Effects of Medium Supplements on Proliferation, Differentiation Potential, and In Vitro Expansion of Mesenchymal Stem Cells

Experimental Overview

MSC cultures were grown in culture medium with test supplements to confluence, at which time the increase in cell number was calculated by cell counting. An aliquot of these cells was replated into test medium again and grown to confluence as before. This was repeated until cells reached senescence. At the end of every second passage, a further aliquot of the cells was taken for flow cytometric analysis of expression of MSC-related cell surface markers, together with two aliquots which were transferred to osteogenic or adipogenic media and cultured for up 14 days to test for differentiation potentials. Osteogenic and adipogenic differentiation of MSCs was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for expression of phenotypic marker genes and for production of mineralization or lipid-containing adipocytes in cultures.

Cell Culture

Primary human MSCs from young male and female adults were purchased from Lonza (Slough, U.K., http://www.lonza.com) (a total of three different cell lines). Cells already at passage 2 when purchased were expanded in culture (this was considered passage 3) in normal growth medium consisting of α-Minimal Essential Medium, penicillin (50 U/ml), streptomycin (50 μg/ml) (all from Sigma-Aldrich, Poole, Dorset, U.K., http://www.sigmaaldrich.com), Glutamax (2 mM) (Invitrogen, Paisley, U.K., http://www.invitrogen.com), and 10% fetal bovine serum (Sigma-Aldrich) and maintained at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. Upon reaching confluence cells were seeded at a density of 2,000 cells per cm² in T-75 flasks and grown in normal medium alone or medium supplemented with 0.2 mM AA (Sigma-Aldrich), 10 ng/ml EGF (Invitrogen), 10 ng/ml FGF-2 (Peprotech, London, U.K., http://www.peprotech.com), 10 ng/ml IL-6 (Invitrogen and Peprotech), 10 ng/ml PDGF-BB (Peprotech), 10 μg/ml transferrin (Sigma-Aldrich), or 50 ng/ml Wnt3a (50 ng/ml) (R&D Systems Inc., Minneapolis, MN, http://www.rndsystems.com) and kept in continuous culture until cells reached senescence. Cell number was counted at each passage using a Z2 Coulter counter (Beckman Coulter, High Wycombe, U.K., http://www.beckmancoulter.com) and subcultured for further expansion at a density of 2,000 cells per cm². After every second passage cells were also analyzed for proliferation (CellTiter 96 AQ_ueous One Solution Cell Proliferation assay; Promega, Southampton, U.K., http://www.promega.com), cell surface marker and gene expression, and differentiation potential.

Differentiation Potential

MSCs were seeded at a density of 5,000 cells per cm² in 12-well plates (Nunc, Fisher Scientific, Loughborough, U.K., http://www.fisherscientific.com). After 24 hours of incubation osteogenic differentiation was induced by replacing the medium with the osteogenic medium (growth medium supplemented with 0.1 μM dexamethasone, 0.05 mM AA, and 10 mM glycerophosphate, all from Sigma-Aldrich). To assess osteogenic differentiation, RNA was isolated after 4 and 14 days of incubation, mRNA expression of markers of differentiation (i.e., Runx-2, alkaline phosphatase [ALP], collagen 1 [Col-1], and osteocalcin [OSC]) was determined by qRT-PCR, and accumulation of calcium deposits was visualized by staining with alizarin red dye. Briefly, cells were fixed (15 minutes with 4% formaldehyde in phosphate-buffered saline [PBS]), stained for 10 minutes with alizarin red S (1:100 dilution in H₂O), and washed (five times) in 50% ethanol and air-dried.

To induce adipogenic differentiation cells were seeded at a density of 40,000 cells per cm² in 12-well plates (Nunc) and incubated for 24 hours before switching to adipogenic medium (growth medium supplemented with 1 μM dexamethasone, 0.25 mM isobutylmethylxanthine, 50 μM indomethacin, and 10 μg/ml insulin, all from Sigma-Aldrich). Adipogenesis was assessed by qRT-PCR analysis of markers of differentiation, that is, peroxisome proliferator-activated receptor (PPAR)-γ, Ccaat enhancer binding protein-α (CEBP-α), fatty acid binding protein 4 (FAB-4), and lipoprotein lipase (LPL), and visualized by light microscopy alone or following staining with Oil Red O dye as described previously [24]. Briefly, cells were fixed (15 minutes with 4% formaldehyde in PBS), stained for 15 minutes, and washed with 60% isopropanol and with PBS.

qRT-PCR Analysis

Total RNA was extracted using TRI reagent (Ambion, Warrington, U.K., http://www.ambion.com) and Phase Lock Gel Heavy tubes (five prime; VWR, Lutterworth, U.K., https://us.vwr.com) according to the manufacturers' instructions. RNA purity and quantity were assessed by Nanodrop (Fisher Scientific) (A₂₆₀/A₂₈₀ 1.8–2 was considered suitable for further analysis), possible contaminating DNA was removed, and cDNA was prepared from 1 μg of RNA using QuantiTect Reverse Transcription Kit (Qiagen, Crawley, U.K., http://www.qiagen.com) according to the manufacturer's instructions. qRT-PCR was performed on a Rotor-Gene 6000 thermal cycler (Qiagen) using Brilliant III Ultra-Fast SYBR Green qPCR Master Mix (Stratagene, Agilent Technologies, Stockport, U.K., http://www.stratagene.com) and primer pairs as listed in supplemental online Figure 1. PCR conditions consisted of 1 cycle of 95°C for 3 minutes and 40 cycles of 95°C for 10 seconds and 60°C for 10 seconds followed by melting analysis of 1 cycle with gradual increase from 65°C to 95°C. RPL13a was used as an invariant housekeeping gene. The quantitative expression of gene of interest relative to the housekeeping gene was calculated, and this ratio for basal or untreated cells was assigned a value of 1. Amplification of the right product was confirmed by monitoring the dissociation/melt curve (at the end of the PCR, samples were subjected to stepwise increase in temperature from 60°C to 95°C, with fluorescence measurements taken throughout this range). Template and reverse transcription negative controls were also included in all amplification experiments.

Flow Cytometry

MSC surface marker expression was assessed using four-color flow cytometry analysis, with the FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com). Briefly, 150,000 cells were incubated with Fc block (BioLegend, Cambridge, U.K., http://www.biolegend.com) in 50 μl of fluorescence-activated cell sorting (FACS) buffer for 15 minutes at room temperature. Direct staining was performed by adding an antibody cocktail of the following: A, CD34-PerCP/Cy5.5 (clone 4H11), CD105-fluorescein isothiocyanate (FITC) (clone 43A3), CD146-phycoerythrin (PE)/Cy7 (clone SHM-57), and TNAP-PE (clone W8B2); or B, CD45-PerCP/Cy5.5 (clone HI30), CD44-FITC (clone BJ18), CD90-allophycocyanin (APC) (clone 5E10), and CD71 (clone A015). Appropriate isotype controls were also included. All antibodies and isotype controls were obtained from BioLegend U.K. (Cambridge Bioscience) and used at the recommended concentration. Cells were incubated for 15 minutes at room temperature in the dark. After being washed twice, cells were resuspended in 300 μl of FACS buffer and immediately analyzed. A total of 10,000 events were acquired for each sample, and data analysis was performed using DIVA software (BD Biosciences).

Data Analysis

Statistical comparisons between means were made by one-way analysis of variance (SPSS 17; SPSS, Chicago, IL, http://www.spss.com) and post hoc analyses using the Tukey test to evaluate the differences among the mean values between groups. If comparisons were made only between two groups Student's t test (SPSS 16; SPSS) was used. A p value of less than .05 was considered statistically significant.

The number of cell doublings at each passage was calculated using the formula Cell doublings = Log₂ (Final cell number/Initial cell number), and the total number of cell doublings was calculated by the sum of doublings for each passage.

MSC Expansion

To compare the stimulatory effect of various cytokines on the total proliferation capacity of MSCs in long-term culture, bone marrow-derived MSCs were grown in parallel cultures with or without AA, EGF, FGF-2, IL-6, PDGF-BB, transferrin, and Wnt3a on three separate occasions. Cells were grown until the fastest cultures reached confluence, which typically took approximately 13–14 days, and then all cultures were passaged. An increased proliferation rate was observed in cells expanded in the presence of FGF-2, AA, PDGF-BB, EGF, and Wnt3a with population doubling times of 2.5, 2.7, 2.8, 3.05, and 4.2 days, respectively, compared with 8.1, 7.8, and 7.6 days for cells expanded in normal growth medium, IL-6, or transferrin at the initial passage. Microscopic images of MSCs expanded in the presence of each factor are shown in supplemental online Figure 2. The proliferation was maintained at approximately the same rate for the first four passages but gradually slowed thereafter and eventually leveled off at the last passage when cells reached senescence. The rate of MSC population doubling for each factor is shown in Figure 1. The total cell doublings prior to senescence were estimated as up to 25.5, 21.1, 20.2, 15.6, and 16.4 in presence of FGF-2, PDGF-BB, AA, EGF, and Wnt3a, respectively, in comparison with 10.0 for MSCs cultured in normal medium alone. IL-6 or transferrin supplementation had no significant effect on cell doubling time or the total number of population doublings. Thus, supplementation with AA, FGF-2, and PDGF-BB resulted overall in a greater than 1,000-fold increase in total cell numbers compared with controls (for controls 2^10.05 = 895 cells; AA, 2^20.27 = 1,267,893 cells; FGF-2, 2^25.59 = 50,542,945 cells). Furthermore, an even greater proliferation rate was observed when a combination of AA, FGF-2, and PDGF-BB was added to the culture. In particular, the combination of FGF-2 with either AA or PDGF-BB or the combination of the three factors together resulted in, respectively, one and two more cell doublings compared with each factor alone after a couple of passages (supplemental online Fig. 3). Cells cultured in the presence of FGF-2, AA, and PDGF-BB also showed enhanced proliferative capacity even when subcultured in normal growth medium for 1 week, with up to 1.5-, 1.7-, and 1.4 (p < .01)-fold increase in comparison with those subcultured from normal medium (supplemental online Fig. 4).

MSC Surface Phenotype Expression

To determine whether MSC surface marker expression alters with long-term culture or in the presence of cytokine supplements, we carried out four-color flow cytometry analysis of panels of known negative and positive markers of MSCs. As shown in Table 1, MSCs were 90%–100% positive for CD105, CD90, CD44, CD71, and CD146 but lacked the HSC marker (0%–5%) CD34 and CD45 expression. Among the positive markers CD90 and CD44 expression remained more or less constant (95%–100% positive), and both negative markers stayed negative throughout the duration of culture and with all culture conditions. Expression of ALP was elevated from 29.8% at first to 39.3% at the third passage of culture in normal medium but returned back to approximately 30% thereafter. Long-term culture was associated with slight reduction in expression of CD105, CD146, and CD71 cells (from, respectively, 95%, 92%, and 76% in initial culture to 75%, 78%, and 60% at the final passage), although this was not statistically significant. MSCs cultured with FGF-2 showed significant reduction in cells positive for CD146 at every passage (40.9%, 52%, 65%, and 61% in comparison with 92%, 91%, 91%, and 78% in control at passages 4, 6, 8, and 10) examined. AA, EGF, and PDGF-BB cultures also showed a lower level of CD146 positive cells in comparison with control. ALP expression was significantly lower in cells grown in FGF-2 (by less than half in passages 4 and 6), whereas it was enhanced in cells cultured with AA, EGF, and PDGF-BB (49%, 52%, and 40%, respectively, in comparison with 30% in control at passage 4). The reduction seen in CD146 expression following FGF-2 treatment was found to be reversible, and the expression level was restored to control levels following a week of incubation in the absence of FGF-2. ALP expression was also partly restored but still remained lower when compared with cells grown entirely in normal growth medium (supplemental online Fig. 5.1).

We also investigated the effect of differentiation on cell surface marker expression. Neither osteogenesis nor adipogenesis altered the expression of negative markers (CD34 and CD45) or positive markers CD44, CD71, CD90, and CD146 following a week of differentiation (supplemental online Fig. 5.2). Only CD105 was reduced significantly following both osteogenic and adipogenic differentiation. As expected the expression of ALP was significantly increased following osteogenesis, but it also increased upon adipogenic differentiation (supplemental online Fig. 5.2).

MSC Differentiation Potential

Next, we tested whether the long-term culture or incubation with cytokine and growth factors was associated with changes in MSC multipotential capacity. MSCs grown in presence of different factors or normal growth medium for more than 100 days of culture (eight passages) were induced to differentiate to osteoblasts or adipocytes at every second passage, and changes in markers of osteogenesis or adipogenesis were analyzed by qRT-PCR and phenotype changes by alizarin red and Oil Red O staining. MSCs were capable of differentiating to osteoblasts and adipocytes, as exhibited by increase in expression of marker genes and accumulation of calcium deposits and lipid droplets by the cells (Figs. 2, 3). The maximal differentiation capacity was observed at initial passage and was significantly reduced at late passages. MSCs cultured in osteogenic medium showed 5- and 10-fold increases in expression of Runx-2 and ALP mRNA at initial passage, but the level was reduced to as low as 2.5 and 1.5 (p < .01), respectively, in subsequent passages (Fig. 2A, 2B). We also observed approximately 60% and 80% (p < .01) reduction in expression of Col-1 and OSC mRNA with cultivation time (Fig. 2C, 2D). Furthermore, the deposition of calcium induced by osteogenic medium was significantly reduced by increase in culture time and at the last passage (passage 10 [P10]) was almost absent (Fig. 3A). The time taken for cells to undergo matrix mineralization also gradually increased from approximately 21 days at the initial passage to up to 40 days at late passages, and even complete loss of mineralization capacity was observed for some cultures (Fig. 3A). Similarly and even more strikingly, the adipogenic differentiation potential of MSCs was reduced by time in culture. As shown in Figure 2E and 2F the expression of PPAR-γ and CEBP-α was induced in presence of adipogenic medium to approximately 190- and 770-fold at the initial passage, but it significantly decreased (p < .01) with prolonged cultivation to 30- and 36-fold for PPAR-γ, 458- and 50-fold for CEBP-α at subsequent passages. Cultivation was also associated with reduced expression of FAB-4 and LPL by more than 75% and 85% (p < .01), respectively (Fig. 2G, 2H). Accordingly, the ability of cells to accumulate lipid following differentiation was reduced gradually and eventually completely inhibited at last passage as shown microscopically after staining with Oil Red O (Fig. 3C).

Parallel analysis was also carried out on the osteogenic and adipogenic potential of cells cultured with various supplements that induce proliferation of MSCs. Except with IL-6 and transferrin, which had little or no effect on either osteogenesis or adipogenesis (data not shown), the effects were vastly diverse between other cytokines, although reduced differentiation was apparent with all factors at the last passage. Cells grown in cultures supplemented with FGF-2 showed an increase in expression of Runx-2 but a reduction in ALP (Fig. 4A, 4B). These cells when induced to differentiate to osteoblasts also showed a reduction in ALP level, although no significant change was observed with other osteogenic genes. Supplementation of medium with AA resulted in a significant increase in Runx-2 and ALP expression under basal conditions, but a reduction in expression of Runx-2 was observed following osteogenic differentiation upon long incubation of cells with AA. EGF also induced the expression of ALP but had no significant effect on osteogenic potential following treatment of cell with differentiation medium. Supplementation of cultures with Wnt3a reduced the osteogenic potential of MSCs, with reduction in expression of Runx-2 and ALP. PDGF-BB had little effect on osteogenic potential at early passages (Fig. 4A, 4B). Despite the effect on mRNA expression of osteogenic genes by AA, EGF, FGF-2, PDGF-BB, and Wnt3a, supplementation of these factors over six passages completely inhibited the ability of cell to undergo mineralization, as shown by reduced alizarin staining of calcium deposits (Fig. 3A).

Analysis of adipogenic genes showed that supplementation of culture with FGF-2 or PDGF-BB enhanced (p < .01) the expression of “master switch ” gene PPAR-γ but not CEBP-α or FAB-4 expression under basal conditions (Fig. 5A–5D). Surprisingly, FGF-2-treated and, to a lesser extent, PDGF-BB-treated cells showed a reduction in adipogenic gene expression during the differentiation process, and phenotypic reduction in lipid containing cells was more apparent in cells grown with FGF-2 (Fig. 3B). Addition of AA in culture also significantly reduced (p < .01) the expression of genes involved in adipogenesis both under basal conditions and during the differentiation, as well as reducing the number of lipid-filled adipocytes (Figs. 3B, 5). Similar to the reduction seen with osteogenic genes and mineralization level, the potential for adipogenesis was reduced at the last passage for the cells grown in AA, EGF, FGF-2, PDGF-BB, and Wnt3a as shown by decreased gene expression and lack of lipid accumulation (Fig. 3B).

MSC Stemness, Cell Cycle, Senescence, and DNA Repair Gene Expression

Time in culture (number of cell doublings) or supplementation with cytokines was shown to alter the proliferative rate, self-renewal ability, and differentiation capacity of MSCs. Therefore, we investigated the expression of genes involved in maintenance of stem cell phenotype, cell cycle, senescence, and DNA repair. Human MSCs expressed “stemness” genes Nanog, Oct-4, and Klf2, but Sox-2 and TRET genes were barely detectable. Consistent with the alteration in MSC stem cell properties (i.e., differentiation potential) there was a significant reduction in expression of Nanog, Oct-4, or Klf4 with long cultivation time (Fig. 6A). Supplementation with FGF-2 and AA, however, resulted in enhanced expression (p < .01) of Oct-4 and Klf4 (Fig. 6E) only at the initial passage. Cells grown in the presence of AA also expressed more CDK2 mRNA, and CDK4 expression was higher in cultures treated with FGF-2 and EGF (p < .01). No significant change was observed in expression of cyclin D or cyclin E in the presence of growth factors. Long-term culture, however, resulted in reduction in expression of cell cycle-related genes, that is, CDK2, CDK4, cyclin D, and cyclin E (Fig. 6B) and increased expression of senescence gene P16 (p < .01) and was even further induced in the presence of FGF-2, PDGF-BB, and AA only at the last passage. Expression of other senescence-related genes, such as P21 and Rb2, remained unchanged during cultivation but was induced in the presence of AA at the last passage. Similarly, the DNA repair genes Pold3, Ercc1, and Mre11a were unaffected by culture time, although higher expression of Pold3 was observed in cells cultured with AA, EGF, FGF-2, and PDGF-BB, and Ercc1 and Mre11a expression was higher in cells treated with AA (Fig. 6G).