title: Characteristic differences among osteogenic cell populations of rat bone marrow stromal cells isolated from untreated, hemolyzed or Ficoll-treated marrow.
abstract: OBJECTIVE
  Although bone marrow (BM) stromal cells (SC; BMSC) isolated from adherent cultures of untreated BM are known to contain both committed and uncommitted osteogenic cells, it remains unknown whether BMSC isolated either by hemolysis or Ficoll centrifugation also contain both of these populations.
  
  
  METHODS
  Differences in the osteogenic cell populations of rat BMSC isolated from untreated, hemolyzed or Ficoll-treated BM were analyzed by in vivo transplantation, flow cytometry, alkaline phosphatase (ALP) assay, real-time polymerase chain reaction (PCR) and alizarin red staining.
  
  
  RESULTS
  Transplantation of non-cultured samples indicated that the Ficolled BMSC contained the lowest number of committed osteogenic cells. Flow cytometric analysis of cultured, non-induced samples showed that the percentage of ALP-positive cells was significantly lower in Ficolled BMSC. Quantitative ALP assays confirmed that the lowest ALP activity was in the Ficolled BMSC. Hemolyzed BMSC also contained lower numbers of committed osteogenic cells than untreated BMSC, but still more than Ficolled BMSC. Interestingly, the Ficolled BMSC showed the greatest levels of osteogenic ability when cultured in osteogenic induction medium.
  
  
  CONCLUSIONS
  These findings suggest that, although Ficolled BMSC rarely contain committed osteogenic cells, they are able to show comparable or even greater levels of osteogenic ability after induction, possibly because they contain a greater proportion of uncommitted stem cells. In contrast, induction is optional but recommended for both untreated and hemolyzed BMSC before use, because both these groups contain both committed and uncommitted osteogenic cells. These findings are of significant importance when isolating BMSC for use in bone tissue engineering.
authors.affiliation: Tissue Engineering Research Group, Division of Molecular Therapy, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan. agata@ims.u-tokyo.ac.jp
authors.forename: Hideki
authors.initials: H
authors.lastname: Agata
authors.forename: Mika
authors.initials: M
authors.lastname: Yamazaki
authors.forename: Mariko
authors.initials: M
authors.lastname: Uehara
authors.forename: Akiko
authors.initials: A
authors.lastname: Hori
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Sumita
authors.forename: Arinobu
authors.initials: A
authors.lastname: Tojo
authors.forename: Hideaki
authors.initials: H
authors.lastname: Kagami
mesh_terms: D000469:Alkaline Phosphatase / Q000032:analysis; D000818:Animals; D001853:Bone Marrow / Q000378:metabolism; D016026:Bone Marrow Transplantation; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D005362:Ficoll / Q000494:pharmacology; D005434:Flow Cytometry; D006461:Hemolysis; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects*; D051381:Rats
chemical_list: D005362:Ficoll; D000469:Alkaline Phosphatase