Mesenchymal stem cells (MSCs) are of great therapeutic potentials due to their multilineage differentiation capabilities. Before transplantation, in vitro culture expansion of MSCs is necessary to get desired cell number. We observed that cell contact accelerated replicative senescence during such process. To confirm the finding as well as to investigate the underlying mechanisms, we cultured both human bone marrow-and umbilical cord blood-derived MSCs under noncontact culture (subculture performed at 60-70% of confluence), or contact culture (cell passage performed at 100% of confluence). It was found that MSCs reached cellular senescence earlier in contact culture, and the doubling time was significantly prolonged. Marked increase of senescence-associated β-galactosidase-positive staining was also observed as a result of cell contact. Cell cycle analysis revealed increased frequency of cell cycle arrest after contact culture. It was noted, however, that the telomere length was not altered during contact-induced acceleration of senescence. Moreover, cell cycle checkpoint regulator P53 expression was not affected by cell contact. Marked increase in intracellular reactive oxygen species (ROS) and a concomitant decrease in the activities of antioxidative enzymes were also observed during contact-induced senescence. Importantly, increased p16 INK4a following Ras upregulation was found after contact culture. Taken together, cell contact induced accelerated senescence of MSCs, which is telomere shortening and p53 independent. ROS accumulation due to defective ROS clearance function together with Ras and p16 INK4a upregulation play an important role in contact-induced senescence of MSCs. Overconfluence should therefore be avoided during in vitro culture expansion of MSCs in order to maintain their qualities for clinical application purposes. The contact-induced senescence model reported in this study will serve as a useful model system that allows further study of the molecular mechanisms of senescence in MSCs.

Polyhydroxyalkanoates (PHAs), intracellular carbon and energy reserve compounds in many bacteria, have been used extensively in biodegradable plastics. PHA formation is influenced by nutrient limitations and growth conditions. To characterize the PHA accumulation in a new denitrifying phosphorusremoving bacterium Brachymonas sp. P12, batch experiments were conducted in which the electron acceptor (oxygen or nitrate) was varied and different concentrations of carbon (acetate), nitrogen (NH 4 Cl), and phosphorus (KH 2 PO 4 ) were used. Polyhydroxybutyrate (PHB) was the dominant product during PHA formation when acetate was the sole carbon source. The PHB content of aerobically growing cells increased from 431 to 636 mg PHB g -1 biomass, but the PHB concentration of an anoxic culture decreased (-218 mg PHB g -1 biomass), when PHB was utilized simultaneously with acetate as an electron donor for anoxic denitrification. The specific PHB production rate of the carbon-limited batch, 158.2 mg PHB g -1 biomass h -1 , was much greater than that of batches with normal or excess carbon. The effects of phosphorus and nitrogen concentrations on PHB accumulation were clearly less than the effect of carbon concentration. According to the correlation between the specific PHB production rate and the specific cell growth rate, PHB accumulation by Brachymonas sp. P12 is enhanced by nutrient limitation, is growth-associated, and provides additional energy for the biosynthesis of non-PHB cell constituents to increase the cell growth rate beyond the usual level.

Change of actin filament organization at the early stage of cell differentiation directs cell fate commitment of mesenchymal stem cells (MSCs). Thymosin beta-4 (Tb 4 ), a major G-actin sequestering peptide, is known to regulate the cytoskeleton. The study investigated the ways in which Tb 4 regulates cell fate determination in MSCs upon differentiation induction. It was found that Tb 4 decreased F-actin formation, reduced the F-actin/G-actin ratio, and inhibited osteogenic differentiation; such actin reorganization was not associated with the change of Runt-related transcription factor 2 gene expression during early osteogenic induction. Besides, Tb 4 reciprocally facilitated adipogenic differentiation. Tb 4 treatment was found to up-regulate gene as well as promote surface expression of adipocyte adhesion molecule during early adipogenic differentiation, which accompanied acceleration of adipocyte phenotypic maturation but was not associated with differential expression of peroxisome proliferator-activated receptor gamma during the first week of adipogenic induction. In summary, Tb 4 initiated cell fate determination of MSCs through biophysical effects exerted by cytoskeleton reorganization and altered cellcell adhesion rather than direct regulation of lineage-determining transcriptional factors. Such findings suggest that Tb 4, a ubiquitous peptide, may be involved in osteoporosis when its intracellular concentration is elevated. Further investigation of targeting Tb 4 for future osteoporosis treatment is warranted. ß