The work involving human material has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. Use of human material has been approved by the Medical Ethical Committee II in Mannheim. Informed consent has been obtained prior to cells/tissue collection.
Preparation of platelet derivates: pHPL and tPRP
Buffy coat-derived pooled platelet concentrates (pools of four healthy blood donors, all fulfilling the criteria for blood donor eligibility), were prepared in AB-plasma from one donor . To further minimize donor variability, each batch was prepared by pooling two platelet concentrates. To assess variability between batches, eight different pooled batches have been analyzed.
Each batch was then split into two parts. To yield the whole platelet lysate pHPL, one part was processed by a freezing and thawing step at −30°C and 37°C. The other part was activated with 100U/ml thrombin from human plasma (Merck) for 60 min under gentle agitation to derive the releasate tPRP, containing only the released platelet proteins in plasma. Here, in contrast to the pHPL, a fibrin clot was formed and depleted by centrifugation affecting the relative composition of platelet and plasma factors compared to pHPL. Further processing was standardized for both derivatives: samples were centrifuged at 2000 g, 20 min at RT, supernatant was aliquoted and cryopreserved at −30°C. Prior to use, derivatives were gently thawed and centrifuged at 2000 g for 10 min. To prevent clotting, DMEM medium (Lonza, supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin and 4 mM L-glutamine (all from PAA) was added with 5 IU/ml heparin (Ratiopharm). The respective supplement was added at the indicated concentration and then the medium filtered through a 0.45 μm filter. pHPL- and tPRP was always prepared fresh and used within 24 hours.
Isolation and expansion of MSC
Adipose tissue was collected from three female donors undergoing elective tumescence liposuction of the abdomen and hip/thigh region. BM aspirates were obtained from three healthy donors in the department of Internal Medicine V at the University Hospital of Heidelberg by puncturing the iliac crest. LA-MSC and BM-MSC were isolated by collagenase digestion or density gradient centrifugation (Ficoll-Paque Plus =1.078 g/ml, GE Healthcare) respectively as described previously [10, 11, 13, 47]. Primary cells were seeded in 10% pHPL, tPRP and FBS (PromoCell), respectively. At a confluence of 70-80%, cells were passaged using trypsin-EDTA (PAA) and either replated at a density of 200 cells per cm2 for all passages or used for experiments. MSC were characterized regarding the proliferation capacity, immune phenotype, adipo- and osteogenic differentiation potential (Additional file 1: Figure S1), according to previously described methods [11, 13]. Chondrogenic differentiation was not performed.
To deplete abundant proteins, pHPL and tPRP, each pooled from two batches (a total of 16 donors and plasma from 4 donors), were pretreated with Albumin/IgG Depletion Kit (GE Healthcare; ∅ Alb/IgG) according to the manufacturer’s instruction (Figure 1A). Samples were mixed with prefrozen acetone/methanol (v/v 8:1) for 3 h at −30°C and subsequently centrifuged at 10000 g for 1 h at 4°C. After washing with 500 μl ice-cold acetone and centrifugation (10000 g, 10 min, 4°C), the pellet was air dried and resuspended in rehydration buffer (6 M urea, 2 M thiourea, 2% CHAPS (all Roth), 0.002% bromophenol blue (Sigma-Aldrich).
Two-dimensional difference gel electrophoresis
To separate, quantify and identify differential protein signatures and protein species in ∅ Alb/IgG pHPL and in ∅ Alb/IgG tPRP, multiparametric 2D-DIGE (two-dimensional difference gel electrophoresis) was performed [32, 33, 48, 49]. Thus, 50 μg protein of each sample was labelled with CyDye DIGE Fluors (Cy3, Cy5, respectively, GE Healthcare) according to the Minimal labeling protocol (GE Healthcare). For an internal standard, equal amounts of pHPL and tPRP were mixed and 50 μg from this mix labelled with Cy2. Labelled platelet derivate samples and internal standard were pooled equally to a final protein quantity of 150 μg. Rehydration buffer with 32 mM DTT (Roth) and 0.5% IPG buffer (pH 4–7, BioRad) was added to a final volume of 450 μl. After 24 h rehydration, the 24 cm IPG-strips (pH 4–7, BioRad) were subjected to isoelectric focussing on an IPGphor (GE Healtcare) for a total of 58000 V/h. This specific range of pH (pH 4–7) was chosen, since most of the proteins seem to occur in this isoelectric window, after testing IPG-strips with a range from pH 3–10 (data not shown). Then each strip was equilibrated in 15 ml equilibration buffer (6 M urea, 30% glycerol, 2% SDS, 50 mM Tris (pH 8.8), 0.002% bromophenol blue, all Serva), first with 32 mM DTT for 20 min, second with 135 mM iodoacetamide (Roth). Equilibrated strips were transferred onto a homogenous precast 12% bis-tris polyacrylamide gel (1 mm, Serva) and sealed with agarose (1% agarose, 0.002% bromophenol blue, Roth). SDS-PAGE was performed with constant 17 W per gel and a running time until the blue front ran out of the gels (Ettan DALT II system; GE Healthcare). Three replicates were run.
Advanced image warping and quantitative protein analysis
Visualization of labelled protein spots was performed with a fluorescence scanner (FujiFilm FLA-5100). Therefore, gels were scanned with three different fluorescence filters (Cy2: Ex. 488 nm, Em. 520 nm, Filter Y520/LBB; Cy3: Ex. 532 nm, Em. 580 nm, Filter O580/DGR1; Cy5: Ex. 633 nm, Em. 670 nm, Filter R665/LPR). Sensitivity was set on 400 V with 50 μm pixel/resolution. Spot detection, warping, and statistical analysis of stained or labeled samples were performed using Delta2D 3.4 software (Decodon) as described previously . For significant differential protein spot detection, sensitivity, background settings, and spot size were chosen according to the manufacturer’s instruction. Images were matched using stringent warping strategy. Regulation factor was set to 1 +/− 0.5 (red spots >1.5; green spots <0.8; and yellow spots <1.5, >0.8).
Mass spectrometry and protein identification
Preparative gels were run (250 μg protein/gel), fixed with methanol/acetic acid/H2O (40/10/50%), stained with Sybro Ruby (GE Healthcare) and scanned at 618 nm. In addition, a sensitive coomassie G250 blue staining was performed. Matched protein spots of interest were picked manually and subjected to an in-gel tryptic digestion as described previously . Briefly, after washing (100 μl H2O, 8 min, 37°C, 600 rpm), picked spots were incubated in 200 μl 40 mM NH4HCO3/CAN (acetonitrile) (v/v 1:1) for 15 min (25°C, 600 rpm). After incubation with ACN (1 min, 600 rpm, RT), protein spots were air dried for 5 min. For peptide mass fingerprint mapping 10 μl trypsin (3.5 μg/μl, Promega) was added for 30 min at 4°C. Excessive trypsin was removed and 10 μl 40 mM NH4HCO3 added and incubated over night at 37°C. Samples were stored at −30°C.
Before MALDI analyses of digested human platelet proteins, peptides were concentrated and manually desalted using ZipTip® pipette C18 columns (Millipore). Peptides were eluted with 1 mL α-cyano-4-hydroxycinnamic acid diluted 1:7 in 1:1 ACN/0.1% trifluoracetic acid solution and spotted on a MALDI 800–384 anchor chip target plate and analyzed automatically on a Bruker Ultraflex II using flex control, flex analysis, and Bruker BioTools software (batchmode). For external calibration, peptide calibration standard II was used (Sigma; m/z ratio from 700 to 3500) . Database searches were performed using MS ion search against all entries for Homo sapiens in the Swiss-Prot database, applying the following: tryptic digest, up to one missed cleavage; fixed modifications, carbamidomethyl (C); variable modification, and oxidation at methionine (M). Mass tolerance was set to 7100 ppm. Identification was taken as unambiguous if the Mowse score was at least 56 (with p-value < 0.05). In addition, PMF identification was verified comparing molecular weight and pI of the identified protein with gel position of the according protein spot.
Data verification by Western Blot analysis
Proteins were lysed in RIPA buffer (Santa Cruz Biotech). Protein concentration was measured by Bradford protein assay (Bio-Rad) and adjusted to equal concentrations. Protein lysates were mixed with loading buffer (Fermentas), separated on 12% Bis-Tris gels and then transferred onto a PVDF membrane (GE Healthcare). Primary antibodies used were: α-Fibrinogen-Biotin pAB (IMS06-038-312, Agrisera), α-Apolipoprotein A1 pAB (178422, Calbiochem), α-CLIC-1 (chloride intracellular channel 1; 356.1, Santa Cruz Biotech). HRP-conjugated secondary reagents used were α-Mouse IgG, α-Rabbit IgG (both Dako) and Streptavidin (Calbiochem). All antibodies were dissolved in TBS, 5% skimmed milk powder, 0.05% Tween-20 (all Roth). Band detection was performed using enhanced chemiluminescence reagent (Amersham™ ECL™ Select Western Blotting Detection Reagent, GE Healthcare). Loading concentrations were assessed by Ponceau S staining and reference controls (Serva dual color protein standard III, Serva).
Proliferation and stimulation assay
To determine the proliferation rate of MSC, CellTiter-Glo™ assay (Promega) quantifying ATP was performed. MSC in passage 2 with 70% confluence were trypsinized and seeded at a density of 3000 cells for 3 days into a black 96 well plate (Iso plate, PerkinElmer). Culture medium was either supplemented with each 10%, 7.5%, 5%, 2.5% pHPL, tPRP or FBS, respectively. Stimulation experiments utilized 5% of the supplements and the following substances at the indicated concentrations: fibrinogen, apolipoprotein A1 (apoA1), adenosine triphosphate (ATP) (Roche) and in a second set of experiments hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1) (all Calbiochem), and basic fibroblast growth factor (bFGF) (Miltenyi Biotec).
Enzyme-linked immunosorbent assay (ELISA)
In order to detect cytokines, described to induce MSC proliferation, platelet derivates (pHPL, tPRP n = 6; FBS n = 2) as well as medium of MSC (BM-, LA-MSC in FBS, pHPL, tPRP, each n = 3), conditioned for 24 h, were tested for PDGF-AA, AB, BB; IGF-1; HGF, bFGF (all Quantikine ELISA kit, R&D Systems) by ELISA according to the manufacturer’s instructions.
To assess the expression of cytokine receptors, flow cytometric analyses were performed. MSC were double-stained in 100 μl cell wash (BD) with the following phycoerythrin- (PE) and allophycocyanin- (APC) labelled antibodies: PDGFR-α-PE (16A1), PDGFR-β-APC (18A2, both biolegend), TGF-βRII-PE (FAB2411P; R&D systems), IGFR-1-APC (1H7; eBioscience), HGFR-APC (FAB35821), FGFR-PE (FAB684P; both R&D systems). 7-Aminoactinomycin D was used for dead cell exclusion (Beckman Coulter). Stained cells were analyzed with the FACS Canto II from BD.
Statistical tests were performed using SigmaPlot 11.0 (Systat Software Inc). Data was tested for normality and equal variance before analysis. Statistical differences between three samples were calculated using analysis of variance (ANOVA, Holm-Sidak method; or ANOVA on ranks if equal variance testing failed, Tukey method). Comparing two samples paired or unpaired t-tests, respectively, were used. The symbols indicate: (*) significant difference before and after stimulation; (+) significant difference between supplements; (#) significant difference between MSC sources.