title: ROCK inhibitor Y-27632 maintains the proliferation of confluent human mesenchymal stem cells.
abstract: OBJECTIVE
  The transplantation of cell sheets of mesenchymal stem cells (MSCs) is expected to be the next generation of periodontal regenerative therapy. An adequate method of multilayering MSCs has yet to be established. When cell sheets proliferate, they usually contract and detach from culture dishes and then the proliferation of cells in the contracted areas is arrested. ROCK-mediated contractile force causes cell contraction. Although multilayer formation medium (MFM) stimulated the proliferation of growth-arrested confluent MSCs, MSCs detached from the culture dish. Therefore, we investigated the effects of ROCK inhibitor Y-27632 on the proliferation and detachment of confluent MSCs, and examined the ability of cells to differentiate within the cell sheets.
  
  
  METHODS
  Confluent MSCs were cultured in MFM containing transforming growth factor-β1, ascorbic acid and fetal bovine serum either with or without Y-27632. Cell proliferation was examined by bromodeoxyuridine incorporation assays and total DNA measurement. Sheet contractions were examined by light microscopy and stereomicroscopy. Multilayer formations and focal adhesion assembly were observed with confocal microscopy. Characteristic of cells were examined by flow cytometric analysis. Osteoblast lineage differentiation was observed with alkaline phosphatase and alizarin red S staining. Adipocyte lineage differentiation was observed with oil red O staining.
  
  
  RESULTS
  The addition of Y-27632 to MFM prevented the cell sheets from detaching and did not inhibit MSC growth. The cell numbers cultured with MFM/Y-27632 were significantly higher than that obtained with MFM-only on day 4. Cell sheets detached from the culture dish on day 4, and the number of bromodeoxyuridine-positive cells in the detached area decreased. Cells in the cell sheets had similar characteristics to primary MSCs, and differentiated into osteoblast and adipocyte lineages.
  
  
  CONCLUSIONS
  Y-27632 both prevented the MSC sheets from detaching and maintained the multilayered proliferation of confluent MSCs by MFM, and then cells in the sheets had differentiation potency.
authors.affiliation: Department of Periodontology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Nakamura
authors.forename: A
authors.initials: A
authors.lastname: Yoshimura
authors.forename: T
authors.initials: T
authors.lastname: Kaneko
authors.forename: K
authors.initials: K
authors.lastname: Sato
authors.forename: Y
authors.initials: Y
authors.lastname: Hara
mesh_terms: D017667:Adipocytes / Q000502:physiology; D000469:Alkaline Phosphatase / Q000032:analysis; D000577:Amides / Q000494:pharmacology*; D002448:Cell Adhesion / Q000187:drug effects; D002452:Cell Count; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D019070:Cell Lineage / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D048430:Cell Shape / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D022001:Focal Adhesions / Q000187:drug effects; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000187:drug effects* / Q000502:physiology; D010006:Osteoblasts / Q000502:physiology; D011725:Pyridines / Q000494:pharmacology*; D013997:Time Factors; D023822:Tissue Engineering / Q000295:instrumentation; D054457:Tissue Scaffolds; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors*
chemical_list: D000577:Amides; D004791:Enzyme Inhibitors; D011725:Pyridines; C108830:Y 27632; D054460:rho-Associated Kinases; D000469:Alkaline Phosphatase
keywords: ROCK inhibitor; cell proliferation; cell sheets; mesenchymal stem cells (MSCs)