CD146/MCAM Surface Marker for Identifying Human Periodontal Ligament-derived Mesenchymal Stem Cells

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  • Saito Yoko
    Nihon University Graduate School of Dentistry
  • Watanabe Eri
    Laboratory of Diagnostic Medicine, The Institute of Medical Science, The University of Tokyo
  • Mayahara Kotoe
    Department of Orthodontics, Nihon University School of Dentistry Dental Research Center, Nihon University School of Dentistry
  • Watanabe Nobukazu
    Laboratory of Diagnostic Medicine, The Institute of Medical Science, The University of Tokyo
  • Morokuma Masakazu
    Nihon University Graduate School of Dentistry
  • Isokawa Keitaro
    Dental Research Center, Nihon University School of Dentistry Department of Anatomy, Nihon University School of Dentistry
  • Shimizu Noriyoshi
    Department of Orthodontics, Nihon University School of Dentistry Dental Research Center, Nihon University School of Dentistry
  • Masaki J. Honda
    Dental Research Center, Nihon University School of Dentistry Department of Anatomy, Nihon University School of Dentistry

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Abstract

Identification of the specific subset of periodontal ligament-derived mesenchymal stem cells (PDLSCs) will permit the development and improvement of current periodontal regeneration therapy. A popular approach for the isolation of a subset of PDLSCs would be to use cell surface markers to identify these cells, which are effectively enriched in order to isolate stem cells. The CD146 marker is most commonly used to isolate PDLSCs from human periodontal ligament tissues (hPDL). Previous studies have shown that CD146-positive (CD146+) cells possess potencies of high self-renewal and multi-lineage differentiation. However, the capability and potency of mesenchymal stem cells (MSCs) in hPDL-derived CD146-negative (CD146-) cells have not been well established.<br>In this study, CD146+ and CD146- cells were isolated from hPDL, and their properties, including their MSCs potential, were characterized. hPDL was obtained from healthy premolars (n = 10) extracted for orthodontic reasons. Flow cytometry analysis revealed that the average proportion of CD146+ cells was about 50%. An approximately 20% fraction of cells with the highest CD146 expression was sorted as CD146+ cells and an approximately 20% fraction with the lowest CD146 expression was sorted as CD146- cells using fluorescence-activated cell sorting. Cell cultures were assessed for their colony-forming efficiency, proliferation and differentiation into osteoblasts, adipocytes and chondrocytes. Approximately 20% of CD146+ cells had replicative potential and formed single-cell colonies. The colony-forming efficiency of CD146+ cells was approximately twofold higher than for CD146- cells. Cell proliferation measured by cell-cycle analysis and cell counting showed that the proliferative potential of CD146+ cells was higher than that of CD146- cells. Cell differentiation potential in vitro was determined by real-time PCR and cell staining with alkaline phosphatase and Alizarin Red S for osteogenic differentiation, Oil Red O for adipogenic differentiation, and Alcian Blue for chondrogenic differentiation. The levels of osteogenic and adipogenic differentiation were significantly higher in CD146+ cells than in CD146- cells under appropriate conditions. The level of glycosaminoglycan and gene expression of cartilage oligomeric matrix protein in CD146- cell pellets were higher than in CD146+ cell pellets. These results suggest that CD146+ cells possess bi-potent differentiation potential whereas CD146- cells possess unipotent differentiation potential.

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