title: Cell contact accelerates replicative senescence of human mesenchymal stem cells independent of telomere shortening and p53 activation: roles of Ras and oxidative stress.
abstract: 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.
authors.affiliation: Graduate Institute of Clinical Medicine, Taipei Medical University, Taipei, Taiwan.
authors.forename: Jennifer H
authors.initials: JH
authors.lastname: Ho
authors.forename: Yu-Fan
authors.initials: YF
authors.lastname: Chen
authors.forename: Wei-Hsien
authors.initials: WH
authors.lastname: Ma
authors.forename: Tzu-Ching
authors.initials: TC
authors.lastname: Tseng
authors.forename: Ming-Hsiang
authors.initials: MH
authors.lastname: Chen
authors.forename: Oscar K
authors.initials: OK
authors.lastname: Lee
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000293:Adolescent; D000328:Adult; D000975:Antioxidants / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology; D002448:Cell Adhesion; D059447:Cell Cycle Checkpoints; D002478:Cells, Cultured; D016922:Cellular Senescence; D018031:Connexin 43 / Q000378:metabolism; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism; D016193:G1 Phase; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000201:enzymology; D008875:Middle Aged; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism; D016192:Resting Phase, Cell Cycle; D059506:Telomere Shortening; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D015854:Up-Regulation; D055815:Young Adult; D018631:ras Proteins / Q000378:metabolism*
chemical_list: D000975:Antioxidants; D018031:Connexin 43; D019941:Cyclin-Dependent Kinase Inhibitor p16; D017382:Reactive Oxygen Species; D016159:Tumor Suppressor Protein p53; D000255:Adenosine Triphosphate; D018631:ras Proteins