2. Materials and Methods 1. Materials
2.2. Methods
2.2.1. Culture of human bone-marrow (hBM-MSCs) and umbilical-cord derived stromal cells (hUC-MSCs)
Cells were cultured in MSCBM + 2% FCS (Lonza Cologne AG, Germany) and cultured at 37 °C in a 5% CO2 humidified atmosphere. After reaching a confluency of 80%, cells were seeded at a density of 1000 cells/cm² into T-75 culture flasks (Greiner BioOne, Germany) for passaging.
2.2.2. Differentiation of hMSCs
Osteogenic medium for primary hMSCs consists of MSCBM medium (Lonza Cologne AG, Germany) containing 10 mM ß-glycerol phosphate, 50 µg/ml ascorbic acid 2-phosphate, 100 nM dexamethasone and 2% FCS (Lonza Cologne AG, Germany).
Primary hMSCs were plated in 24-well plates (Greiner BioOne, Germany) and medium was changed every two days for 21 days. To analyze osteogenesis of hMSCs induced by osteogenic medium mineralization deposits were stained by alizarin red. Alizarin red staining was performed as stated in 2.2.8.1.
Adipogenic medium for primary hMSCs consists of DMEM High Glucose (4,5 g/l) supplemented with 10% FCS (Lonza Cologne AG, Germany), 1% Pen/Strep, 1 µM Dexamethasone, 500 µM IBMX, 1 µg/ml Insuline and 100 µM Indomethacin. Control medium for adipogenic differentiation consists of DMEM High Glucose (4,5 g/l) supplemented with 10% FCS (Lonza Cologne AG, Germany) and 1% Pen/Strep.
Medium was replaced every 2-3 days. Adipogenic differentiation was analyzed by Oil red o staining after 13 days as stated in 2.2.8.2.
Chondrogenic medium for primary hMSCs consists of DMEM High Glucose (4,5 g/l) supplemented with 1% Penicillin/Streptomycin, 50 µg/ml l-ascorbic acid-2-phosphate, 100 nM dexamethasone, 100 µg/ml pyruvate, 40 µg/ml l-proline and 1% Insulin-transferrin-sodium selenite media supplement. TGF-β3 (10 ng/ml) was added with every medium change. The negative controls were cultured in chondrogenic differentiation medium without TGF-β. Chondrogenic differentiation and analysis was performed as stated in 2.2.8.3.
2.2.3. Isolation, culture, and maintenance of bone-marrow-derived stromal cells
Isolation of BMSC was performed as previously described (Soleimani & Nadri 2009).
Briefly, C57BL6 mice were sacrificed using cervical dislocation and rinsed with 70%
ethanol. The two tibias and femurs of each animal were cleaned of skin and transferred into alpha-MEM (Genaxxon) supplemented with 1% Pen/Strep. After removing the connective tissue in a sterile bench the ends of each bone were clipped off and the bone marrow was flushed out with a 26 G needle containing culture medium. The samples were stored on ice. Cells were separated with a 70 µm filter mesh followed by centrifugation (1000 g; 5 min) and resuspended in 1 ml of BMSC culture medium consisting of α-MEM medium (Genaxxon) supplemented with fetal calf serum (15%, Hyclone). 4x106 cells/cm2 were plated (Greiner) and incubated at 37% and 5% CO2 in a humidified incubator. The culture medium was replaced after 3, 8 and 16 hours. 72 hours after isolation, the cells were washed with phosphate buffered saline (PBS). Afterwards, the medium was changed every 2-3 days until the cells were 70% confluent. Cells were split using Versene plus 0.5% trypsin (Invitrogen) and cultured in a 25 cm2 flask or a 6 cm culture dish (Greiner). Cells were maintained in culture medium for further studies and passaged at a confluence of 6700 cells/cm2 every 2-3 days if not mentioned otherwise. The number of population doublings (PD) was calculated using the formula PD = (ln nch − ln ncs)/ln 2, where nch
is number of cells harvested and ncs is number of cells seeded (Kurz et al. 2004).
2.2.4. Immunofluorescence and microscopy
BMSCs or Fucci2-HeLa were grown on collagen-coated coverslips, washed with PBS, fixed in 4% paraformaldehyde at room temperature for 15 min, washed, permeabilized with 0.1% Triton-X 100 (5 min, RT) and blocked with blocking buffer (5% FBS in PBS) for 30 min. The cells were incubated with for example with DAPI (1 µg/ml) for 15 min, washed, incubated for example with Alexa-488 labelled phalloidin diluted in blocking buffer for 1 hour, washed, mounted in Fluormount G (Southern Biotechnology, AL, USA) and analyzed using the Cellobserver (Zeiss, Germany).
Cells were imaged with a Plan-Apochromat 20x/0.8 DIC objective lens.
Confocal images were taken using the LSM 710 (Zeiss). DAPI was excited at 405 nm using the diode laser, mAG was excited at 488 nm, and mKO2 was excited at 514 nm using a multiline argon laser. Alexa633 was excited at 633 nm using the Helium
neon laser. Images were taken with 40 or 63 times magnification (oil) in a 10 stack 4x4 tile scan. Pictures were analyzed using ZEN, Image J and Adobe Photoshop.
2.2.5. Beta-galactosidase staining
mBMSCs were stained with the senescence beta-galactosidase staining kit (Cell Signalling) according to manufacturer’s guidelines. Briefly, cells were rinsed once with PBS. Then 1 ml of 1x fixative solution was added to each well. The cells were fixed for 10-15 minutes at room temperature. After that cells were rinsed two times with PBS. After that 1 ml of the beta-galactosidase staining solution was added to each well and incubated at 37°C overnight in a dry incubator. While the beta-galactosidase staining solution was still on the plate, images were taken at a light microscope to detect development of blue colour.
2.2.6. Propidium iodide staining
1*106 HeLa cells were seeded in 6 cm culture dishes. Cells were trypsinized, washed with cold PBS and resuspended in 1 ml of cold PBS. 1 ml of the cell suspension was then added to 9 ml 70% EtOH while vortexing to ensure minimal cell clumping. Cells were washed with PBS and resuspended in 500 µl PI-staining solution (474 µl PBS, 25 µl PI (1 mg/ml), 0,5 µl RNAase A (20 mg/ml). Cells in staining solution were incubated at 37°C for 30 minutes under light protection and measured afterwards.
2.2.7. Flow cytometry
Flow cytometry analysis of PI stained HeLa cells was performed using the MACS Quant Analyzer. MACS Quantify software was used to analyze fluorescence intensity.
Linear gating between FL height and FL area ensured exclusion of clumped cells.
Flow cytometry analysis of mouse BMSC and human BM- and UC-MSC was performed by Georg Siegel and colleagues (Institute of Clinical and Experimental Transfusion Medicine (IKET), University Hospital Tübingen, Tübingen, Germany).
Flow cytometry analysis of mMSC populations was performed at passage 8, passage 23 and passage 43 and for hMSC at passage 1-4 employing a FACScan instrument (BD Biosciences) and BD CellQuest Pro software. Cells were incubated with (secondary) PE-labeled mouse or human-specific antibodies (anti-CD4, -CD8a, -CD9,
CD11b, CD11c, CD14, CD29, CD31, CD43, CD44, CD45R, CD71, CD73, CD80, CD86, CD90, CD105, CD106, CD117, CD135, CD140a, CD144, -CD184, -CD195, -H-2K/H2D, -IA/IE, -SCA1 and -VEGFR2 (BD Biosciences); -CD34 (Abd Serotec); CD39 (R&D); CD140b (Epitomics); CD146 (Bio Legend) and -CD271 (STEMCELL Technologies)) for 60 min on ice. Unspecific antibody binding was prevented by using the Mouse BD Fc Block™ (BD Biosciences) according to the manufacturer’s instructions. PE-conjugated or non-labeled IgG, IgG2aκ, IgG1κ, IgG1λ1, IgG2cκ, IgG2bκ, IgG2bκ and IgG1 antibodies (BD Biosciences) were used as isotype matched controls. As secondary antibodies mouse rat Ig, donkey goat IgG, goat rat Ig, rat mouse IgG2a+b (BD Biosciences) or goat anti-rabbit Ig (Biotrend) were used. Dead cells were excluded by uptake of 7-Aminoactinomycin D. Analysis of percentage of antigen positive cells and fluorescence intensity was performed using FlowJo software. The respective isotype control was subtracted from all samples to compensate unspecific antibody binding.
2.2.8. Differentiation of mBMSCs
2.2.8.1 Osteogenic differentiation
BMSCs were grown to 90-100% confluence in 24-well-plates and the culture medium was then replaced with osteogenic medium (α-MEM supplemented with 15% FCS plus 1% Penicillin/Streptomycin, 100 nM dexamethasone, 50 µg/ml ascorbate-phosphate, and 10 mM beta-glycerol phosphate). The medium was changed every 2-3 days. Osteogenic differentiation was assessed by Alizarin Red staining 21 days after initial osteogenic induction. In brief, cells were washed with PBS and allowed to dry for 5 to 10 min. Then cells were fixed with 50% ethanol for 20 min. The fixed cells were then stained with 1% Alizarin red (Roth) at pH 6.4 for 30 min under continuous shaking. Subsequently, cells were rinsed three times with H2O, and transmitted light pictures were taken. As a negative control cells grown in culture medium for 21 days were used.
2.2.8.2 Adipogenic differentiation
BMSCs were grown to confluence on Permanox 4-well chamber slides (Thermo Scientific). Adipogenic differentiation medium (α-MEM supplemented with 15% FCS
plus 1% Penicillin/Streptomycin, 1 µM dexamethasone, 500 µM IBMX, 10 µg/ml human insulin and 100 µM indomethacin) was added and renewed every 2-3 days.
12 days after initial adipogenic induction cells were washed with PBS and fixed for 10 min in 4% Histofix (Roth). Then, cells were rinsed once with H2O and incubated in 60% isopropanol for 5 min. Subsequently, the cells were incubated for 10 min with Oil Red O. Afterwards the cells were washed once with 60% isopropanol followed by H2O. Nuclei were counterstained with hemalaun. As a negative control cells grown in culture medium for 12 days were used.
2.2.8.3 Chondrogenic differentiation
2.5*105 BMSC were centrifuged (1000 g; 5 min) in a 15 ml Falcon tube, the supernatant was discarded and 1 ml of chondrogenic differentiation medium (α-MEM plus 1% Penicillin/Streptomycin, 50 µg/ml l-ascorbic acid-2-phosphate, 100 nM dexamethasone, 100 µg/ml pyruvate, 40 µg/ml l-proline and 1% Insulin-transferrin-sodium selenite media supplement) was added to the cell pellet. TGF-β3 (10 ng/ml) was added with every medium change. The negative controls were cultured in chondrogenic differentiation medium without TGF-β3. Medium was changed twice a week for 27 days. After fixing the pellets for two hours in 4% Histofix (Roth), they were embedded in paraffin in cassettes using the automated system Shandon Citadel 1000 (Thermo Scientific). The cassettes with the pellets were transferred to a heating chamber at 60°C after the program of the automated system was finished.
The melted paraffin was removed from the cassettes and the cell pellets were transferred into a metal form filled with liquid paraffin. The paraffin solidified on a cooling plate. The obtained solid paraffin block containing the pellet was then sliced with a rotary microtome RM 2145 (Leica).
The slices were deparaffinized using a descending sequence of xylol-ethanol. After 3 min in 0.1 N HCl the slices were stained with 1% Alcian Blue (Roth) dissolved in 0.1 N HCl (pH 1.0) for 30 min. Two washing steps 0.1 N HCl followed by H2O, were performed to remove the unspecific bound dye. The slices were counterstained with nuclear fast red for 4 min. To dehydrate the pellet slice ascending concentrations of ethanol were used. Isomount (LABOnord) was used to fix the slices on the object slides.
2.2.9. Colony-forming-unit fibroblast assay
The proliferative induction capability of the M2 cell line was evaluated by colony-forming-unit fibroblast (CFU-F) analysis. M2 BMSCs were seeded into 100 cm2 cell culture dishes at an initial density of three cells per cm2 in 12 ml medium. Cells were incubated for 7 days in a humidified 5% CO2 incubator at 37°C. On day 7, cultures were simultaneously fixed and stained with 0.1% crystal violet in 20% methanol by incubating them at room temperature for 30 min and then washed twice with water.
Crystal-violet stained colonies with a minimum surface area of 1 mm2 were counted and then photographed. CFU-F frequency was calculated by dividing the number of colonies by the number of cells seeded. The experiment was performed in triplicates.
2.2.10. Single clone analysis
BMSCs (P42) were seeded with a concentration of 0,5 cells/well in a 96-well plate in pre-conditioned sterile filtered α-MEM supplemented with 15% FCS. The wells were analyzed microscopically to record the single cell seeding events one day after plating. Single clone colonies were passaged after 17 days. 24 clones were analyzed for osteogenic and adipogenic differentiation.
2.2.11. Nucleofection
BMSCs (P20-P24) were nucleofected with Amaxa nucleofection kit for MSC (Lonza) according to the manufacturer’s instructions. Briefly, 5*105 cells were resuspended in 100 µl nucleofector solution, 1 µM siRNA (ON-Targetplus SMART pool Mouse Runx2 (12393, Dharmacon) or the non-target plus smartpool control) was added and the cell suspension was transferred to a Nucleofector cuvette. Cells were nucleofected using program U23 of the Amaxxa Nucleofector (Lonza). Subsequently, the cell suspension was transferred into culture medium and incubated at 37°C and 5% CO2. Successful knockdown was verified by qPCR two days post nucleofection.
2.2.12. Quantitative real-time PCR
RNA was extracted using the RNeasy Kit (Qiagen) according to the manufacturer’s instructions and concentration was measured using a NanoDrop (Peqlab). The
260/280 and 260/230 OD ratios were measured to assess the purity of RNA samples.
One-step real-time RT-PCR was applied to quantitatively analyze mRNA levels. In brief, the reaction was performed on the CFX96 Touch qPCR System (BioRad) platform using QuantiTect SYBR Green RT-PCR Kit (Qiagen). The PCR mix contained 10 µl Quantitect SYBR Green PCR Mix, 0,2 µl reverse transcriptase, 2 µl of the respective QuantiTect Primer Assay consisting of a mixture of predesigned forward and reverse primer for the gene of interest and 100 ng RNA in 7,8 µl RNAse free H2O. PCR was performed under the following conditions: 50°C for 30 min, 95°C for 15 min, followed by 40 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 30 s. A melt curve analysis step was added with a melting profile of 65.0°C to 95.0°C (Increment 0.5°C) Quanti-Tect Primer Assays (Qiagen) were used for Osteocalcin (Mm_Bglap_1_SG), Osteopontin (Mm_Spp1_1_SG), Integrin-binding-sialo-protein (Mm_Ibsp_1_SG), Runx2 (Mm_Runx2_1_SG), GAPDH (Mm_Gapdh_3_SG), PParע (Mm_Pparg_1_SG), Fabp (Mm_Fabp4_1_SG) and DLK1 (Mm_Dlk1_1_SG). The relative expression was calculated by normalization to GAPDH using the ∆Cq or the
∆∆Cq method.
2.2.13. Western blot analysis
2.5*105 cells were harvested via the hot lysis method. Briefly, supernatants were aspirated and adherent cells lysed by addition of 300 µl of hot (95°C) laemmli buffer (125 mM tris (pH 6.8), 10% glycerol, 4% SDS, 0.02% bromophenol blue, 10% beta-mercapto-ethanol) followed by incubation at 95°C for further 10 min and sonication.
Freshly prepared bone marrow cells (BM) were lysed by resuspension in 300 µl hot laemmli buffer. After centrifugation, supernatants were collected and 15 µl of each protein samples subjected to a 4-12% gradient Bis-Tris SDS-PAGE (Life Technologies) blotted onto nitrocellulose membranes and incubated with primary antibodies for example a p53-specific antibody (1C12; Cell Signalling). Equal loading was monitored by reprobing membranes with an antibody against actin or Tubulin (Sigma-Aldrich). HRP conjugated secondary antibodies were from Jackson Immunoresearch Laboratories. Bands were visualized applying chemiluminescence SuperSignal®detection system (PIERCE). Alternatively secondary infrared labelled antibodies (LiCor) were used. Bands were visualized using the Odyssey-Reader (Li-Cor). Bands were quantified using ImageJ.
2.2.14. Anchorage-independent growth
1000 BMSCs were seeded in 50 µl of α-MEM supplemented with 15% FCS in the lid of a 100 mm culture dish. The lid was then inverted to create hanging drops. Images were captured with a 45 times magnification at a sz61 binocular (Olympus). The diameter of the spheres was monitored at day 3, 6, 8, 12, and 18.
2.2.15. In vivo transplantation
The in vivo experiments with mBMSCs were performed by Dr Sacchetti (Department of Molecular Medicine, Sapienza University, Rome, Italy).
In order to assess the osteogenic potential of the M2 cell line in vivo, mBMSC M2 cell strains in early (P8-10) or late (P40-44) passages were seeded onto osteoconductive material (hydroxyapatite/tricalcium phosphate particles, HA/TCP) as reported previously (Kuznetsov et al. 1997; Krebsbach et al. 1997; Bianco et al. 1998). All animal procedures were approved by the relevant institutional committees. Briefly, 2x106 cells were loaded onto hydroxyapatite/tricalcium phosphate particles (40 mg, 100–200 µm; Zimmer, Warsaw IN) and embedded in a fibrin gel to generate carrier-cell constructs. The constructs and the carrier-cell-free carrier, as control, were subcutaneously transplanted in the backs of 6-15 weeks-old female SCID/beige mice (CB17.Cg-Prkdcscid Lystbg/Crl; Charles River Laboratories International, Inc., Wilmington, MA). In brief, operations were performed under sterile conditions under anaesthesia achieved by intramuscular injection of a mixture of Zoletil 20 (Virbac, 5 µl/g of body weight) together with Rompun (Bayer, 1ml/ Zoletil 20 bottle). The mouse back was disinfected with betadine and midlongitudinal skin incisions of about 1 cm in length were made on the dorsal surface of each mouse. Subcutaneous pockets were formed by blunt dissection. A single transplant was placed into each pocket with up to four transplants per animal. The incisions were closed with surgical staples.
2.2.16. Histology
Histology of M2 transplants was performed by Dr Sacchetti (Department of Molecular Medicine, Sapienza University, Rome, Italy).
Heterotopic transplants were harvested after 8 weeks, fixed in 4% formaldehyde, decalcified in 10% ethylenediaminetetraacetic acid at pH 7.2, and embedded in paraffin. Subsequently, the deparaffinized and rehydrated sections were stained with hematoxylin and eosin.
2.2.17. Statistical analysis
Data are expressed as means ± SEM. A one-way ANOVA test followed by a Bonferroni posttest was used in statistical analysis for comparison, and P < 0.05 was used as the criterion for statistical significance.
2.2.18. Synchronization of Fucci-BMSCs and Fucci2-HeLa cells
2.2.18.1 RO 3306
RO3306 is a selective CDK1 inhibitor that reversibly arrests proliferating human cells at the G2/M phase border (Vassilev 2006). Fucci-BMSCs were treated with RO3306 for 24 hours. Cells were released from synchronization by changing the medium to culture medium.
2.2.18.2 Aphidicolin block
Aphidicolin is a specific inhibitor of DNA polymerase in eukaryotic cells and can be used to synchronize cells at the G1/S boarder (Pedrali-Noy et al. 1980).
Synchronization of Fucci-BMSC was performed using 5 µg/ml aphidicolin for 24 hours. Fucci2-HeLa cells were treated with descending concentrations from 5 µg/ml down to 0,3 µg/ml. If not stated otherwise 0,3 µg/ml aphidicolin were used for Fucci2-HeLa cells for 18 hours. Cells were released by an initial medium change to remove aphidicolin and a second medium change 15 minutes later to ensure that residual aphidicolin is removed.
2.2.18.3 Thymidine-Nocodazole block
2 mM of thymidine were added to HeLa cells at 40 % confluency for 24 hours. After washing cells were released in culture medium for 3 hours. Subsequently, cells were
treated with 100 ng/ml nocodazole for 12 hours. Cells were released by washing cautiously and new culture medium was added. Cells progressed synchronously through mitosis.
2.2.18.4 Serum deprivation
Fucci-BMSC were seeded in 24-well plates and cultured with αMEM without serum for 24 hours. After addition of serum cells were fixed at different time points.
2.2.19. Live Cell Imaging
Live cell imaging of Fucci-BMSCs and Fucci-MEFs was performed at the Cell Observer. 20.000 cells were seeded in collagen coated 4well glass bottom chambers.
Images were taken every 10 minutes for up to 27 hours with 10 x magnification. Used filtersets were 43 HE Cy3, shiftfree (E), 38 HE eGFP shift free (E) and transmitted light. Time lapse images were analyzed using Image J plugin MTrack J.
Live cell imaging of Fucci2-HeLa cells was performed at the Cell Observer. The Colibri-System was used to excite mCherry and mVenus. Additionally transmitted light was used. The MatLab based Fucci Analysis software, developed by Matthias Lorenzen (IST, Stuttgart), was used to analyze imaging data obtained with Fucci2-HeLa cells.
2.2.20. PKDreporter assay
PKDrep, a PKD activity reporter, and G-PKDrep, the Golgi targeted variant (Czondor et al. 2009; Fuchs et al. 2009), were transfected using Turbofect according to manufacturer’s instructions. Briefly, 8*105 cells were seeded in a 6 cm dish. 7 µg DNA, 700 µl Optimem and 14 µl Turbofect were mixed and incubated at room temperature for 20 min. Subsequently, the transfection mix was added to the growth medium on the cells. The pSer294-specific antibody was used to detect the phosphorylation state of the substrate sequence and GFP antibody was used to normalize phosphorylation to expression levels of the construct.