Isolation of meniscus-derived mesenchymal stem cells (MMSCs) and bone marrow stem cells (BMSCs)
Ten female New Zealand white rabbits (8–10 week-old, 3.0 - 4.0 kg) were used in all experiments. The protocol for use of the animals was approved by the IACUC of the Nanjing Medical University. The rabbits were fully sedated by intra-muscular injection of Ketamine (10 mg/kg) and Xylazine (3 mg/kg) and were then sacrificed using pentobarbital (120 mg/kg). After sacrifice, rabbit medial and lateral menisci were dissected from bilateral knee joints. For isolation of MMSCs, the parameniscus tissues were removed, and the whole meniscus was then weighed and minced into small pieces (1 mm × 1 mm × 1 mm). Each 100 mg tissue sample was digested with 3 mg collagenase type I (Worthington Biochemical Corporation, Lakewood, NJ) and 4 mg dispase (StemCell technologies Inc., Vancouver, BC, Canada) in 1 ml phosphate buffered saline (PBS) at 37 °C for 1 hr. The suspensions were centrifuged at 1,500 g for 15 min, and the supernatant was discarded. The remaining cell pellet was re-suspended in growth medium consisting of Dulbecco’s modified Eagle’s medium (DMEM; Lonza, Walkersville, MD) supplemented with 20% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA), 100 μM 2-mercaptoethanol (Sigma-Aldrich, http://www.sigmaaldrich.com), 100 U/ml penicillin and 100 μg/ml streptomycin (Atlanta Biologicals, Lawrenceville, GA) to make a single-cell suspension. It was then cultured in either tissue culture flasks or plates at 37 °C with 5% CO2.
For BMSCs, two milliliters of bone marrow was aspirated with an 18-gauge needle that was fastened to a 5-ml syringe containing 0.2 ml of heparin (1,000 units/ml). The aspirates were washed twice with phosphate-buffered saline (PBS) and centrifuged at 1,500 rpm for 5 min. The supernatant was discarded. The cells were re-suspended in growth medium and incubated in either tissue culture flasks or plates at 37 °C in a humidified 5% CO2–95% air atmosphere. The cell colonies were stained with methyl violet (Sigma-Aldrich, http://www.sigmaaldrich.com). Colony numbers were counted manually. Each colony was trypsinized locally under microscopic visualization in order to detach stem cell colonies and detached cells were collected.
The total cell numbers from each colony were counted using a hemocytometer and transferred to T25 flasks for further culture.
Expression of stem cell markers of MMSCs and BMSCs
Immunocytochemistry was used to assay for expression of the following stem cell markers: nucleostemin, Nanog, SSEA-4, CD34, CD44, CD90 and STRO-1. To perform immunostaining, MMSCs and BMSCs at passage 1 were seeded in 12-well plates at the density of 3 × 104/well and cultured with growth medium for three days.
After removing the medium, the cells were washed with PBS once. MMSCs and BMSCs were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature and treated with 0.1% Triton X-100 for 30 min for Nanog and nucleostemin staining. After washing the cells with PBS, either mouse anti-Nanog (1:350, Santa Cruz Biotechnology, Inc., cat. # SC-33759, Santa Cruz, CA) or goat anti-nucleostemin (1:400, Neuromics, Cat. # GT15050, Edina, MN) was applied to the cells. In order to stain for SSEA-4 and strol-1, fixed cells were incubated either with mouse anti- SSEA-4 antibody (1:500, Invitrogen, Cat. # 414000, Frederick, MD), or mouse anti-strol-1 antibody (1:400, Cat. #398401, Invitrogen, Carlsbas, CA). After 2 hours reaction at room temperature, the cells were washed with PBS for three times, and either Cy-3-conjugated goat anti-mouse IgG antibody (1:500 for Nanog, SSEA-4 and strol-1, Millipore, Cat. # AP124C, Billerica MA) or Cy3-conjugated donkey anti-goat IgG antibody (1:500 for nucleostemin, Millipore, Cat. # AP180C, Billerica, MA) was applied for 1 h at room temperature.
In addition, stem cell surface markers CD34, CD44 and CD90 were stained in parallel by immunocytochemistry. Briefly, fixed cells were incubated with fluorescein isothiocyanate (FITC)-conjugated mouse anti-CD34, or FITC-conjugated mouse anti-CD44, or phycoerythrin (PE)-conjugated mouse anti-CD90 antibodies (1:400) at room temperature for 1 hour. Antibodies were purchased from BD Pharmingen (BD Biosciences; http://bdbiosciences.com), Stem cell Technologies (Vancouver, BC) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Fluorescent images of the stained cells were taken by a CCD camera on an inverted fluorescent microscope (Nikon eclipse, TE2000-U) using SPOT™ imaging software (Diagnostic Instruments Inc., Sterling Heights, MI). A total of 36 views from 3 wells of a 12-well plate were randomly chosen for each stem cell marker and the number of positively stained cells was manually counted. The percentage of each stem cell marker expression was determined by dividing the number of positively stained cells by the total number of cells stained by the nuclear staining reagent Hoechst fluorochrome 33342 (1 μg/ml; Sigma, St. Louis, MO).
Multi-differentiation potential of MMSCs and BMSCs in Vitro
Multi-differentiation potential of MMSCs and BMSCs in vitro were tested for adipogenesis, chondrogenesis, and osteogenesis. Both types of cells at passage 2 were seeded either on plastic surfaces in 6-well plates at a density of 2.4 × 105 cells/well or in 24-well plates at a density of 6 × 104 cells/well in basic growth medium consisting of low glucose DMEM, 10% heat inactivated FBS, 100U/ml penicillin, and 100 μg/ml streptomycin. To test adipogenic potential, cells were cultured in adipogenic induction medium (Millipore, Billerica, MA) consisting of basic growth medium added with dexamethasone (1 μM), insulin (10 μg/ml), indomethacin (100 μM), and isobutylmethylxanthine (0.5 mM). As a test of chondrogenic potential, two kinds of MSCs were cultured in basic growth medium supplemented with prolin (40 μg/ml), dexamethasone (39 ng/ml), TGF-β3 (10 ng/ml), ascorbate 2-phosphate (50 μg/ml), sodium pyruvate (100 μg/ml), and insulin transferrin-selenious acid mix (50 mg/ml) (BD Bioscience, Bedford, MA). Finally, the osteogenic potential of MMSCs and BMSCs was tested by culturing them in osteogenic induction medium (Millipore, Billerica, MA) consisting of basic growth medium supplemented with dexamethasone (0.1 μM), ascorbic 2-phosphate (0.2 mM), and glycerol 2-phosphate (10 mM).
Histochemical analysis
After culturing for 21 days, MMSCs and BMSCs grown in 24-well with various differentiation media were stained using Oil Red O for adipogenesis, Safranin O for chondrogenesis, or Alizarin Red S for osteogenesis, respectively. The stained samples were examined using an inverted microscope as we depicted above. The ratio of positive staining was calculated by dividing the stained area by the view area. The values of all views from three duplicate wells were averaged to obtain the percentage of positive staining, which represents the extent of cell differentiation in the respective induction medium.
Quantitative real-time PCR (qRT-PCR)
The specific gene expression of differentiated MMSCs and BMSCs were determined using qRT-PCR. Total RNA was extracted using a RNasy Mini-Kit with an on-column DNase I digest (Qiagen). First-strand cDNA was synthesized in a 20 μl reaction of 1 μg total RNA through reverse transcription with Super-Script II (Invitrogen). The conditions for the cDNA synthesis were: 65 °C for 5 min and cooling for 1 min at 4 °C, then 42 °C for 50 min, and finally 72 °C for 15 min. The qRTPCR was carried out using QIAGEN QuantiTect SYBR Green PCR Kit (Qiagen) [11]. In a 25 μl PCR reaction mixture, 2 μl cDNA (total 100 ng RNA) were amplified in a Chromo 4 Detector (MJ Research). Rabbit-specific primers for differentiated cells were used for collagen type II, peroxisome proliferators-activated receptor γ (PPARγ), Sox9, osteocalcin, and Runx2. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The forward and reverse primer sequences and the resultant products were designed according to published methods, and are listed in Additional file 1: Table S1 [12-15]. All primers were synthesized by Invitrogen (Carlsbad, CA). The relative gene expression levels were calculated from 2-∆CT, where ∆CT was determined by the formula: ∆CT = (CTtarget -CTGAPDH)differentiation-(CTtarget -CTGAPDH)control. In the formula, CTtarget and CTGAPDH are the cycle thresholds of target gene and GAPDH gene, respectively, for each RNA sample. The standard deviation (SD) of the ∆CT was determined from at least three parallel tests.
Western blot
Two kinds of MSCs were seeded in 6-well plates at a density of 2.4 × 105 per well and cultured with adipogenic, osteogenic and chondrogenic induction media for 21 days, respectively. Then MMSCs and BMSCs were lysed using a mammalian protein extraction reagent cocktail (Pierce, Rockford, Illinois) containing 1.5% protease inhibitors (Sigma-Aldrich). After centrifugation at 12,000 rpm for 10 minutes, the protein concentrations of the supernatants were determined using a BCA Protein Assay kit (Pierce). Equal amounts of total protein were run on 12% SDS polyacrylamide gels (Bio-Rad) at a constant voltage of 100 V for 60 minutes. Proteins were blotted to a nitrocellulose membrane using a Semi-Dry transfer module (Bio-Rad) at 200 mA for 90 minutes. The membrane was blocked in a 5% dry milk/TBS-Tween 20 solution for 1 hour at room temperature and then probed for 5 hours with a mouse monoclonal anti-adiponectin antibody (Millipore; Cat #MAB3604) at a dilution of 1:1000; mouse monoclonal anti-osteocalcin (Abcam; Cat #ab13418) at a dilution of 1:1000 for 5 hours; and mouse anti-collagen II (Millipore; Cat # MAB8887) at a dilution of 1:500 in a 1% dry milk/PBS-Tween 20 solution. Incubation with the primary antibody was followed by a horseradish peroxidase-conjugated goat anti-mouse antibody (Millipore; Cat #12-349) at a dilution of 1:2000 in a 1% dry milk/PBS solution. The targeted protein bands were detected using an ECL (enhanced luminol-based chemiluminescence) detection kit (Amersham Biosciences, Piscataway, New Jersey), followed by exposure of the membrane to X-ray film. Membranes were also re-probed for mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EMD Millipore, Merck KGaA, Darmstadt, Germany; Cat # MAB374) to verify equal protein loading in the gels. The band intensity was quantified by image J software (http://rsb.info.nih.gov/nih-image/).
Histochemical analysis of wounded meniscus sections
The menisci were obtained aseptically from female New Zealand white rabbits within 12 hours of death. A wound with 1 mm diameter was created in the center of each meniscus by a biopsy punch (Miltex, Inc., Cat. #REF33-31AA, York, PA). Either BMSCs or MMSCs at passage 2 were seeded in these defects, culturing with 10% FBS-DMEM for 6 weeks. The culture medium was changed every 3 days.
Six weeks later the wounded meniscus samples were harvested and placed in pre-labeled base molds filled with frozen section medium (Neg50; Richard-Allan Scientific; Kalamazoo, MI). The tissue samples in base mold filled were then quickly immersed in liquid nitrogen cold-2-methylbutane and allowed to solidify completely. Then the tissue blocks were placed on dry ice and subsequently stored in the −80 °C freezer until histological analysis was carried out.
The tissue block was cut into 10 μm thick sections and placed on glass slides, and then these glass slides were left over night at the room temperature to dry. The sections were rinsed 3 times with PBS and fixed with 4% paraformaldehyde for 30 min, then washed with PBS for another 3 times. The chondrogenesis differentiation of BMSCs and MMSCs on wounded rabbit meniscus was tested by histochemical staining. The sections were stained with alcian blue, toluidine blue, safranin O and fast-green. All images were taken using a CCD camera as we depicted above.
In vivo implantation experiments
Four female nude rats (10 weeks old; 200 – 250 g) were used to test the differentiation of two kinds of MSCs in vivo. Rats were housed individually on a 12 h : 12 h light–dark cycle and were cared for in accordance with the Guide to the Care and Use of Experimental Animals. The stem cells at passage 2 with the density of 6 × 104 cells/0.1 ml were mixed with 0.5 ml of Matrigel (BD 201 Biosciences, Cat. # 354234, Bedford, MA) in a 24-well plate and overnight cultured with DMEM-10% FBS at 37 °C and 5% CO2. In the next day, the medium was removed from each well and the Matrigel with cells were used for implantation into nude rat skin.
The nude rats were placed under general anesthesia using ketamine hydrochloride (75 mg/kg body weight) and xylazine hydrochloride (5 mg/kg body weight), administrated by intramuscular injection. Wounds (1 cm diameter/each wound) were created on the back of each rat, and three pieces of cell-Matrigel were placed in three distinct wounds on each side of the rat’s back. The approximate distance between two wound sites was 1.5 cm, with a total of six cell-Matrigel composites implanted into each rat. Each group had two rats and a total of four rats were used for two groups. At 3 weeks after implantation, tissue samples were harvested and frozen according to the measure we described above.
The tissue block was cut into 10 μm thick sections, which were then placed on glass slides and allowed to dry overnight at room temperature. The multi-differentiation potentials of two kinds of MSCs in vivo were tested by immunostaining on tissue sections of nude rats after implantation. The tissue sections were fixed with 4% paraformaldehyde for 15 min and reacted with mouse anti-adiponectin (1:300, Millipore; Cat. #MAB3604; Temecula, CA), mouse anti-osteocalcin (1:200, Abcam, Cat #13418; Cambridge, MA), mouse anti-collagen type I (1:100, Millipore, Cat. #MAB1340; Temecula, CA), and mouse anti-collagen type II (1:100, Millipore, Cat. #MAB1330, Temecula, CA) at room temperature for 2 hours. FITC-conjugated goat anti-mouse IgM (1:500, Santa Cruz Biotechnology, Cat. #sc-2082, Santa Cruz, CA) was used as the secondary antibody to detect adiponectin and Cy-3 conjugated goat anti-mouse IgG (1:500, Jackson ImmunoResearch Laboratories, Inc., Cat. #115-165-146, West Grove, PA) was used as the secondary antibody to detect collagen type I, collagen type II and osteocalcin at room temperature for 2 hrs. The tissue sections were also treated with Hoechst 33342 (Sigma, Cat. #B2261, St. Louis, MO) to stain nuclei.
Statistical analysis
Data is presented as mean plus and minus standard deviation (SD). At least three replicates for each experimental condition were performed, and the presented results are representative of these replications. One-way analysis of variance (ANOVA), followed by either Fisher’s predicted least-square difference (PLSD) for multiple comparisons or two tailed student t-test wherever applicable, were used for statistical analysis. Differences between two groups were considered significant when the p-value was less than 0.05.
