Chordin knockdown enhances the osteogenic differentiation of human mesenchymal stem cells

Human MSCs were obtained from two sources, the first one being a commercially available bone marrow aspirate from a 19-year-old male donor (Clonetics-Poietics, Walkersville, MD, USA). This was plated at 10 μl aspirate/cm² on a 150 cm² tissue culture plate (Costar, Cambridge, MA, USA) and cultivated until confluency in 25 ml of basal medium (BM), Dulbecco's modified Eagle's medium (DMEM) containing 1,000 mg/l glucose (Invitrogen Life Technologies, Carlsbad, CA, USA), supplemented with 10% foetal bovine serum (FBS; Stem Cell Technologies, Vancouver, Canada), which had been commercially screened from carefully selected lots, and 1% antibiotic-antimycotic (Invitrogen). MSCs were selected from the marrow aspirate on the basis of their ability to adhere to tissue culture plastic. Nonadherent, haematopoietic cells were removed during the first medium replacement after 3 days in culture. Primary culture human MSCs were subsequently detached using 0.25% trypsin/1 mmol/l EDTA (Invitrogen), replated at 1,000 cells/cm², and cultured until confluency to generate first passage MSCs. These were frozen in Recovery Cell Culture Freezing Medium (Invitrogen), stored in liquid nitrogen, and further expanded for osteogenic differentiation studies at the appropriate time.

The second source of human MSCs was an Institutional Review Board approved aspiration of the medullary cavities of femora of four patients undergoing hip hemiarthroplasty, as described previously [16]. There were three female patients aged 71, 76 and 78 years, and a male aged 77 years. Briefly, the marrow cells were layered on Ficoll (Sigma-Aldrich) and centrifuged for 30 minutes at 400 g. The mononuclear cells collected from the Ficoll-supernatant interface were cultured in BM at a density of 5 × 10⁷ per 75 cm² flasks (Becton Dickinson, Franklin Lakes, NJ, USA). After 2 weeks in primary culture, cells were passaged at seeding densities between 100 and 1,000 cells/cm², trypsinised and stored in Recovery Cell Culture Freezing Medium in liquid nitrogen.

Before osteogenic differentiation, 6,000 cells/cm² of human MSCs were seeded in BM in each well of 24-well plates (Becton Dickinson Labware, Franklin Lakes, NJ, USA). Each experimental condition was repeated in quadruplicate. On day 1 of culture, approximately 24 hours after cells were seeded, BM was removed and immediately replaced with an appropriate volume of osteogenic differentiation medium (OM) consisting of DMEM with 4,500 mg/l glucose (Invitrogen) supplemented with 10% FBS (Stem Cell Technologies), 1% antibiotics, and the osteogenic stimulants 100 nmol/l dexamethasone, 50 μg/ml ascorbate phosphate and 3 mmol/l β-glycerophosphate. First to third passage cells were used for osteogenic differentiation studies. Media of all cultures was changed every 3 days.

Osteogenic differentiation of human MSCs was evaluated after 10 and 21 days in BM or OM. Medium was aspirated directly from the wells. Cultures were quantitatively compared on the basis of the expression of alkaline phosphatase (ALP) activity and incorporation of calcium in the extracellular matrix. The deposition of a mineralized matrix was further examined by staining with Alizarin Red.

ALP activity was determined by measuring the conversion of p-nitrophenyl phosphate to p-nitrophenol. The substrate solution was prepared by dissolving 4-nitrophenyl phosphate disodium salt hexahydrate into a substrate buffer consisting of 50 mmol/l glycine and 1 mmol/l MgCl₂ at a pH of 10.5. Cultures were washed twice with phosphate-buffered saline (PBS) and 250 μl of the substrate solution was added to each well. Following a 15-minute incubation, the resulting solution was transferred to tubes containing the same volume of 1 mol/l NaOH. The cells were immediately washed with PBS twice and solubilized with a cell lysis buffer for protein assay. The cell lysis buffer consisted of 50 mmol/l Tris, 150 mmol/l NaCl and 0.2% Triton X-100 (Fisher Biotech, Fairland, NJ, USA; pH 7.2).

The quantity of p-nitrophenol liberated from the substrate was determined by comparison to a standard curve. The absorbance was read at 405 nm on a UVmax Kinetic Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). Readings were obtained within the linear range of the standard curve. ALP activity was normalized to the number of cells (as determined by the WST-1 assay described below) and expressed as nitrophenol product/minute/absorbance.

The calcium deposition assay was based on that of Hanada and coworkers [17]. Briefly, cultures were washed twice with PBS. Mineral was then collected after dissolution with 300 μl of 0.6 mol/l hydrocholoric acid at room temperature overnight and the samples assayed the following day. Incorporation of calcium in the extracellular matrix was quantified using the commercial diagnostic kit QuantiChrom Calcium Assay Kit (DICA-500; Bio Assay Systems, Hayward, CA, USA), in accordance with the manufacturer's instructions. Absorbances were compared with curves prepared using standard solutions of calcium. Calcium deposition was expressed as μmoles per tissue culture well (μmol/well).

Human MSCs were plated and cultured as described above. After culturing for 10 days, the media were aspirated and the cells washed twice in PBS. A commercially available kit (Sigma-Aldrich) was used for the cytochemical demonstration of ALP activity with Neutral Red Solution counterstian. Representative pictures at 10× magnification were taken with a Leica Microscope DM IL (Leica Microsystems, Bannockburn, IL, USA) and a Kodak DC 290 200 M digital camera (Eastman Kodak, Rochester, NY, USA).

Mineral deposition was also assessed by staining with Alizarin Red, after 21 days exposure to test the media. Monolayers in the 24-well plates were washed with PBS and fixed in 10% (vol/vol) formaldehyde (Sigma-Aldrich) at room temperature for 15 minutes. The monolayers were then washed twice with excess distilled water before addition of 250 μl of 40 mmol/l of Alizarin Red solution (pH 4.1) per well. The plates were incubated at room temperature for 10 minutes with gentle shaking. After aspiration of the unincorporated dye, the wells were washed four times with 1 ml distilled water while shaking for 5 minutes. The plates were then left at an angle for 2 minutes to facilitate removal of excess water and reaspirated.

BLOCK-iT Fluorescent Oligo (Invitrogen) was used to assess the transfection efficiency of siRNA. It is a fluorescein-labelled, double-stranded RNA duplex with the same length, charge and configuration as standard siRNA. The sequence of the BLOCK-iT Fluorescent Oligo is not homologous to any known gene, ensuring against nonspecific cellular events caused by the introduction of the oligonucleotide into the cells. Human MSCs were seeded under the same experimental conditions and transfected with BLOCK-iT Oligo at varying concentrations. After 24 hours, the proportion of cells transfected was determined using a Cytomics FC500 MPL flow cytometer (Beckman Coulter, Fullerton, CA, USA) and Flow Jo flow cytometry analysis software (Tree Star, Ashland, OR, USA).

The most effective chordin siRNA used was targeted to the sequence 5'-CAG GTG CAC ATA GCC AAC CAA-3'. The sense sequence of the siRNA used was r(GGU GCA CAU AGC CAA CCA A)dTdT and the antisense sequence was r(UUG GUU GGC UAU GUG CAC C)dTdG. This was generated by the Qiagen HiPerformance siRNA Design Algorithm. A scrambled siRNA nucleotide sequence (catalog number 1022076; Qiagen) with no significant homology to any mammalian gene (sense: UUC UCC GAA GUC ACG UdTdT; and antisense: ACG UGA CAC GUU CGG AGA AdTdT) was used as a negative control.

Human MSCs were transfected with siRNA 24 hours after plating onto 24-well plates. On the day of the transfection, the culture medium in each well was replaced with 400 μl of Opti-MEM I Reduced Serum Medium (Invitrogen) without FBS or antibiotics. The siRNA was prepared according to the manufacturer's instructions and it was added to each well at a final concentration of 300 nmol/l. Lipofectamine 2000 at 2% (vol/vol) and chordin-siRNA were used in a total transfection volume of 511 μl/well. Appropriate cell, Lipofectamine 2000 and negative siRNA controls were established in parallel. The cells were incubated at 37°C in a humidified carbon dioxide incubator for 16 to 24 hours and replaced with either BM or OM. The cells transfected with siRNA were examined 3 and 10 days after transfection and used for RNA expression assays.

Human MSC proliferation was assessed as a function of metabolic activity using the WST-1 assay (Roche Applied Science, Indianapolis, IA, USA) [18]. Briefly, 1,000 cells were plated in each well of a 96-well plate. After an overnight incubation, the cells were exposed to the relevant test media, as described above. Following treatment, media were removed from each well and replaced with 100 μl fresh DMEM with 4,500 mg/l glucose, to which 10 μl WST-1 test solution was then added. After 4 hours of incubation, absorbance was measured using a UVmax Kinetic Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) at a test wavelength of 450 nm and reference wavelength of 610 nm. The cell-free blank value was then subtracted from each value. Results are reported as the average of OD from the four wells point ± standard deviation.

To analyze gene expression under a given experimental condition, human MSCs were seeded at the same density as above in BM in three wells of a 12-well plate. After an overnight incubation, the cells were exposed to the relevant media and appropriate conditions. To test the efficacy of chordin siRNA in ablating the expression of chordin transcripts, human MSCs were transfected with scrambled (control siRNA) or chordin siRNA for 24 hours and then exposed to the relevant media. At the appropriate time, total RNA was collected by scraping each well with a Fisherbrand cell scraper (Fisher Scientific, Pittsburgh, PA, USA) in PBS, pelleting the cells and collecting the RNA in RNA Later solution (Ambion, Austin, TX, USA) at -80°C.

To extract RNA, samples were first suspended in 1 ml cold Trizol (Invitrogen) and choloroform added before centrifugation to enable phase separation. RNA was precipitated by addition of isopropanol to the aqueous phase, followed by further centrifugation. Precipitated RNA pellets were washed in 75% ethanol and re-suspended in distilled RNAse-free water before being quantified using a NanoDrop ND-1000 spectrophotometer (Labtech International, East Sussex, UK). All samples were treated with DNAse I (Invitrogen) to remove any potential contaminating DNA in the specimen. The DNAse was subsequently inactivated by the addition of EDTA, before reverse transcription.

RT-PCR was performed to evaluate the expression of BMP-2 and chordin, using the housekeeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as an internal control. cDNA was generated using the High Capacity cDNA reverse transcription Kit (Applied Biosystems, Warrington, UK). PCR was performed using the Invitrogen Platinum Taq (Invitrogen) method, in accordance with the manufacturer's instructions. The PCR primers were designed on the basis of the published human gene sequences (Table 1; Invitrogen). PCR amplication was performed using an MJ Research PTC-2000 Peltier thermal cycler (GMI, Ramsey, MN, USA) in the following sequence: denaturation at 94°C for 90 seconds followed by a specific number cycles of denaturation (Table 1) at 94°C for 30 seconds, annealing at the temperature shown below for each specific primer for 30 seconds, and extension at 72°C for 6 seconds followed by a last extension at 72°C for 10 minutes (Table 1). A 20 μl aliquot of the PCR product was added to 10 μl loading buffer (consisting of 50% glycerol, 0.2 mol/l EDTA [pH 8.0] and 0.05% bromophenol blue), and the resulting solution electrophoresed on 2% agarose gel stained with GelRed nucleic acid stain (Cambridge Biosciences, Cambridge, UK). As well as the tested samples, a 100 base pair DNA ladder (Hyperladder IV; Bioline, London, UK), a positive control prepared from the reverse transcription of human total RNA (Promega UK, Southampton, UK) and a negative control of molecular biology grade water were also included on each gel. Photographs of the gel were taken under ultraviolet light with a Syngene high-resolution camera (Synoptics, Cambridge, UK) and images acquired and stored with a Genesnap software (Syngene, Cambridge, UK).

The data are expressed as mean ± standard deviation and were analyzed using the statistical package SPSS 13.0 for Windows (SPSS Inc., Chicago, IL, USA). Statistical analyses were performed using analysis of variance, followed by the Gabriel post hoc test, with P < 0.05 considered to be statistically significant.

MSCs were cultured in BM or OM for 20 days. The usual change in phenotype of MSCs from spindle-shaped cells (when cultured in BM) to more cuboidal-shaped cells (when cultured in OM) was noted. The osteogenic phenotype of MSCs was confirmed by the increase in ALP activity at day 10 (Figure 1) and positive Alizarin Red staining at day 21 for cells cultured in OM, but not BM (Figure 1). These data confirm the ability of human MSCs to undergo osteogenesis in response to OM.

BMP-2 expression was weakly detectable in MSCs exposed to 5 and 10 days of BM (Figure 2a). Osteogenic differentiation induced an increase in the expression of BMP-2 (Figure 2a). Chordin mRNA expression was not detectable by RT-PCR when the MSCs were cultured in BM. However, there was detectable expression of chordin transcripts in MSCs cultured in OM, with an apparent increased expression at day 10 compared with day 5 (Figure 2a).

To assess the effect of chordin knockdown on osteogenic differentiation, we first determined whether we could transfect siRNA into human MSCs, by using BLOCK-iT Oligo fluorescent siRNA and detecting its presence using a Cytomics FC500 MPL flow cytometer. As measured by fluorescence-labelled siRNA using a flow cytometer, transfection with 100, 200 and 300 nmol/l oligomucleotides respectively resulted in 51%, 91% and 99% efficiencies of transfection (Figure 3). Based on these data, the dose of 300 nmol/l siRNA was used in further experiments to screen the designed candidate siRNAs.

Subsequently, the cells were transfected with chordin-specific siRNA and were cultured for 3 days (for mRNA analysis), 10 days (for mRNA analysis and ALP assay), and 21 days (for calcium deposition assay). After 3 and 10 days of culture, total RNAs were extracted from the human MSCs. RT-PCR analysis showed that the human MSCs transfected with chordin siRNA had undetectable chordin gene expression, whereas the control siRNA cells exhibited detectable chordin mRNA expression (Figure 2b).