This study uses a factorial design to investigate the effects of donor age and cell passage on BMSC differentiation into 3 mesenchymal lineages. Murine bone marrow derived mesenchymal stem cells were harvested from donors aged 6 days (postnatal), 6 weeks (adult), and 1 year (aged) and cultured through either 1 or 6 passages before differentiation was induced. The harvested and cultured cells used were the adherent population of cells within the bone marrow and are often referred to as either marrow stromal cells or mesenchymal stem cells (a sub-population of marrow stromal cells). For this study, the adherent cell population examined will be referred to as bone marrow derived mesenchymal stem cells or BMSCs.
BMSC harvest and culture
All experiments followed protocols approved by the Animal Experiment and Care Committee of Shanghai Jiao Tong University School of Medicine. Postnatal (6-day old), adult (6-weeks old), and aged (1-year old) transgenic male eGFP C57Bl/6 mice were obtained from a local breeder colony. Mice were euthanized via cervical dislocation and bilateral thoracotomy and then immersed in 75% ethanol for 15 minutes. The bilateral femurs and tibias were aseptically excised, stripped of connective tissues, and the epiphyses and metaphyses were then removed. The remaining diaphyses were placed in a culture dish with 7.5 ml sterile PBS, and the bone marrow was flushed from the shafts via 3–5 minutes of vigorous pipetting of the PBS. The PBS/marrow suspension was then filtered through a 70-μm cell strainer (BD Biosciences, Mississauga, ON, Canada), collected and centrifuged for 5 minutes at 524 × g. After removal of the supernatant, the resulting pellet was resuspended in α-MEM supplemented with 10% fetal bovine serum (FBS, HyClone, Logan, UT), 2 mM L-glutamine and plated at a density of 1.7–2.0 × 104 cells/cm2. Media were changed every 3 days, and cells were passaged when confluent (~5 × 104 cells/cm2) with 0.05% trypsin in 0.02% ethylenediaminetetraacetic acid (Gibco, Canada). Cells were replated at a density of 3 × 103 cells/cm2. First and sixth passage cells were used as indicated for all experiments. Each experiment was performed with cells pooled from 2–7 mice for each age group. Experiments were repeated in triplicate using different batches of marrow isolates.
Cell attachment and proliferation
After harvesting and resuspension, cells from postnatal, adult, and aged mice were plated individually in wells of 6-well tissue culture plates at a density of 1 × 104 cells/cm2. At 1/2, 1, 2, 4, 8, 16, and 24 hours after plating, media were gently aspirated and the remaining cells were fixed with 4% paraformaldehyde (Sigma, St. Louis, MO) in phosphate buffered saline (PBS) for 15 minutes. Cell adhesion was measured by counting the total cell number per well under fluorescence.
For proliferation, first passage cells from postnatal, adult, and aged mice were plated individually at 2 × 103 cells/cm2 in wells of 6-well tissue culture plates. At days 1, 2, 4, 6, and 8 cells were trypsinized and counted using a hemacytometer.
Attachment and proliferation assays were performed in triplicate. Cell doublings were calculated as : doublings = log2 (number of cells at passage/number of cells seeded).
Adipogenic differentiation and characterization
As previously described , the first or the sixth passage cells were seeded in 2 wells each of a 6-well plate at a density of 3000 cells/cm2. Cells were maintained for 24 hours in regular culture media, after which adipogenic differentiation was induced using Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS, 0.5 mM isobutylmethylxanthine, 10 μM insulin, 1 μM dexamethasone, and 200 μM indomethacin (all from Sigma) for 3 weeks with full medium changes performed every 3 days. 2 control wells were maintained in DMEM with 10% FBS over the same time course. Each experiment was repeated in triplicate, resulting in 6 total wells for adipogenic differentiation and 6 control wells.
Differentiated and control wells were stained with Oil Red O. At 3 weeks post-induction, wells were gently rinsed with PBS and then fixed with cold 4% paraformaldehyde for 10 minutes. Wells were then washed with 60% isopropyl alcohol (Sigma), incubated for 5 minutes at room temperature in 2% (w/v) Oil Red O reagent (Sigma), and then washed once with isopropyl alcohol followed by repeated washes with distilled water.
For each well, 3 fields were randomly chosen and examined by light and fluorescence microscopy. The number of total cells per field was determined under fluorescence followed by determination of the number of cells containing Oil Red O stained inclusions. A cell containing a visibly stained vacuole was considered to be positively stained. Additionally, for each view the percent area of positive staining was determined using ImageJ (NIH, Bethesda, MD). The area stained was determined by quantifying the actual area stained rather than the area covered by cells containing stained vacuoles. The average percentage was determined from total 3 views of each well and means plus standard deviation were derived from the average percentage of total six wells.
Chondrogenic differentiation and characterization
As previously described , following monolayer culture until the first or the sixth passage, cells were trypsinized, counted, and centrifuged into pelletted micromass cultures (5 × 106 cells/pellet) in 15 ml conical tubes (BD Biosciences). After centrifugation and culture in regular culture media for 24 hours, chondrogenesis was induced using low glucose DMEM supplemented with 10% FBS, 10 ng/ml TGF-β1, 100 ng/ml IGF (Peprotech, Rocky Hill, NJ), and 10 nM dexamethasone (Sigma). Cells were cultured in induction or control media (low glucose DMEM with 10% FBS only) in incubators with the conical tube lids loosely fastened for 3 weeks and half media were changed every other day. Four pellets (2 for induction, 2 for control) per age/passage batch were cultured, and each batch was repeated in triplicate.
Following culture, 3 pellets from each group were analyzed for type II collagen expression and glycosaminoglycan (GAG) quantification. Immunohistochemical staining was performed as previously described . Briefly, pellets were fixed with 4% paraformaldehyde for 2 h and embedded in Tissue-Tek OCT (Fisher Scientific, Pittsburgh, PA) and sliced into 10 μm thick sections. Samples were blocked and incubated overnight at 4°C in 1:100 diluted mouse anti-Collagen-II (Lab Vision, Fremont, CA) in 1% bovine serum albumin (Sigma) PBS solution. Samples were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (DAKO, Carpinteria, CA) diluted in phosphate buffered solution (PBS, 1:100) for 30 minutes at room temperature and developed with diaminobenzidine tetrachloride (DAB). Cells were counterstained with hematoxylin. Slides were also stained with Safranin O by being fixed for 10 minutes in 10% formalin in PBS, rinsed with distilled water, and then stained for 2 minutes with 6% Safranin O (Sigma) in distilled water.
Sulfated GAG production was measured from the remaining 3 pellets per group using an Alcian blue binding assay as previously described .
Osteogenic differentiation and characterization
As previously described , the first or the sixth passage cells were seeded in 2 wells each of a 6-well plate at a density of 3000 cells/cm2 as similarly performed in 2.3. Cells were maintained for 24 hours in regular culture media, after which they were cultured in low glucose DMEM supplemented with 10% FBS, 0.1 μM dexamethasone, 50 μM ascorbate-2-phosphate, and 10 mM β-glycerophosphate (all from Sigma) to induce osteogenic differentiation. The induction culture was maintained for 3 weeks with full medium changes every 3 days, whereas the control cells were cultured for 3 weeks in low glucose DMEM with 10% FBS. Experiments were performed in triplicate.
Following culture, 3 wells per group were stained with Alizarin Red to visualize calcified deposits. Wells were gently rinsed with PBS and then fixed with 70% ice-cold ethanol for 1 hour. After washing with distilled water, Alizarin Red solution (40 mM Alizarin Red-Tris-HCl, pH 4.1; Sigma) was left at room temperature in wells for 10 minutes. Wells were then extensively washed with distilled water to remove any nonspecific staining, and the stained area was quantified using ImageJ as in 2.4. For the remaining 3 wells per group, calcium cation (Ca2+) concentration was determined via a colorimetric assay (Diagnostic Chemicals, Charlottetown, PEI, Canada) as previously described .
Analyses of variance (ANOVA) were performed using SAS software (SAS Institute Inc., Cary, NC), followed by Tukey's multiple comparison tests to determine pairwise statistical significance within 95% confidence intervals (p < 0.05). All results are reported as means ± standard deviations. Due to quasi-complete separation, binary logistic regression was not performed on the data for collagen II staining of pellets.