title: Evaluating the Safety of Somatic Periosteal Cells by Flow-Cytometric Analysis Monitoring the History of DNA Damage.
abstract: In preparing cell-based products for regenerative therapy, cell quality should be strictly controlled. Methodologies for evaluating cell viability, identity, and purity are established and used routinely, whereas current methodologies for evaluating cell safety, particularly genetic integrity or tumorigenicity, are time-consuming and relatively insensitive. As part of developing a more practical screening system, the authors previously demonstrated that γ-H2AX and p53 were useful markers for evaluating the history of DNA damage. To validate these markers further and develop a more quantitative methodology, single cell-based expression of these markers and two additional candidates have now been examined using flow cytometry (FCM). FCM analysis and immunofluorescent staining demonstrated that γ-ray-irradiation suppressed proliferation, enlarged cells, and cell nuclei, and immediately upregulated γ-H2AX and p21(waf1) in large numbers of cells for up to 12 days. Gamma-H2AX foci were formed in the nuclei of many affected cells. An initial sharp increase in p53 expression declined slowly over 12 days, while Rb expression increased linearly. The present findings suggest that this high-throughput, cell-based, combinational evaluation of protein markers and cell size enables a small number of cells with a history of DNA damage to be detected quickly and routinely from within a very large cell population. Using this screening methodology will improve the ability to verify the quality of cell-based products used in regenerative therapy.
authors.affiliation: 1 Division of Oral Bioengineering, Institute of Medicine and Dentistry, Niigata University , Niigata, Japan .|2 Advanced Research Center, The Nippon Dental University School of Life Dentistry at Niigata , Niigata, Japan .
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Kawase
authors.affiliation: 3 Department of Oral and Maxillofacial Radiology, The Nippon Dental University School of Life Dentistry at Niigata , Niigata, Japan .
authors.forename: Kazuhide
authors.initials: K
authors.lastname: Hayama
authors.affiliation: 3 Department of Oral and Maxillofacial Radiology, The Nippon Dental University School of Life Dentistry at Niigata , Niigata, Japan .
authors.forename: Makoto
authors.initials: M
authors.lastname: Tsuchimochi
authors.affiliation: 4 Division of Oral and Maxillofacial Surgery, Institute of Medicine and Dentistry, Niigata University , Niigata, Japan .
authors.forename: Masaki
authors.initials: M
authors.lastname: Nagata
authors.affiliation: 5 Division of Periodontology, Institute of Medicine and Dentistry, Niigata University , Niigata, Japan .
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Okuda
authors.affiliation: 5 Division of Periodontology, Institute of Medicine and Dentistry, Niigata University , Niigata, Japan .
authors.forename: Hiromasa
authors.initials: H
authors.lastname: Yoshie
authors.affiliation: 6 Midwest Biomedical Research Foundation, Research Service, Department of Veterans Affairs Medical Center , Kansas City, Missouri.
authors.forename: Douglas M
authors.initials: DM
authors.lastname: Burns
authors.affiliation: 7 Bioscience Medical Research Center, Niigata University Medical and Dental Hospital , Niigata, Japan .
authors.forename: Koh
authors.initials: K
authors.lastname: Nakata
mesh_terms: D004249:DNA Damage; D005434:Flow Cytometry; D006801:Humans; D061214:Patient Safety; D010521:Periosteum / Q000166:cytology*