Preparation of adipose-derived stem cells
ADSCs were prepared according to the method developed by Zuk et al. with some modifications. In brief, subcutaneous adipose tissue (3 to 4 g) was obtained from the inguinal regions of male Sprague–Dawley (SD, Vital River, Beijing, China) rats. The adipose tissue was minced and digested with collagenase I (2 mg/ml; Worthington Biochemical Corp, Lakewood, NJ, USA) at 37°C for 30 to 60 minutes. The digested adipose tissue was filtered twice with a 100-μm and then a 25-μm nylon membrane to eliminate the undigested fragments. The cellular suspension was centrifuged at 1,000 g for 10 minutes. The cell pellets were resuspended in cell-culture medium (CCM, DMEM+10%FBS) and cultivated for 24 hours at 37°C in 5% CO2. Unattached cells and debris were removed, and fresh CCM containing 15% fetal bovine serum (FBS, Gibco, arlsruhe, Germany) was added to the adherent cells, which were cultured at 37°C in 5% CO2. Cell-surface markers were measured with flow cytometry (Beckman FC500, CA, USA). Passage 4 cells were used for the following experiments.
Preparation of bone marrow stem cells
BMSCs were isolated from bone marrow as described previously . In brief, the bone marrow was harvested from the femurs and tibiae of the same male SD rats as for the ADSCs. Bone marrow cells were resuspended in phosphate-buffered saline (PBS, Gibco) to a final volume of 10 ml and layered over an equal volume of 1.077 g/ml Percoll solution (Pharmacia, Piscataway, NJ, USA). After centrifugation at 2,000 rpm for 20 minutes, the mononuclear cells were recovered and transferred to a 100-mm culture flask (Corning, Schiphol-Rijk, the Netherlands) and incubated (37°C, 5% humidified CO2) with low-glucose Dulbecco Modified Eagle Medium (DMEM, Gibco) containing 0.2 mmol/ml L-glutamine (Gibco), 100 U/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco), 10 ng/ml epidermal growth factor (EGF, PeproTech, Rocky Hill, NJ, USA), and 10% fetal calf serum (PAA, Pasching, Austria). Nonadherent cells were removed after 24 hours. Cell-surface markers were measured with flow cytometry. Passage 3 or 4 cells were used for the following experiments.
SPIOs were preincubated in CCM and antibiotics for 60 minutes at room temperature. The concentrations of SPIOs in cell-culture medium were 25 μg/ml (Group 1), 50 μg/ml (Group 2), and 100 μg/ml (Group 3), whereas a medium without SPIOs was used for the control group. The two kinds of stem cells were incubated in CCM (37°C, 5% humidified CO2) for 24 hours.
Prussian blue staining
Prussian blue staining was used to detect the presence of iron oxide nanoparticles. Cells of Groups 1 to 3 were fixed in 4% paraformaldehyde for 30 minutes and then detected with Prussian blue staining. In brief, fixed cells were washed 3 times with PBS, incubated for 30 minutes with 2% potassium ferrocyanide in 6% hydrochloric acid, and then rewashed 3 times with PBS. Labeled cells were examined under a light microscope to determine intracellular iron oxide distribution.
Cells labeled as described were washed with culture medium and then washed 3 times with PBS, resuspended in 37% HCl, and incubated at 70°C for 30 minutes. Iron content was determined by using a total iron reagent set. The average iron content per cell was then calculated.
To determine cell viability, cells of each group were initially seeded in 96-well plates at 5,000 cells per well. After incubation for 72 hours, cells of each group were assessed by using a standard 3-(4,5)-dimethylthialzo(−z-yl)-2,5-di-phenyltetrazoliumbromide (MTT) assay (Sigma-Aldrich, St. Louis, MO, USA) for 4 hours. The supernatant fluid was discarded, and 150 μl dimethyl sulfoxide (DMSO, Sigma-Aldrich) was added to every well for 10 minutes with shaking. The light absorption of all cells was measured with an enzyme-linked immunosorbent assay (ELISA) reader (BioTek, VT, USA). Results were expressed as relative ratios versus unlabeled cells.
Adipogenic and osteogenic differentiation were measured to assess the effect of SPIOs on the transdifferentiation potential of cells. Cells in Group 2 and the control group were subjected to two types of induction (adipogenic and osteogenic).
Cells for osteogenic differentiation were seeded in six-well plates at 105 cells per well with CCM. After they reached 60% to 80% confluence, the culture medium was replaced by bone cell-induction culture medium containing 10% FBS, 100 U/ml penicillin/streptomycin, 50 μg/ml L-ascorbate-2-phosphate (Sigma-Aldrich), 0.1 μM dexamethasone, and 10 mM β-glycerophosphate in DMEM. The cells were cultured for 2 more weeks. Alizarin red was used to detect matrix mineralization of osteogenic differentiation . Cells were rinsed in PBS, fixed in 4% formaldehyde, and stained in 1% alizarin red solution (Rowley Biochemical Institute, Danvers, MA, USA) for 3 minutes. Stained cells were observed under a phase-contrast microscope (Olympus, Tokyo, Japan). To quantify the change in osteogenic potential, the activity of alkaline phosphatase (ALP) was detected by using an ALP enzyme activity kit . Expression levels of Bone Gla Protein (BGP)-mRNA osteogenic markers were measured with real-time polymerase chain reaction (RT-PCR) .
Cells for adipogenic differentiation were seeded in six-well plates at 105 cells per well with CCM. After they reached 60% to 80% confluence, the culture medium was replaced by adipose cell-induction culture medium containing 10% FBS, 100 U/ml penicillin/streptomycin (Sigma-Aldrich), 200 mM indomethacin (Sigma-Aldrich), 1 mM dexamethasone (Sigma-Aldrich), 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich), and 10 mg/ml insulin (Sigma-Aldrich) in DMEM. The cells were cultured for 2 more weeks. Cells were rinsed in PBS(PH=7.4), fixed in 4% formaldehyde, and incubated in 2% (wt/vol) Oil Red O (Sigma-Aldrich) for 5 minutes. Stained cells were observed under a phase-contrast microscope (Olympus, Tokyo, Japan). To quantify the change in adipogenic potential, the optical-density (OD) values of lipid droplets stained by Oil Red O were measured. Expression levels of adipocyte Protein 2 (aP2)-mRNA adipogenic markers  were measured with RT-PCR.
Magnetic resonance imaging of SPIO-labeled mesenchyme-derived stem cells in vitro
We then determined the differences between the two kinds of SPIO-labeled cells in MR relaxation time. The two kinds of cells labeled with different concentrations of SPIOs were suspended in 1% agarose before being transferred into 1.5-ml microcentrifuge tubes (Eppendorf, Westbury, NY, USA). Each tube contained 1 × 104 cells. The tubes were imaged with an eight-channel phased-array head coil on a 3.0-Tesla MR scanner (GE Signa Excite; GE Medical Systems, WI, USA). Enhanced T2*-weighted angiography (ESWAN) sequences were used to enhance the T2* effects of the SPIOs. The sequence parameters were: FOV = 20 × 20 mm2; matrix = 240 × 240; TR = 45 msec; NEX = 0.69; slice thickness = 2.0 mm; flip angle = 25 degrees; TE = 5 msec, 9.9 msec, 14.7 msec, 19.6 msec, 24.5 msec, 29.3 msec, 34.2 msec, and 39.0 msec.