Prolonged Induction of Human Mesenchymal Stem Cells Does Not Sustain the Osteogenic Phenotype

Prior to transplantation, mesenchymal stromal/stem cells (MSC) can be osteogenically induced toward the osteoblast phenotype in vitro using a cocktail of osteogenic supplements. However, there is little evidence of induced MSC directly participating in bone formation in vivo, suggesting that MSC may return to an undifferentiated phenotype upon removal of the osteogenic stimulus. As MSC are commonly induced for only a few days in culture, the induction period may be insufficient to sustain the osteoblast phenotype. We hypothesized that increasing the duration of MSC osteoinduction would preserve the osteoblast phenotype after removal of the osteogenic stimulus. MSC derived from three male donors (20-30yrs old) were expanded under standard conditions: α-MEM supplemented with 10% FBS and 1% penicillin (10,000 U/mL) and streptomycin (10 mg/mL). Osteogenic media consisted of growth media supplemented with 50 ¬µg/mL ascorbic acid, 10 mM β-glycerophosphate, and 10

nM dexamethasone. MSC were seeded at 30,000 cells/cm² and cultured in growth media or osteogenic media for 2, 4, and 6 weeks. After each duration, induction was removed from osteogenically induced MSC, which were subsequently cultured in growth media for an additional 5 days (OM/GM). As a positive control, osteogenically induced MSC remained in osteogenic media (OM). MSC cultured in growth media for the full duration (GM) served as the negative control. Osteogenic markers were assessed at 0 (baseline), +1, and +5 days where 0d marks the day OM/GM was switched from osteogenic media to growth media. After 24h following induction removal, OM/GM exhibited a regression in both normalized ALP activity and calcium deposition compared to OM regardless of induction duration: 2 (Fig. 1A), 4 (Fig. 1B), and 6 weeks (Fig. 1C).  The trend in calcium deposition for 6 week regression extended across three donors, wherein the bars span data points and the line signifies the mean (Fig. 1D). After 2 week differentiation, OM/GM decreased to a calcium deposition more similar to that of GM at +5d (Fig. 1A).  Significant differences in normalized ALP activity and calcium deposition between OM/GM and OM were not attributed to significant differences in DNA. In 24h, induction withdrawal from 4 week differentiated MSC (OM/GM) diminished bone sialoprotein (IBSP) expression, thereby demonstrating the implications of induction withdrawal extend to the transcriptional level. Osteogenically induced MSC retained their ability to differentiate toward adipocytes despite mineral production and morphological transition from spindle to cuboidal-shaped. MSC induced in osteogenic media exhibited enhanced colony-forming efficiency (CFE) compared to MSC in growth media. Upon removal of the osteogenic stimulus, MSC secreted similar or greater concentrations of vascular endothelial growth factor (VEGF) compared to cells maintained in growth or osteogenic media for the entire culture period. These data demonstrate osteoinduction withdrawal abrogates the potency of the acquired osteogenic phenotype regardless of induction duration. Our findings underscore the clinical requirement to strike a balance between maximizing the therapeutic potential of MSC while minimizing costly, time-consuming, and potentially deleterious ex vivo manipulation. These data highlight the importance of understanding the merit of 2D soluble cue differentiation for bone tissue engineering applications.