Isolation, characterization, and culture of human tendon stem cells
Human tendon stem cells (hTSCs) were isolated from supraspinatus tendon specimens collected during arthroscopic rotator cuff repair, according to a previous procedure (Fig. 1a) [11]. The study protocol has been approved by the Hospital Ethical Committee (authorization number 2642; Sept 19, 2011; ASL Milano 2, Melegnano, Milan) and patients received and signed an informed consent. Briefly, samples from supraspinatus tendons (4–8 mm wide) were collected from six patients, kept in HypoThermosol (BioLife Solutions) at 4 °C, and processed separately within 24 h, according to the procedure described below. Samples were washed with phosphate-buffered saline (PBS) (Euroclone), cut into small pieces, and digested for 90 min with collagenase type I (3 mg/mL; Worthington) and dispase (4 mg/mL; Gibco, Life Technologies) in PBS at 37 °C. After centrifugation, cell pellets were resuspended in the following culture medium: α-Minimal Essential Medium (α-MEM) (Sigma-Aldrich) supplemented with 2 mM glutamine (Euroclone), 1 % antibiotic-antimycotic mixture (Euroclone) and 20 % (v/v) fetal bovine serum (FBS) (HyClone, Thermo-Fisher Scientific). Cells were then filtered with a cell strainer (70 mM; BD Falcon) and plated in 150-cm2 dishes. Adherent cells were cultured at 37 °C with a humidified atmosphere of 5 % CO2. The medium was changed every 2–3 days. The isolated hTSCs at passage three were characterized by flow cytometry for the expression of key stem cell markers and to test the level of contamination with other cell types (Additional file 1: Figure S1). Flow cytometry analysis was performed on 1 × 105 cells/sample. Briefly, aspecific binding sites were blocked with a blocking solution (50 % 1x PBS, 50 % FBS) for 30 min at room temperature, and washed twice with PBS. Cells were stained with fluorochrome-conjugated mouse anti-human antibodies at the optimal concentration (1:20 dilution) in PBS for 10 min at 4 °C, and washed twice with PBS at 4 °C. Cell characterization was performed using the following antibodies: αCD9 FITC, αCD73 FITC, αHLA-DR FITC, αCD13 PE, αCD29 PE, αCD44 PE, αCD45 PE, αCD90 PE, αCD105 PE, αCD106 PE, αCD34 PerCP-eFluor710, αCD166 PerCP-eFluor710, and αSSEA-4 PE (all from eBioscience); αLineage Cocktail FITC, αCD18 PE, αCD146 PE, and αStro-1 Alexa Fluor 647 (all from BioLegend); and αCD117 PE (Miltenyi Biotech). The respective isotype antibodies were used as controls. Samples were acquired with a Navios flow cytometer (Beckman Coulter), and data were processed with Kaluza 1.2 software (Beckman Coulter). Cells at passage three were used for all the experiments.
Cell differentiation
hTSCs were induced to differentiate toward adipocytes, osteoblasts, and chondroblasts in vitro to test for their multi-lineage potential, according to the following procedures (Fig. 1b).
Adipogenic differentiation
hTSCs were plated at a concentration of 3 × 104 cells/cm2 in normal growth medium, and then switched to DMEM-low glucose (Sigma-Aldrich), 10 % FBS (HyClone, Thermo-Fisher Scientific), 4 mM L-glutamine (Euroclone), 1 % antibiotic-antimycotic mixture (Euroclone), with the addition of the mesenchymal stem cell adipogenesis kit (Millipore) for 21 days, according to the manufacturer’s instructions. At day 21, Oil Red O solution (Millipore) was used to stain lipid droplets of derived adipocytes, according to the manufacturer’s procedures. All photomicrographs were acquired with an Axiovert 40 microscope (Zeiss) equipped with a Moticam 2300 camera (Motic). The adipogenic medium was changed every 2–3 days.
Osteogenic differentiation
hTSCs were plated at a concentration of 3 × 104 cells/cm2 in normal growth medium, and then switched to the osteogenesis induction medium, which was constituted of DMEM-low glucose (Sigma-Aldrich), 10 % FBS (HyClone, Thermo-Fisher Scientific), 4 mM L-glutamine (Euroclone), 1 % antibiotic-antimycotic mixture (Euroclone), supplemented with 0.1 μM dexamethasone, 50 μg/ml L-ascorbic acid-2-phosphate, and 10 mM β-glycerophosphate (all reagents from Sigma-Aldrich) for 17 days. At day 17, Alizarin Red solution (Millipore) was used to detect calcium deposition in derived osteoblasts, according to the manufacturer’s instruction. All photomicrographs were acquired with an Axiovert 40 microscope (Zeiss) equipped with a Moticam 2300 camera (Motic). The osteogenic medium was changed every 2–3 days.
Chondrogenic differentiation
hTSCs were maintained in a 3D culture by growing them in cell pellets (1 × 106 cells/pellet) in AdvanceSTEM chondrogenic differentiation medium (HyClone, Thermo Scientific), according to the manufacturer’s instructions. After 28 days of differentiation, matrix deposition by derived chondroblasts was detected with Alcian Blue staining (Sigma-Aldrich), according to the manufacturer’s instruction. All photomicrographs were acquired with an Axiovert 40 microscope (Zeiss) equipped with a Moticam 2300 camera (Motic). The chondrogenic medium was changed every 2–3 days.
PST® device and cell treatments
The PST® treatment was performed by placing the cell culture plate at the center of PST® device in order to have the magnetic field vector perpendicular to the plate surface (Fig. 2). hTSCs were plated at a concentration of 2.6 × 103 cells/cm2 in normal growth medium. Twenty-four hours after seeding, cells were either treated with PST® for 1 h (PST), or were kept outside the incubator for the same amount of time (control) (Fig. 2c). PST and control cells were then returned to the incubator and grown for other 10, 24, or 48 h.
Cell morphology and proliferation experiments
To assess whether PST® stimulation could affect hTSCs phenotype, cell morphology was examined with a phase-contrast microscope (Axiovert 40 CFL, Zeiss, equipped with a Moticam 2300 camera, Motic) after 0, 10, 24, and 48 h of PST® exposure. For cell viability analyses, hTSCs were subjected to PST® stimulation, as described before. PST and control cells were analyzed at each time point after harvesting with Trypsin-EDTA solution (Sigma-Aldrich) by counting with a Countess Cell Counter (Invitrogen, Life Technologies), according to the manufacturer’s procedure. Cell viability was determined by trypan blue dye exclusion assay. The number of viable cells in each sample was expressed as a percentage of the total untreated cells number at day 0. All assays were carried out in triplicates for each sample.
Cell viability by MTT assay
hTSCs were plated in 12-well plates (1 × 104 cells/well) and were subjected to PST® stimulation, as previously described. At each time point (0, 10, 24, and 48 h), two hours before collection, the reconstituted 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (5 mg/ml in PBS; Sigma) was added to the medium (10 % of the final volume). Following a 2-hour incubation at 37 °C, PST and control cells were lysed by adding an amount of MTT Solubilization Solution equal to the original culture medium volume, gently pipetting to completely dissolve the MTT formazan crystals. The MTT reduction was spectrophotometrically measured at a wavelength of 570 nm.
Cell migration by wound-healing assay
Wound-healing assay was performed as previously described [26]. hTSCs were grown to confluence in 6-well plates and were subjected to PST® stimulation or were kept outside the incubator for the same amount of time (controls). A sterile P200 pipet tip was used to create a scratch across the cell monolayer. Then, cultures were washed once with 1 ml of growth medium to remove the damaged and detached cells. After replacing the medium, hTSCs were allowed to grow for 48 h. At different time points, cell cultures were examined with a phase-contrast microscope (Axiovert 40 CFL, Zeiss, equipped with a Moticam 2300 camera, Motic) and images of the same scratch fields were acquired at time 0 and after 5, 20, 24, and 30 h from the scratch. The gap area between the cells was calculated in each acquired image using software ImageJ. The migration rate was based on the measure of the recovered wound area (experimental data expressed in percentage). All assays were carried out in triplicates for each sample.
Cell apoptosis analysis
Apoptosis was measured by flow cytometry on PST and control cells before a 1-h PST® treatment and then 10, 24 and 48 h post treatment, using Annexin V-FITC Apoptosis detection kit (Enzo Life Sciences), according to the Manufacturer’s protocol. Briefly, adherent cells were trypsinized, washed in PBS by gentle shaking, and resuspended with 200 μl of a specific Binding Buffer (10 mM HEPES/NaOH, pH 7.4; 140 mM NaCl; 2.5 mM CaCl2) containing 5 μl of annexin V-FITC. After incubation for 10 min in the dark at room temperature, cells were washed in PBS, resuspended in 190 μl of Binding Buffer, and then stained with 10 μl Propidium Iodide (20 μg/ml). Samples were acquired with a Navios flow cytometer (Beckman Coulter), and analyzed using Kaluza 1.2 software (Beckman Coulter).
Gene expression analysis
Stem cell, tendon-related marker, and vascular endothelial growth factor expression was tested by Real-Time PCR. Total RNA was extracted from PST and control cells at 0, 10, 24 and 48 h using TRIzol®Reagent (Ambion, Life Technologies) and 1 μg of extracted RNA was reverse transcribed to cDNA using the iScript cDNA synthesis kit (BioRad), according to the Manufacturer’s instructions. Real-Time PCR was performed in a 96-well plate with 10 ng of cDNA as template, 0.2 μM primers, and 1× Power SYBR® Green PCR Master Mix (Applied Biosystems, Life Technologies) in a 20 μl final volume per well, using a StepOnePlus™Real-Time PCR System (Applied Biosystems). The mRNA levels of octamer-binding transcription factor 4 (Oct4), kruppel-like factor 4 (KLF4), Nanog homeobox (Nanog), Tenascin C, collagen type I alpha-1 (COL1A1), and vascular endothelial growth factor (VEGF) were assessed. Somatostatin-14 peptide (S14) was used as the housekeeping gene in quantitative analysis. Primer sequences: S14, forward 5′-GTGTGACTGGTGGGATGAAGG-3′ and reverse 5′-TTGATGTGTAGGGCGGTGATAC-3′; Oct4, forward 5′-AGGAGAAGCTGGAGCAAAA-3′ and reverse 5′-GGTCGAATACCTTCCCAAA-3′; KLF4, forward 5′-GACTTCCCCCAGTGCTTC-3′ and reverse 5′-CGTTGAACTCCTCGGTCTC-3′; Nanog, forward 5′-GGTCCCAGTCAAGAAACAGA-3′ and reverse 5′-GAGGTTCAGGATGTTGGAGA-3′; Tenascin C, forward 5′-CGGGGCTATAGAACACCAGT-3′ and reverse 5′-AACATTTAAGTTTCCAATTTCAGGTT-3′; COL1A1, forward 5′-GGGATTCCCTGGACCTAAAG-3′ and reverse 5′-GGAACACCTCGCTCTCCA-3′; VEGF, forward 5′-CAACATCACCATGCAGATTATGC-3′ and reverse 5′-TCGGCTTGTCACATTTTTCTTGT-3′.
Amplification protocol: an initial denaturation at 95 °C for 3 min, followed by 40 cycles of 5 s each at 95 °C and 30 s at 57 °C. Relative quantification of target genes was performed in triplicates, analyzed using the 2−ΔΔCt method and normalized to the corresponding S14 values.
Statistical analysis
Statistical analysis was performed using GraphPad Prism v 6.0 software (GraphPad Software Inc.). Data were typical results from three replicate experiments for each of the four patients-derived cell lines, and were expressed as the mean ± standard deviation (SD). Paired comparisons were performed by two-tailed t test. When data was not normally distributed, the Wilcoxon matched-paired test was performed. The significance level was set at p value lower than 0.05.