Human adipose stem cells (hASCs) - isolation and identification
Adipose tissue was collected from healthy donors by liposuction. Donors were informed and agreed to participate in this study. The procedure was approved by the Local Bioethics Committee. In order to remove red blood cells, 400 ml fat tissue was mixed 2:1 vol/vol with buffered physiological salt solution (Phosphate-buffered saline- PBS) and shaken every 15 min. Following phases separation, PBS with red blood cells were discarded. Purification process was repeated three times. Afterwards, 0.075% collagenase solution from Clostridium histolyticum (Sigma – Aldrich) in PBS was added to adipose tissue (1:2 vol/vol), shaken and incubated in temp. 37 °C for 1.5 h in order to digest the tissue. The fat and collagenase mixture were shaken every 15 min. After obtaining a homogenous suspension, human albumin (20% concentration) was added (final concentration 2%) to stop the digestion reaction. Mixture was centrifuged (400 g) for 10 min at room temperature (RT). The liquid fat and salt interphases were discarded and the cell pellet was suspended in PBS. Cell suspension was filtered through 100 μm nylon filter, washed in PBS and centrifuged (300 g) for 10 min, RT. The amount and viability of the cells were determined and cells were seeded into plastic flasks at a density of 8 × 104 cells/cm2 for further cultured in growth medium (GM) composed of DMEM-LG (Dulbecco’s modified Eagle’s Medium with low glucose; Sigma-Aldrich) supplemented with fetal calf serum (FCS; 15%; Invitrogen) and antibiotic–antimycotic solution (Penicillin-streptomycin-amfoterycin; 1.5%; Invitrogen) and incubated under standard cell culture conditions (37 °C, 5% CO2, 95% humidity). When primary cultures reached subconfluency, cells were detached by exposure to trypsin (0.25% trypsin with 1 mM EDTA; Invitrogen) and replated at a density of 5.0 × 103 cells/cm2 for subsequent passage. After passage 3 cells were identified by flow cytometry and multilineage differentiation capacity and were frozen in liquid nitrogen. For experiments refrozen hASCs from 6 independent donors at passage 4–7 were used.
In vitro osteogenic differentiation
Osteogenic differentiation was performed at the third passage. Cells were cultured in hMSC Osteogenic Differentiation BulletKit™ Medium (Lonza) for 3 weeks. The medium was changed every 3 days. Osteogenic differentiation was characterized by identification of mineral depositions in extracellular matrix. At 3 weeks, the plated cells were fixed for 15 min with 4% formaldehyde and stained with Alizarin Red (Sigma-Aldrich). After staining, the wells were rinsed with distilled water and visualized by standard light microscopy.
In vitro adipogenic differentiation
Adipogenic differentiation was performed at the third passage. Cells were cultured in hMSC Adipogenic Differentiation BulletKit™ Medium (Lonza) for 3 weeks. Adipogenic differentiation was assessed using Oil Red O (Sigma-Aldrich) stain as an indicator of intracellular lipid accumulation. Prior to staining, plastic-adherent cells were fixed for 45 min with 10% formaldehyde and then for 5 min with 60% isopropanol. After fixation and staining, the wells were rinsed with distilled water and visualized by standard light microscopy.
In vitro chondrogenic differentiation
To induce chondrogenic differentiation, three-dimensional pellet culture was performed. In a 15 ml tube, 3 × 105 cells were pelleted by centrifugation. Unsuspended cell pellets were cultured for 19 days in chondrogenic medium (Lonza) composed of basic medium supplemented with dexamethasone, ascorbate, ITS + supplement, pyruvate, proline, GA-1000, L-glutamine and recombinant human transforming growth factor-β3. For histological analysis, pellets were immersed in paraffin, sectioned and stained with Masson trichrome method.
Flow cytometry analysis
The surface antigen profiles of adipose derived MSCs at the third passage were characterized by flow cytometry. A total of 2,5 × 106 cells were incubated with the following phycoerythrin (PE)-conjugated anti-mouse antibodies: CD29, CD34, CD45, CD73, CD90 and CD105 (Becton Dickinson) for 30 min, RT in the dark. Nonspecific PE-conjugated IgG was substituted as an isotype control. The fluorescence intensity of cells was evaluated using BD FACScalibur flow cytometer equipped with CellQuest Pro software (Becton Dickinson).
Cells were grown in Petri dishes (Ø 3.5, 6 or 10 cm, depending on the experiment). At 80% confluence cells were exposed to growth medium supplemented with human recombinant BMP-12 (Sigma-Aldrich, SRP4572) in the concentrations of 50 ng/ml and/or 100 ng/ml (depending on the test). Cells from the same donors cultured at the same time in standard GM without BMP-12 served as a control. Media were changed every 2 or 3 days. After 7 days cells were harvested by trypsinisation, counted and directed either to RNA/protein isolation, or to functional tests on microplates (proliferation, migration, oxidative stress susceptibility, mixed lymphocyte reaction). If certain test required further culturing, the medium containing or not BMP-12 was used respectively. Experiments were always conducted on cells from each donor separately. The cells from different donors were not pooled in this study. This approach allowed for detection inter-individual variations. Unless it stated differently, all experiments were performed on cells from 6 different donors n = 6. The scheme of study design is presented in Additional file 1.
For gene expression analysis cells were treated for 7 days with/without 100 ng/ml of BMP-12. At least 3 × 105 cells were used for this procedure. Isolation of total RNA was performed using RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA concentration and purity was assessed by spectrophotometer at 260 nm using NanoDrop (ND-1000 Spectrophotometer, NanoDrop Technologies, Inc).
Real-time PCR analysis
Real-Time PCR was performed on ABI Prism 7500 Sequence Detection System using TaqMan® RNA-to-CT™ 1-Step Kit (Applied Biosystems, Foster City, USA). Specific primer and probe set was purchased from Applied Biosystems: Collagen, type I, alpha 1 (Col1α1) Hs00164004_m1, Scleraxis (SCX) Hs03054634_g1, Mohawk homeobox (MKX) Hs00543190_m1, Tenascin (TNC) Hs01115665_m1, Decorin (DCN) Hs00370385_m1, Runt-related transcription factor 2 (RunX) Hs01047973_m1,. GAPDH (4333764 T) gene was used for normalization. Duplicates of each sample were performed. The relative expression of mRNA expression was calculated by 2−ΔΔCt method. The result was presented as a fold change of gene expression in relation to the calibrator. Statistical analysis was performed by comparison of dCt values using non-parametric test for related data (control versus treated cells from the same population).
To assess the effect of BMP-12 treatment on expression of collagen type I and type III ICC staining was performed. For this analysis cells were seeded on Nunc™ Lab-Tek™ II CC2™ 8-Chamber Slide System. First, cells were cultured for 7 day with or without 50 or 100 ng/ml BMP-12. For ICC quantification, the incubation time of was shortened to 5 days in order to avoid full confluence which would hinder subsequent analysis). At the end of experiment, hASCs were fixed with 4% paraformaldehyde (10 min, RT), permeabilized with 70% methanol (15 min, -20 °C), treated with blocking solution composed of 5% normal donkey serum, 1% of bovine serum albumin in PBS and probed overnight in 4 °C with Rabbit polyclonal Anti-Collagen I antibody (Abcam, ab34710, 1:300) or Rabbit polyclonal Anti-Collagen III antibody (Abcam, ab7778, 1:150) followed by secondary Alexa Fluor 594- conjugated Donkey Anti- Rabbit antibody (1:150, Jackson ImmunoResearch, 1 h, rt). The nuclei were visualized with DAPI staining (20 ng/mL of DAPI solution for 4 min, RT). The result was evaluated with fluorescence microscopy (Olympus IX51 and CellSens™ Microscope Imaging Software). The expression of collagens (type I and III) was measured as the area of specific fluorescence per cell [μm2]. At least 900 cells per well were analyzed from 10 randomly selected fields of view. Immunocytochemistry was performed on hASCs from two different donors.
Western blot (WB)
Human ASCs were cultivated with or without BMP-12 (100 ng/ml) for 7 days on Ø 100 mm. culture dishes. Collected cell pellets were lysed with RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% Na-deoxycholate, and 1 mM PMSF) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma-Aldrich) for 30 min at 4∘C in order to isolate protein extracts. Lysates were cleared for 20 min at 14000 rpm, and supernatants were collected. The total protein concentration was determined using Bio-Rad protein assay dye reagent according to the producer’s instructions (Bio-Rad Laboratories Inc., Hercules, CA, USA). Proteins (35 μg of total protein per well) were resolved by SDS-PAGE and transferred onto PVDF membrane (Sigma-Aldrich). For immunostaining membranes were blocked with 5% nonfat dry milk in TBS (20 mM Tris-HCl, 500 mM NaCl) containing 0.5% Tween20. The membranes were incubated with Rabbit polyclonal Anti-Collagen I antibody (Abcam, ab34710, 1:500) or Rabbit polyclonal Anti-Collagen III antibody (Abcam, ab7778, 1:1000) or Rabbit polyclonal Anti-Mohawk antibody (LSBio, aa46-75, 1:1000) or Goat polyclonal Anti-Actin (Santa Cruz Biotechnology, C-11, sc1615, 1:1000) primary antibodies. Next the blots were washed three times for 15 min and incubated with appropriate secondary antibodies conjugated with IR fluorophores: IRDye 680 or IRDye 800 CW (purchased from LICOR Biosciences; Lincoln, NE, USA) at 1:5000 dilution. Odyssey Infrared Imaging System (LI-COR Biosciences) was used to analyze the protein expression. Scan resolution of the instrument was set at 169 μm and the intensity at 5. Quantification of the integrated optical density (IOD) was performed with the analysis software provided with the Odyssey scanner (LI-COR Biosciences). Immunoblot analysis for cells from each donor was performed on samples from three independent electrophoreses. For the purpose of publication the color immunoblot images were converted into black and white images in the Odyssey software.
Mixed lymphocyte reaction (MLR)
Ten milliliters of venous blood was collected in heparinized tubes from healthy blood donors after obtaining informed consent. Separation of peripheral blood mononuclear cells (PBMCs) was performed within 2 h of withdrawal of blood. Blood samples were taken into preservative-free heparin (20 units/ml) tubes, and PBMCs were isolated by centrifugation on Histopaque-1077 (Sigma-Aldrich) of the blood diluted 1:1 with Sodium Chloride 0.9% (0.9% NaCl, Fresenius Kabi). PBMCs were taken up in Parker medium (Biomed) supplemented with 2 mM L-glutamine (Sigma-Aldrich), 0.1 mg/ml gentamycin (KRKA), β-mercaptoethanol (Sigma), 0.23% Hepes (Sigma) and 10% fetal bovine serum (FBS, Gibco). Half of the isolated PBMCs were inactivated by gamma-irradiation for 90 min.
hASCs after 7 days culture with BMP-12 were collected and seeded onto 96-well flat-bottom plate (Greiner) in a concentration of 0.8 × 104/well. Each time, cells from the same donor cultured in parallel without BMP-12 were seeded on the same plate in the identical scheme. Cells were left overnight to attach. For the MLR, 2 × 105 PBMCs (1 × 105 cells/well from a first donor and 1 × 105 cells/well from the second donor) were co-seeded with hASCs in the following combinations: XXir, YYir, XYir, YXir (X – first donor’s PBMCs, Xir – irradiated first donor’s PBMCs, Y – second donor’s PBMCs, Yir – irradiated second donor’s PBMCs). PBMC cultures without hASCs were used as controls. Cells were cultured for 5 days at 37 °C in a humidified atmosphere with 5% CO2. After 5 days cells were pulsed with 1 μCi/well of 3H-thymidine (113 Ci/nmol, NEN) for the last 18 h of the incubation and harvested with an automated cell harvester (Skatron). The amount of 3H-thymidine incorporated into the cells was measured using a Wallac Microbeta scintillation counter (Wallac), giving the level of radioactivity as ‘Corrected Counts per Minute’ (CCPM). For this experiments hASCs from 5 different donors were used (n = 5), but the influence of hASCs from each donor was tested on PBMCs from two different blood donors (X and Y). Therefore, 10 separate experiments were performed, each in triplicate.
Cell proliferation assay
Cell proliferation assay was conducted using a colorimetric BrdU proliferation ELISA immunoassay (Roche). hASCs after 7 days culture with or without BMP-12 (50 or 100 ng/ml) were collected and seeded onto 96-well flat-bottom plate in a concentration of 0.8 × 104/well in experimental or control medium respectively. The cells were allowed to grow at 37 °C and 5% CO2 for the next 36 h. Afterwards, BrdU labeling reagent was added to each well. Then, the cells were further incubated for 12 h, and the pyrimidine analogue BrdU was incorporated in place of thymidine into the DNA in proliferating cells. Next, the immunostaining was performed according to manufacturer instructions. The absorbance was measured at wavelength 450 nm (BioTek PowerWave XS). Experiments were performed in triplicates for each sample.
Cell migration assay
The migration assay was performed using trans-well inserts with 8 μm pore membrane (BD Biosciences, San Jose, CA, USA). The wells of the 24-well glass- bottom plates (SensoPlate, Grainer) was filled with 1 ml of different culture medium: standard GM, GM containing 50 ng/mL BMP-12, or GM containing 100 ng/mL BMP-12. Human ASCs after 7 days culture with or without BMP-12 were seeded to the upper compartment of the cell culture inserts at a density 1.5 × 104 cells per insert and then inserts were placed into the proper wells. To allow cell migration from the inserts to the wells, plates were incubated at 37 °C and 5% CO2 for the next 72 h. Afterwards, medium was removed and the inserts were peeled off the cells that migrated to the bottom side of the membrane using 0.25% tripsin- EDTA solution (Sigma). After 24 h, when the cells adhere to the bottom of the wells, hASCs were fixed with 70% methanol and the nuclei were visualized with DAPI staining (20 ng/mL of DAPI solution for 4 min). Results were assessed with a cell imaging multi-mode microplate reader Cytation™ 3 (BioTek) which allowed to specify the number of objects giving a signal of blue fluorescence (DAPI, wavelength: 377–477 nm). Experiments were performed in duplicates for each sample.
hASCs viability under oxidative stress in vitro
hASCs after 7 days culture with or without BMP-12 (100 ng/ml) were collected and seeded onto 96-well flat-bottom plate in a concentration of 0.8 × 104/well in respective experimental or control medium. Cells were left to attach and after 24 h were exposed to oxidative stress by adding a medium with hydrogen peroxide in increasing concentrations (0, 750, 1000, 1500 μM). After 22 h of incubation, 20 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma-Aldrich) at the concentration of 5 mg/ml in PBS were added and incubated for another 2 h. The medium was discarded and 100 μl of DMSO was added. The plate was shaken for 10 min using microplate shaker. Absorbance, corresponding to the mitochondrial dehydrogenases activity of viable cells was measured colorimetrically at wavelength 570 nm (BioTek PowerWave XS).
Like in other experiments, hASC were cultured with or without BMP-12 (100 ng/ml) for 7 days. For the last 48 h the serum was withdrawn from the medium and cells were incubated with DMEM-LG supplemented with 4% bovine serum albumin and antibiotics (1%) with or without BMP-12 respectively. At the end of incubation period, the supernatants were collected and frozen in -80 °C. Luminex multiplex assays (Procarta) were used for measuring concentration of the following molecules: IL-6, TNF-α, IL-10, EGF, VEGF, MMP-1, MMP-8 and MMP-13. TGF-β1 concentration was evaluated with ELISA kit (R&D) according to the manufacturer’s instruction. All experiments were done in duplicates.
For data analysis STATISTICA software (StatSoft®Polska) was used. Data are presented as medians, quartiles and min-max or means ± SEM. Differences between groups were analyzed by non-parametric Wilcoxon test for related data or U Mann-Whitney if non-related data were compared. Student T-test was used if in compared groups consisted of at least 10 values with normal distribution confirmed by Shapiro-Wilk test. The p value less than 0.05 was considered as statistically significant.