Effects of medium supplements on proliferation, differentiation potential, and in vitro expansion of mesenchymal stem cells

Abstract

Mesenchymal stem cells (MSCs) possess great potential for use in regenerative medicine. However, their clinical application may be limited by the ability to expand their cell numbers in vitro while maintaining their differential potentials and stem cell properties. Thus the aim of this study was to test the effect of a range of medium supplements on MSC self-renewal and differentiation potential. Cells were cultured until confluent and subcultured continuously until reaching senescence. Medium supplementation with fibroblast growth factor (FGF)-2, platelet-derived growth factor (PDGF)-BB, ascorbic acid (AA), and epidermal growth factor (EGF) both increased proliferation rate and markedly increased number of cell doublings before reaching senescence, with a greater than 1,000-fold increase in total cell numbers for AA, FGF-2, and PDGF-BB compared with control cultures. Long-term culture was associated with loss of osteogenic/adipocytic differentiation potential, particularly with FGF-2 supplementation but also with AA, EGF, and PDGF-BB. In addition FGF-2 resulted in reduction in expression of CD146 and alkaline phosphatase, but this was partially reversible on removal of the supplement. Cells expressed surface markers including CD146, CD105, CD44, CD90, and CD71 by flow cytometry throughout, and expression of these putative stem cell markers persisted even after loss of differentiation potentials. Overall, medium supplementation with FGF-2, AA, EGF, and PDGF-BB greatly enhanced the total in vitro expansion capacity of MSC cultures, although differentiation potentials were lost prior to reaching senescence. Loss of differentiation potential was not reflected by changes in stem cell surface marker expression.

Figure 1.

Effect of supplements on mesenchymal stem cell (MSC) cumulative population doubling. Calculated cell doubling number for MSCs cultured with or without AA, EGF, FGF-2, IL-6, PDGF-BB, transferrin, and Wnt3a from passage 3 to passage 10 (mean of three experiments). Note that error bars have been omitted for clarity of reading the figure; standard deviations for these mean values are shown in tabular form in supplemental online Figure 6. Abbreviations: AA, ascorbic acid; EGF, epidermal growth factor; FGF, fibroblast growth factor; IL, interleukin; PDGF, platelet-derived growth factor.

Figure 2.

Differentiation potential of mesenchymal stem cells in long-term culture. Osteogenic and adipogenic differentiation was induced for 4 and 14 days after every second passage of cultivation. Expression of osteogenic markers Runx-2, ALP, Col-1, and OSC (A–D) and adipogenic markers PPAR-γ, CEBP-α, FAB-4, and LPL (E–H) mRNA was analyzed by quantitative reverse transcription-polymerase chain reaction. (Mean ± SEM of three experiments of duplicates.) *, p < .05, **, p < .01, ***, p < .001 compared with P4. Abbreviations: ALP, alkaline phosphatase; CEBPα, Ccaat enhancer binding protein-α; Col-1, collagen 1; OSC, osteocalcin; FAB-4, fatty acid binding protein 4; LPL, lipoprotein lipase; P, passage; PPAR, peroxisome proliferator-activated receptor.

Figure 3.

Representative photograph of mesenchymal stem cells (MSCs) differentiated to osteoblasts and adipocytes following long-term culture or cultivation with various factors. MSCs expanded in presence or absence of AA, EGF, FGF-2, IL-6, PDGF-BB, transferrin, and Wnt3a. The ability for lineage-specific differentiation was assessed for osteogenesis by staining with alizarin red for calcium deposition at P6–P10 (A), and for adipogenesis by staining with Oil Red O for lipid accumulation (magnification, ×200) (B) and with Oil Red O staining in cells expanded with various factors (at P6) and with Oil Red O staining (C) with long-term cultivation (P6–P10). Abbreviations: AA, ascorbic acid; C, control; EGF, epidermal growth factor; FGF, fibroblast growth factor; IL, interleukin; P, passage; PDGF, platelet-derived growth factor; Trans, transferrin.

Figure 4.

Osteogenic marker mRNA expression in mesenchymal stem cells expanded in presence or absence of AA, EGF, FGF-2, IL-6, PDGF-BB. (A, B): Expression of Runx-2 (A) and ALP (B) in undifferentiated cells (day 0) or following 4 and 14 days of osteogenic differentiation. (C, D): Col-1 (C) and OSC (D) expression after 14 days of osteogenesis. Data (mean ± SEM) are from three experiments in duplicate. *, p < .05, **, p < .01 compared with control. Abbreviations: AA, ascorbic acid; ALP, alkaline phosphatase; C, control; EGF, epidermal growth factor; FGF, fibroblast growth factor; IL, interleukin; P, passage; PDGF, platelet-derived growth factor.

Figure 5.

Adipogenic marker mRNA expression in mesenchymal stem cells expanded in presence or absence of AA, C, control; EGF, FGF-2, IL-6, and PDGF-BB. (A, B): Expression of PPAR-γ (A) and CEBP-α (B) in undifferentiated cells (day 0) or following 4 and 14 days of adipogenic differentiation. (C, D): FAB-4 (C) and LPL (D) expression after 14 days of adipogenesis. Data (mean ± SEM) are from three experiments in duplicate. *, p < .05, **, p < .01 compared with control. Abbreviations: AA, ascorbic acid; C, control; EGF, epidermal growth factor; FAB-4, fatty acid binding protein 4; FGF, fibroblast growth factor; IL, interleukin; LPL, lipoprotein lipase; P, passage; PDGF, platelet-derived growth factor.

Figure 6.

Expression of stemness, cell cycle, senescence, and DNA repair marker mRNA in mesenchymal stem cells (MSCs) following long-term culture or cultivation with various factors. (A–D): Expression of mRNA for stemness (Nanog, Oct-4, and Klf4) (A), cell cycle (CDK2, CDK4, cyclin D, and cyclin E) (B), DNA repair (Plod3, Ercc1, and Mre11a) (C), and senescence (P16, P21, and Rb2) (D) genes in MSCs cultivated for 10 passages. Data (mean ± SEM) are from three experiments in duplicate. *, p < .05, **, p < .01 compared with P4. (E–G): mRNA expression of Nanog, Oct-4, and Klf4 (E), CDK2, CDK4, cyclin D, and cyclin E (F), and Plod3, Ercc1, and Mre11a (G) genes in MSCs cultivated in presence of AA, EGF, FGF-2, and PDGF-BB at initial passage (P4). (H): Expression of P16, P21, and Rb2 mRNA in MSCs cultivated in presence of AA, EGF, FGF-2, and PDGF-BB at the last passage (P10). Data (mean ± SEM) are from three experiments in duplicates. *, p < .05, **, p < .01 compared with control at the same time point. Abbreviations: AA, ascorbic acid; C, control; EGF, epidermal growth factor; FGF, fibroblast growth factor; P, passage; PDGF, platelet-derived growth factor.