title: Proliferation and differentiation of human adipose-derived mesenchymal stem cells (ASCs) into osteoblastic lineage are passage dependent.
abstract: OBJECTIVE
  The effect of in vitro expansion of human adipose-derived stem cells (ASCs) on stem cell properties is controversial. We examined serial subcultivation with expansion on the ability of ASCs to grow and differentiate into osteoblastic lineages.
  
  
  METHODS
  Flow cytometric analysis, growth kinetics, cell population doubling time, light microscopy and confocal analysis, and osteogenesis induction were performed to assess growth and osteogenic potential of subcultivated ASCs at passages 2 (P2), P4 and P6.
  
  
  RESULTS
  Flow cytometric analysis revealed that ASCs at P2 express classical mesenchymal stem cell markers including CD44, CD73, and CD105, but not CD14, CD19, CD34, CD45, or HLA-DR. Calcium deposition and alkaline phosphatase activity were the highest at P2 but completely abrogated at P4. Increased passage number impaired cell growth; P2 cultures exhibited exponential growth, while cells at P4 and P6 showed near linear growth with cell population doubling times increased from 3.2 at P2 to 4.8 d at P6. Morphologically, cells in various subcultivation stages showed flattened shape at low density but spindle-like structures at confluency as judged by phalloidin staining.
  
  
  CONCLUSIONS
  Osteogenic potential of ASCs is impaired by successive passaging and may not serve as a useful clinical source of osteogenic ASCs past P2.
authors.affiliation: Division of Rheumatology, Royal Victoria Hospital, McGill University Health Center, Montreal, QC, Canada.
authors.forename: Jiovanni A
authors.initials: JA
authors.lastname: Di Battista
authors.forename: Wassim
authors.initials: W
authors.lastname: Shebaby
authors.forename: Ozge
authors.initials: O
authors.lastname: Kizilay
authors.forename: Eva
authors.initials: E
authors.lastname: Hamade
authors.forename: Raghida
authors.initials: R
authors.lastname: Abou Merhi
authors.forename: Saida
authors.initials: S
authors.lastname: Mebarek
authors.forename: Dina
authors.initials: D
authors.lastname: Abdallah
authors.forename: Bassam
authors.initials: B
authors.lastname: Badran
authors.forename: Fady
authors.initials: F
authors.lastname: Saad
authors.forename: Eddie
authors.initials: E
authors.lastname: K Abdalla
authors.forename: Wissam H
authors.initials: WH
authors.lastname: Faour
mesh_terms: D000273:Adipose Tissue / Q000166:cytology*; D000328:Adult; D000368:Aged; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D008875:Middle Aged; D010006:Osteoblasts / Q000166:cytology*; D010012:Osteogenesis
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