Differentiation of Human Umbilical Cord-derived Mesenchymal Stem Cells, WJ-MSCs, into Chondrogenic Cells in the Presence of Pulsed Electromagnetic Fields

Materials and Methods

Isolation of MSCs from Wharton's jelly. Sections of umbilical cord were obtained, with authorization from the Department of Obstetrics and Gynecology Hospital of the Second University of Naples, following caesarean section, at the end of the gestation period.

MSCs were extracted from the Wharton's jelly according to the following protocol: sections of umbilical cord were washed with phosphate buffer saline (PBS) (Lonza, Verviers, Belgium) supplemented by antibiotics (penicillin-streptomycin; Gibco, Grand Island, NY, USA). After washing with PBS, the sections of umbilical cord were dissected in order to separate the gelatin from the rest of the cord. The gelatin, in small fragments, was immersed in an solution of collagenase 1 mg/ml (300 l of collagenase concentrated to 0.1 g/ml in 30 ml of Hanks' balanced salt solution) (HBSS; Gibco).

After incubation for 20 h at 37°C, 25 ml of sterile PBS were added to reduce the viscosity of the liquid; after sedimentation the supernatant was transferred to a new tube to be centrifuged at 1500 rcf ×g for 10 min, then the supernatant was discarded.

The pellet was then resuspended in 10 ml of sterile PBS and a second centrifugation was carried out at 1000 rcf ×g for 6 min. Finally, the supernatant was removed leaving only the pellet with 1.5 ml of liquid to which 5 ml of culture medium were subsequently added. The cells were then seeded in a 25 cm² flask and placed in moist incubator when 5% CO₂, at 37°C.

The culture medium used for the isolated cells was Dulbecco's modified Eagle's medium (DMEM, Lonza Verviers, Belgium) to which 10% Fetal bovine serum (FBS; Hyclone, Logan, UT, USA) was added, with 50 mg/ml penicillin-streptomycin and 2 mM L-glutamine.

Characterization of WJ-MSCs by flow cytometric assay. To confirm that we isolated MSCs, flow-cytometric analysis was performed on the cells extracted. The data were acquired with a FACScan flow cytometer (Becton Dickinson, Milan, Italy) equipped with an argon laser with a wavelength of 488 nm. For the characterization, approximately 1×10⁶ MSCs were detached, washed and incubated with the monoclonal antibodies against surface molecules: in particular against: CD44, CD105, CD45 (Sigma-Aldrich, Milan, Italy) and CD73 (Lifespan Bioscience, Seattle, Washington, USA). Fluorescein isothiocyanate (FITC) was used as fluorochrome, linked to secondary antibodies goat anti-mouse IgG. For each sample, a minimum of 10,000 events were collected .

PEMF treatment. PEMF treatment was performed as previously described (21). Briefly, the culture plates with WJ-MSCs were placed inside an incubator at equal distance from two solenoids arranged in parallel with one another and perpendicular to the support base, thus respecting the principle of focus signal in order to maintain constant physical characteristics of the stimulation signal. The external device produced an electromagnetic pulse signal of the square wave type with a frequency of 75Hz ±5% and an intensity of 18-30 Gauss.

The cells were cultured in six-well culture plates (BD Falcon TM, BD Biosciences, Mississauga, ON, Canada) and exposed to a PEMF continuously for 8 h each day during the 21 days of culture period. Control experiments were performed on the MSCs isolated from the same umbilical cord Wharton's jelly in a separate CO₂ incubator that had no PEMF device. The incubators were alternated for the treatment and control experiments.

The treatment was performed in double using BIOSTIM SPT (Igea, Carpi, Italy) and Osteoplus (Fisiokinetec, Naples, Italy). Results obtained from the two systems were not different.

Chondrogenic differentiation. MSCs isolated from the umbilical cord Wharton's jelly were induced in the following chondrogenic medium: DMEM, supplemented with 10% FBS, 2 mM L-glutamine, 50 μg/ml penicillin-streptomycin, 50 μg/ml ascorbic acid and 1 ng/ml human recombinant transforming growth factor β1 (TGF-β1).

Immunostaining. Immunostaining was performed as previously described (16, 17). Briefly, MSCs were fixed with a solution of 4% paraformaldehyde in PBS for 30 min, washed in PBS and then incubated with PBS containing 1% bovine serum albumin for 15 min. before the application of the primary antibody. Incubation with the primary antibody to collagen II (rabbit polyclonal) from Abcam (Cambridge, UK), was performed for 75 min at room temperature and the secondary antibody was applied for 1 h at room temperature. After incubation with both the primary and the secondary antibody, cells were washed three times in PBS. After the final washing step, cells were mounted with Glycergel Mounting Medium (C0563) (Dako, Milan, Italy) and then stored in the dark at 4°C. Microscopic images were taken using a confocal microscope (SP1 UV CLSM; Leica, Wetzlar, Germany) and processed using Adobe Photoshop Software 7.0.

Glycosaminoglycan staining (GAG). To analyze the secretion of cartilage proteoglycans, Alcian blue staining was performed as previously described (1, 18). Briefly, the medium where MSCs were grown during the differentiating process, was removed and cells were then washed with PBS and fixed for 10 min in 4% paraformaldehyde in PBS. Cells were stained in 1% Alcian blue 8 GX reagent (Sigma, St. Louis, MO, USA) in 3% glacial acetic acid (pH 2.5) for 30 min at room temperature. Cells were washed before microscopic observation .

Reverse transcriptase-polymerase chain reaction (RT-PCR). RT-PCR was performed as previously described (16, 19, 20, 21). Briefly, total cellular RNA obtained from WJ-MSCs treated with or without PEMF using the RNeasy kit (Qiagen, Valencia, CA, USA) was converted to cDNA. The resulting cDNA was subjected to PCR amplification using rapid cycling in glass capillaries with a thermocycler (Light-cycler; Roche Molecular, Milan Italy). The reaction was performed in a total volume of 50 μl containing 2.5U Taq DNA polymerase, 50 pmole of each primer and 200 μM dNTPs in 1 × PCR buffer (10 mM Tris pH 8.3, 50 mM KCl and 1.5 mM MgCl₂). Specific oligonucleotide primers for type II collagen were used (sense: 5’-ATTTCAAGG CAATCCTGGTG-3’, antisense: 5’-GGCCTGGATAACCTCTG TGA-3’). The following conditions were used for PCR amplification: initial denaturation at 94°C for 1 min, followed by 25-35 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 50 s and extension at 72°C for 30 s, followed by a final extension at 72°C for 5 min. Each PCR was carried out three times. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control.

Statistical analysis. Each experiment was performed in triplicate. The results are expressed as means±standard deviations (SD). Student's t-test was used to determine statistical differences between the means, and p<0.05 was considered a significant difference.