Morphology of the isolated expanded human bone marrow mesenchymal stem cells Cell morphology was then observed, and pictures were taken under phase contrast microscope. After 2-4 days in culture, a heterogeneous population of mononuclear cells (with adherence capacity) including fibroblast-like spindle-shape cells, monocytes, macrophages, endothelial cells, and multinucleated osteoclasts attached to the surface of the dishes. As reported by others[1-5, 13], serial observation of cell shape and size showed that only mesecnhymal stem cell-derived fibroblast-like cells could be proliferated to form colony-forming unit fibroblast colonies (Figure 2). Small fibroblastoid colonies formed at 5-7 days. At the end of the incubation period (13 days), colonies were clearly formed (Figure 2)."

Multipotential of isolated human bone marrow mesenchymal stem cellsThe differentiation potential of the human bone marrow mesenchymal stem cells from three adult donors was observed by culturing the cells under conditions that favorable for adipogenic or osteogenic differentiation. After 1-3 weeks in lineage-specific culture conditions, the expanded cells were highly differentiated (the phenotype of lineage-specific cell types was easily distinguished) without evidence of the other lineages (Figure 3)."

Effects of short-time hydrostatic pressure on the differentiation of human bone marrow mesenchymal stem cells Human bone marrow mesenchymal stem cells cultured in Dulbecco’s modified Eagle’s medium and stimulated with 40 kPa pressure for 4 hours showed a significant increasing in the expression of the osteogenic marker gene of core binding factor α1 and osteocalcin, and a significant decreasing in the expressions of the adipogenic marker genes of peroxisome proliferator- activated receptor γ2 and adipsin, when compared with controls (P < 0.05). Human bone marrow mesenchymal stem cells subjected to 80 kPa pressure for 4 hours showed a decreasing in core binding factor α1 expression (P < 0.05). Except for a decreasing in adipsin expression, there were no significant differences between cells cultured in osteogeneic supplemented medium in the presence of hydrostatic pressure and control cells. When extracellular signal-regulated kinase 1/2 activity was blocked by U0126, a significant decreasing in core binding factor α1 expression was observed in cells pressurized with 40 kPa for 4 hours, although core binding factor α1 expression was still higher than that in the non-pressurized control cells. Furthermore, pressurized cells with extracellular signal-regulated kinase 1/2 inhibition showed a decreasing in osteocalcin expression, reaching levels of expression showed no statistically significant difference from that of nonpressurized control cells. In contrast, pressurized cells with extracellular signal-regulated kinase 1/2 inhibition showed a significant increasing of peroxisome proliferator-activated receptor γ2 expression and adipsin expression (Figure 4)."

Effects of long-term hydrostatic pressure on the differentiation of human bone marrow mesenchymal stem cells (Figure 5) Hydrostatic pressure of 40 kPa for 4 hours per day could promote the expression of the osteogenic marker genes core binding factor α1 and osteocalcin after 7 days. Interestingly, the same hydrostatic pressure conditions continued for up to 14 days had a very different effect on cells, it could increase the expressions of adipogenic marker genes of peroxisome proliferator-activated receptor γ2 and adipsin. There was no synergistic effect under hydrostatic pressure and osteogenic induction."

Qualitative and quantitative analysis of alkaline phosphatase after long-term hydrostatic pressure stressHydrostatic pressure or osteogenic supplemented medium alone could increase the activity and expression of alkaline phosphatase in human bone marrow mesenchymal stem cells, but this effect was not observed when two stimulus combined (Figure 6)."