title: Platelet-derived growth factor (PDGF)-AA/AB in human serum are potential indicators of the proliferative capacity of human synovial mesenchymal stem cells.
abstract: BACKGROUND
  For expansion of human mesenchymal stem cells (MSCs), autologous human serum is safer than fetal bovine serum in clinical situations. One of the problems with the use of autologous human serum is that its proliferative effect on MSCs varies widely between donors. The threefold goals of this study were: (1) to demonstrate an improved method for preparing human serum; (2) to identify growth factors predictive of proliferative potential; and (3) to identify a cytokine to promote MSC proliferation in human serum.
  
  
  METHODS
  Fresh blood was collected using a closed bag system containing glass beads. The bag was shaken at 20 °C for 30 minutes for rapid preparation, or kept stationary at 4 °C for 24 hours for slow preparation. Passage 0 synovial MSCs derived from four donors were cultured with 10 % conventional rapid preparation serum or modified slow preparation serum from four different donors. To perform the colony-forming unit assay, synovial MSCs were cultured in these serums. The protein expression profile in serum was analyzed using cytokine array. The candidate proteins were speculated from the correlation between the colony-forming ability and protein expression. As an evaluation of the candidate proteins, proliferation ability, surface marker phenotype and differentiation capability of synovial MSCs were examined.
  
  
  RESULTS
  Compared with rapid preparation serum, slow preparation serum resulted in a significantly higher total colony number and twofold higher expression levels of nine proteins (angiopoietin-1, BDNF, EGF, ENA-78, IGFBP-2, platelet-derived growth factor (PDGF)-AA, PDGF-AB/BB, RANTES and TfR). Colony number was positively correlated with PDGF-AA/AB concentrations. Exogenous PDGF-AA significantly promoted proliferation of synovial MSCs, whereas PDGF receptor (PDGFR) inhibitor decreased it. Addition of PDGFs or PDGFR inhibitor did not affect surface epitopes of synovial MSCs. Pretreatment with PDGFs or PDGFR inhibitor did not affect chondrogenic, adipogenic, or calcification potentials of synovial MSCs.
  
  
  CONCLUSIONS
  Slow preparation serum contained higher concentrations of PDGF-AA/AB and increased the colony formation number of synovial MSCs. PDGF-AA/AB were indicators of the proliferative potential of human serum. Exogenous PDGF-AA increased proliferation of synovial MSCs without alteration of surface epitopes and differentiation potentials.
authors.affiliation: Center for Stem Cell and Regenerative Medicine, Tokyo Medical and Dental University, Tokyo, Japan. mizuno.arm@tmd.ac.jp.
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Mizuno
authors.affiliation: Center for Stem Cell and Regenerative Medicine, Tokyo Medical and Dental University, Tokyo, Japan. katano.arm@tmd.ac.jp.
authors.forename: Hisako
authors.initials: H
authors.lastname: Katano
authors.affiliation: Center for Stem Cell and Regenerative Medicine, Tokyo Medical and Dental University, Tokyo, Japan. otabe.arm@tmd.ac.jp.
authors.forename: Koji
authors.initials: K
authors.lastname: Otabe
authors.affiliation: Center for Stem Cell and Regenerative Medicine, Tokyo Medical and Dental University, Tokyo, Japan. komori.arm@tmd.ac.jp.
authors.forename: Keiichiro
authors.initials: K
authors.lastname: Komori
authors.affiliation: Center for Stem Cell and Regenerative Medicine, Tokyo Medical and Dental University, Tokyo, Japan. matsumoto.arm@tmd.ac.jp.
authors.forename: Yukie
authors.initials: Y
authors.lastname: Matsumoto
authors.affiliation: Center for Stem Cell and Regenerative Medicine, Tokyo Medical and Dental University, Tokyo, Japan. fujii.arm@tmd.ac.jp.
authors.forename: Shizuka
authors.initials: S
authors.lastname: Fujii
authors.affiliation: Center for Stem Cell and Regenerative Medicine, Tokyo Medical and Dental University, Tokyo, Japan. ozeorj@tmd.ac.jp.
authors.forename: Nobutake
authors.initials: N
authors.lastname: Ozeki
authors.affiliation: Department of Cartilage Regeneration, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan. tsuji.orj@tmd.ac.jp.
authors.forename: Kunikazu
authors.initials: K
authors.lastname: Tsuji
authors.affiliation: Department of Joint Surgery and Sports Medicine, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan. koga.orj@tmd.ac.jp.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Koga
authors.affiliation: Department of Joint Surgery and Sports Medicine, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan. muneta.orj@tmd.ac.jp.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Muneta
authors.affiliation: Department of Bioresources for Drug Discovery, National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan. akifumi-matsuyama@umin.ac.jp.
authors.forename: Akifumi
authors.initials: A
authors.lastname: Matsuyama
authors.affiliation: Center for Stem Cell and Regenerative Medicine, Tokyo Medical and Dental University, Tokyo, Japan. sekiya.arm@tmd.ac.jp.
authors.forename: Ichiro
authors.initials: I
authors.lastname: Sekiya
mesh_terms: D000328:Adult; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D003470:Culture Media / Q000378:metabolism; D016207:Cytokines / Q000097:blood; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000378:metabolism*; D013583:Synovial Membrane / Q000166:cytology* / Q000378:metabolism*
chemical_list: D003470:Culture Media; D016207:Cytokines; D010982:Platelet-Derived Growth Factor; C075278:platelet-derived growth factor A; C095477:platelet-derived growth factor AB