Human Mesenchymal Stem Cells Protect Dorsal Root Ganglia from the Neurotoxic Effect of Cisplatin

Materials and Methods

All procedures on animals were performed under anesthesia and in accordance with the European Communities Council Directive 86/609/EEC.

Drug. Cisplatin was purchased from Sigma Chemical Co. (St. Louis, MO, USA). It was dissolved in sterile water to make a stock solution of 1 mg/ml, which was diluted with medium to obtain the different working concentrations.

DRG explants. DRG from 15-day-old embryonic Sprague-Dawley rats were aseptically removed and cultured onto a single layer of rat-tail collagen surface in 35-mm dishes (4 ganglia/dish). DRG were incubated in AN₂ medium (Minimum Essential Medium plus 10% calf bovine serum, 50 μg/ml ascorbic acid, 1.4 mM L-glutamine, 0.6% glucose) plus 5 ng/ml Nerve Growth Factor (NGF) for 2 hours in a 5% CO₂ humidified incubator at 37°C. After 2 h, the DRG were treated with different concentrations of cisplatin (3.5 and 5 and 7.5 μg/ml) for 24 h and the medium was then replaced with fresh medium. The AN₂ medium was changed every 3 days. At different time points, DRG were photographed (48 h, and 1, 2, 3 and 4 weeks). For each DRG, the longest neurite and the area of death were measured using ImageJ (NIH, Bethesda, MD, USA). These measurements were compared with a calibration grating photographed under identical conditions. The percentage area of death was determined as: (dead area/total DRG area) ×100.

hMSC Culture. After obtaining informed consent, hMSCs were isolated from bone marrow harvested from the iliac crest of six healthy donors as described previously (13) and cultured in Dulbecco's modified Eagle's medium-low glucose (DMEM-LG; Lonza, Verviers, Belgium) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 250 mg/ml fungizone (Lonza), and 10% defined fetal bovine serum (FBS; Hyclone, Logan, UT, USA). Cells were plated in culture flasks at a high density and were maintained at 37°C in a humidified atmosphere containing 5% CO₂. The cells were used from the 4th to the 8th passages.

Direct co-culture. hMSCs were added to DRG explants at a density of 80,000 cells/dish. Co-cultures were treated with AN₂ medium supplemented with 5 ng/ml NGF and then photographed at different time points to analyze the progressive neurite elongation (48 hours and 1, 2, 3 and 4 weeks). The AN₂ medium was changed every 3 days. Each experiment was performed three times to validate the results.

hMSC-conditioned medium (CM). A total of 80,000 hMSCs were seeded onto collagen-coated dishes and cultured in AN₂ medium 10% Calf Bovine Serum plus NGF. After 24 h, the medium was collected and used as CM at a 1:1 ratio with fresh AN₂ medium plus NGF. The medium was changed every 3 days. Each experiment was performed three times.

Sham CM. In a parallel experiment, to verify whether the effect of hMSCs CM on neurite elongation was specific, the culture medium from untreated DRG was used at a 1:1 ratio with fresh AN₂ medium plus NGF as sham CM.

Immunofluorescence experiments. hMSCs were added to DRG explants as described above and a fluorescence study was performed using anti-Neurite Outgrowth Inhibitor (NOGO)-A/B/C (1:50; Millipore, Billerica, MA, USA), and anti-Myelin Associated Glycoprotein (MAG) (1:2000; Abcam, Cambridge, UK). Phalloidin (1:20; Invitrogen, Carlsbad, CA, USA) was used to visualize actin filaments of hMSCs. For the fluorescence studies, the cultures were washed with Phosphate-buffered saline (PBS) and then fixed in 4% paraformaldehyde. Non-specific binding was blocked with 3% Bovine Serum Albumine in PBS for 1 h, then cells were incubated overnight at 4°C with the primary antibodies. After washing with PBS, the cultures were incubated with secondary antibodies for 1 hour at room temperature. Then cells were washed and coverslips were mounted using mounting medium with DABCO (Sigma-Aldrich, St. Louis, MO, USA). To study the expression of NOGO and MAG, cells were examined using confocal laser microscopy, carried out with a Radiance 2100 confocal microscope (Bio-Rad, Hercules, CA, USA) equipped with a krypton/argon laser. Noise reduction was achieved by Kalman filtering during acquisition.

Western blot analysis. Cells were washed twice with PBS, and solubilized in lysis buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 5 mM EGTA] containing protease and phosphatase inhibitors (4 mM phenylmethylsulfonyl fluoride, 1% aprotinin, 10 mM sodium orthovanadate, 20 mM sodium pyrophosphate) (Sigma Chemical Co.). Total proteins were measured with a Coomassie® Protein Assay Reagent Kit (Pierce, Rockford, IL, USA). Protein aliquots (15 μg) were solubilized in Laemmli buffer 5X, boiled for 5 min and run on 10% SDS-polyacrylamide gels.

Immunoblotting analysis was performed according to the manufacturer's instructions [anti-NOGO-A/B/C, 1:1000 (Millipore); anti-MAG, 1:500 (Abcam); anti-α-actin, 1:500 (Santa Cruz Biotechnology, Santa Cruz, CA, USA)]. The immunoreactive proteins were visualized using ECL Prime (GE Heathcare Piscataway, NJ, USA). All data were normalized by actin immunoblotting.

siRNA. NOGO and MAG siRNA were prepared following the manufacturer's protocol (Santa Cruz Biotechnology). hMSCs were cultured with NOGO and MAG siRNA for 2 days and every day the medium was collected and used as siRNA hMSC CM. Down-regulation by NOGO and MAG siRNA was assessed by western blot analysis.

A statistical analysis was carried-out with the one-way ANOVA and Tukey post hoc test with the statistical package GraphPad Prism (GraphPad Software, San Diego, CA, USA). A p-value of less than 0.05 was considered statistically significant.