlabeled MSCs with exposition to magnetic fields immediately after seeding into the 75 cm2 culture flasks and 6-well plates and before adhesion to the plastic surface (MSC + m(0 h)), SPIO-labeled MSCs without exposition to magnetic fields (JPR-MSC - m), SPIO-labeled MSCs with exposition to magnetic fields 24 hours after seeding into the 75 cm2 culture flasks and 6-well plates (JPR-MSC + m(24 h)), and SPIO-labeled MSCs with exposition to magnetic fields immediately after seeding into the 75 cm2 culture flasks and 6-well plates and before adhesion to the plastic surface (JPR-MSC + m(0 h)). The exposition to the magnetic fields continued until the end of the respective experiments.
Proliferation assays and determination of viability
Proliferation assays were performed in 75 cm2 culture flasks positioned with direct contact above the magnets. Labeled and control cells were seeded at 3 × 105 cells/flask in standard medium (α-MEM, 100 I.U./ml Penicillin, 100 μg/ml Streptomycin and 10% Fetal Calf Serum). The incubation was carried out in the incubator at standard conditions (37°C, 5% humidified CO2). The labeled and unlabeled MSCs were exposed to magnetic field immediately after seeding, 24 hours after seeding into the 75 cm2 culture flasks or there was no exposure (control group).
Medium was changed every 3 days. At subconfluence (90%) the cells were detached with Accutase (PAA Laboratories, Cölbe, Germany) and counted with a CASY®2 Analyser (CASY®2-Cell Counter and Analyser System, Model TT, Roche Diagnostics, Mannheim, Germany). The experiments were performed in triplicates.
Cellular viability after the different incubation conditions was examined with the help of a CASY®2 Analyser according to the ECE method described by Lindl et al [45] and the viability-SOP of the manufacturer.
Quantification of the total iron load (TIL)
After reaching subconfluence MSC + m(24 h), MSC + m(0 h) and (MSC - m), all Resovist®/jetPEI™ labeled, were washed three times with PBS, harvested with Accutase and counted with a CASY®2 Analyser. A cell pellet containing 1 × 105 cells was dried for 2 hours at 80°C. Then samples were incubated overnight at room temperature and another 2 hours at 60°C in perchloric and nitric acid at a 3:1 ratio to completely digest the cells and expose iron oxide from the dextran coated nanoparticles. For photometric determination of the TIL, a Ferrozine-based spectrophotometric assay (Eisen Ferene S Plus®, Rolf Greiner Biochemica, Flacht, Germany) was used. Fe2+ forms a blue complex with Ferene which can be measured at 595 nm. The extinction of the sample relates directly to the iron concentration, calculated with the help of a defined standard. All experiments were performed in triplicates.
Transmission electron microscopy
To assess the uptake and localization of the SPIO nanoparticles and their possible influence on the cellular ultrastructure, electron microscopy was performed. Cells grown as described above in the proliferation section and treated as indicated were fixed in 2.5% glutaraldehyde (Paesel-Lorei, Frankfurt, Germany) buffered with 0.1 M cacodylate buffer (pH 7.4), postfixed in 1% OsO4 in 0.1 M cacodylate buffer and scraped off the plastic. The pellet was then dehydrated in an ethanol series (50%, 70%, 96%, 100%). The 70% ethanol was saturated with uranyl acetate for contrast enhancement. Dehydration was completed in propylene oxide. The specimens were embedded in Araldite (Serva). Ultrathin sections were produced on a FCR Reichert Ultracut ultramicrotome (Leica, Bensheim, Germany), mounted on pioloform-coated copper grids, contrasted with lead citrate and analyzed and documented with an EM 10A electron microscope (Zeiss, Oberkochen, Germany).
In vitro MRI
An agar matrix was used as suitable environment for imaging SPIO labeled MSCs. The agar solution (1%) was boiled and embedded in nonferromagnetic boxes before becoming stable. By using a special stamp a series of identical cone shaped cavities was created in the agar block. For MR measurement cell numbers from one thousand to two hundred thousand Resovist® labeled MSCs were used (JPR-m; JPR + m(24 h); JPR + m(0 h)). Cells were centrifuged at 200 × g for 5 minutes, dissolved in 8% gelatine (20 μl) and implanted into the cone shaped cavities within the agar matrix. After solidification of the gelatine the hollows were closed with agar. Thus, it was possible to achieve a homogenous distribution of the target cells in a defined volume of 20 μl within a homogenous agar block. Cells were scanned in a clinically used MR scanner at 3.0 T (Magnetom Trio, Siemens Healthcare, Erlangen, Germany) using the wrist coil of the manufacturer. Imaging was performed with a conventional 3D gradient echo sequence with a Field-of-View (FoV) of 83 × 120 and a matrix size of 176 × 256, resulting in a resolution of 0.21 mm. The echo time (TE) was chosen as the variable parameter and was varied between 5 ms and 15 ms in steps of 5 ms with a repetition time (TR) of 70 ms and a flip angle of 14° according to the T1-value of the agarose gel of 2000 ms. Further parameters were: readout bandwidth (BW) 220 Hz/Px, 32 slices with a slice thickness of 0.5 mm. The acquisition time was then for one average about 6.5 minutes. Signal extinction was described by measuring the dimensions of the signal artifacts in all directions.
Migration assays
The migratory ability of labeled and control MSCs was investigated with cells which finished the proliferation assay. Every six conditions were analyzed (JPR-MSC - m; JPR-MSC + m(24 h); JPR-MSC + m(0 h) and MSC - m; MSC + m(24 h); MSC + m(0 h). There was no magnetic exposure during migration assay.
The MSCs were tested in 24-well compound chambers (Falcon, Becton Dickinson, Franklin Lakes, NJ, USA) with 8 μm pore membrane inserts. Cells were washed three times with PBS, harvested and finally seeded at a density of 20.000 cells in the membrane inserts with 0.5 ml cell culture medium containing 20% FCS. 0.8 ml medium containing 20% FCS and 25 ng/ml PDGF-BB (R&D Systems), serving as a chemoattractant, was added to the lower compartment of the plate. After incubating the plates for 24 h at 37°C in a humidified 5% CO2 atmosphere, the membrane inserts were fixed (ethanol 70%, formaldehyde 3.5%, 10 min. respective) and stained with Coomassie blue after mechanically removing the cells attached to the upper surface. The cells on the lower side of the inserts were counted with the help of a 100× magnification light microscope (Zeiss, Göttingen, Germany) using a Fuchs-Rosenthal counting-chamber. For each condition 5 membranes have been investigated.
Colony forming assays
The clonogenic activity of the cells was determined with the help of colony forming assays. There were two different setups, under exposition to magnetic field and after exposition to magnetic field. Every six conditions were analyzed (JPR-MSC - m; JPR-MSC + m(24 h); JPR-MSC + m(0 h) and MSC - m; MSC + m(24 h); MSC + m(0 h).
The cells were seeded at 250 cells/well in 2 ml medium in 6-well plates (Falcon), resulting in 12 wells per condition. The plates were incubated at 37°C in a 5% CO2 atmosphere without changing medium. After 10 days, the assays were stopped and the cells were fixed with 3.5% formaldehyde and 70% ethanol and subsequently stained with Coomassie blue. The total number of colonies exceeding 50 cells per colony was counted by light microscopy (Zeiss). Because of the intense inter-donator variation of the clonogenic activity of the MSCs, data are given as percentage with respect to the controls.
Investigations on gene and protein expression
Gene expression analyses were performed with Agilent Whole Human Genome Microarrays, 4 × 44 K, Two Color (Miltenyi Biotec).
The MSCs were seeded at 1 × 105 cells/well in 6-well plates (Falcon). The plates were incubated at 37°C in a 5% CO2 atmosphere, as far as they reached subconfluency (90%), and labeled with standard protocol above, following exposure to magnetic field for 24 h (JPR-MSC - m; JPR-MSC + m(0 h) and MSC - m; MSC + m(0 h)). Cells were washed three times with PBS, harvested with Accutase, frosted in liquid nitrogen and stored at -80°C until RNA isolation
The microarrays were performed following the manufacturer's protocol. RNA was isolated using standard RNA extraction protocols (NucleoSpin® RNA II, Macherey-Nagel, Düren, Germany). The RNA samples were quality-checked via the Agilent 2100 Bioanalyzer platform (Agilent Technologies, Waldbronn, Germany). The quality of the isolated RNA was checked in a gel image and an electropherogram using the Agilent 2100 Bioanalyzer expert software. In addition to the visual control, the software allows the generation of a RNA Integrity Number (RIN) to check integrity and overall quality of total RNA samples. All samples showed sufficient quality for gene expression profiling experiments.
For the linear T7-based amplification step, 1 μg of each total RNA sample was used. To produce Cy3- and Cy5-labeled cRNA, the RNA samples were amplified and labeled using the Agilent Low RNA Input Linear Amp Kit (Agilent Technologies) following the manufacturer's protocol. Yields of cRNA and the dye-incorporation rate were measured with the ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, USA).
The hybridization procedure was performed according to the Agilent 60-mer oligo microarray processing protocol using the Agilent Gene Expression Hybridization Kit (Agilent Technologies). Briefly, 825 ng of the corresponding Cy3- and Cy5-labeled fragmented cRNA were combined and hybridized overnight (17 hours, 65°C) to Agilent Whole Human Genome Oligo Microarrays 4 × 44 K using Agilent's recommended hybridization chamber and oven. Finally, the microarrays were washed once with 6× SSPE buffer containing 0.005% N-lauroylsarcosine for 1 min at room temperature followed by a second wash with pre-heated 0.06× SSPE buffer (37°C) containing 0.005% N-lauroylsarcosine for 1 min. The last washing step was performed with acetonitrile for 30 sec.
Fluorescence signals of the hybridized Agilent Oligo Microarrays were detected using Agilent's DNA microarray scanner (Agilent Technologies).
The Agilent Feature Extraction Software (FES) was used to read out and process the microarray image files. The software determines feature intensities and ratios (including background subtraction and normalization), rejects outliers and calculates statistical confidences (p-values). For determination of differential gene expression FES derived output data files were further analyzed using the Rosetta Resolverâ gene expression data analysis system (Rosetta Biosoftware, Seattle, USA). This software offers the possibility to visualize the results of the data analysis in a double-log scatter plot.
FACS analysis of CD93 and Cadherin7 (CDH7) epitope pattern was performed for the conditions JPR-MSC - m; JPR-MSC + m(0 h) and MSC - m; MSC + m(0 h). 10 × 105 cells were incubated for 30 min with unconjugated anti-CD93 antibody (R × D Systems) or anti-CDH7 antibody (Sigma), followed by a respective Alexa488 secondary antibody staining. A FACScan (BD Biosciences) and BD CellQuest Pro software were used.
To evaluate the differences in differentiation potential of the MSCs, adipogenic, osteogenic and chondrogenic differentiation of unlabeled and labeled MSCs with and without exposition to magnetic fields was performed.
Differentiation assays under magnetic exposition were carried out in 75 cm2 culture flasks for adipogenic and osteogenic differentiation, and in 15 ml Falcon tubes for chondrogenic differentiation, all directly above the magnets.
Labeled and control cells were seeded at 3 × 105 cells in standard medium for two days. The incubation was carried out in the incubator under standard conditions. The labeled and unlabeled MSCs were exposed to magnetic field immediately after seeding, or there was no exposure (control group). After two days the medium was replaced with differentiation media, as described above. Medium was changed every 3 days. After 21 days the cells were detached with Accutase, counted with a CASY®2 Analyser, and stored in RLT-buffer (Qiagen, Hilden, Germany) at -80°C.
To quantify the tri-lineage differentiation potential of the MSCs, quantitative Reverse Transcription-Polymerase Chain Reaction Analysis (RT-PCR) was performed as described previously [17]: Briefly, total ribonucleic acid (RNA) was extracted from adipogenic, osteogenic and chondrogenic differentiated MSCs using RNeasy mini spin columns (Qiagen). Reverse transcription was performed by Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics), using anchored-oligo(dT)18 primer. The expression of lineage-specific genes was determined by ready-to-use amplification primer mixes for RT-PCR (search-LC, Heidelberg, Germany) and the LightCycler™ Instrument (Roche Diagnostics). In addition, the expression of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was determined in the same way in all samples. For relative quantification of the gene expression, the expression of each target gene was normalized to the expression of GAPDH in the same sample.
Statistics/Data analysis
Following assessment of normal distribution, statistical significance was tested by Student's t test. The data are presented in mean ± standard error of mean (SEM), p < 0.05 was considered significant (*).
