title: Efficient derivation of osteoprogenitor cells from induced pluripotent stem cells for bone regeneration.
abstract: OBJECTIVE
  There has been great interest in the use of induced pluripotent stem cells (iPSCs) in bone regenerative strategies. To generate osteoprogenitor cells from iPSCs, the most widely used protocol relies on an intermediate using embryoid body (EB) formation. We hypothesized that an osteoprogenitor cell population could be efficiently generated from iPSCs by employing a "direct-plating method" without the EB formation step.
  
  
  METHODS
  Murine iPSC colonies were dissociated with trypsin-EDTA, and obtained single cells were cultured on gelatin-coated plates in MSC medium and FGF-2. Adherent homogeneous fibroblast-like cells obtained by this direct-plating technique were termed as direct-plated cells (DPCs). Expression levels of Oct-3/4 mRNA were analysed by real-time PCR. DPCs were evaluated for cell-surface protein expression using flow cytometry. After osteogenic induction, osteogenic differentiation ability of DPCs was evaluated.
  
  
  RESULTS
  The expression level of Oct-3/4 in DPCs was significantly down-regulated compared to that observed in iPSCs, suggesting that the cells lost pluripotency. Flow cytometry analysis revealed that DPCs exhibited cell-surface antigens similar to those of bone marrow stromal cells. Furthermore, the cells proved to have a high osteogenic differentiation capacity, which was confirmed by the significant increase in alkaline phosphatase activity, the expression levels of osteogenic genes, and calcium mineralization after 14-day osteogenic induction.
  
  
  CONCLUSIONS
  These findings indicate that our novel direct-plating method provides a clinically applicable, simple, and labour-efficient system for generating large numbers of homogeneous iPSC-derived osteoprogenitor cells for bone regeneration.
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Japan, 650-0017.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Dogaki
authors.forename: Sang Yang
authors.initials: SY
authors.lastname: Lee
authors.forename: Takahiro
authors.initials: T
authors.lastname: Niikura
authors.forename: Takashi
authors.initials: T
authors.lastname: Iwakura
authors.forename: Etsuko
authors.initials: E
authors.lastname: Okumachi
authors.forename: Takahiro
authors.initials: T
authors.lastname: Waki
authors.forename: Kenichiro
authors.initials: K
authors.lastname: Kakutani
authors.forename: Kotaro
authors.initials: K
authors.lastname: Nishida
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Kuroda
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kurosaka
mesh_terms: D000818:Animals; D001861:Bone Regeneration / Q000502:physiology*; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D058732:Embryoid Bodies / Q000166:cytology; D000939:Epitopes; D005434:Flow Cytometry; D066298:In Vitro Techniques; D051379:Mice; D010012:Osteogenesis / Q000502:physiology*; D039904:Pluripotent Stem Cells / Q000166:cytology*; D016133:Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology*
chemical_list: D000939:Epitopes