title: Vasculogenic conditioning of peripheral blood mononuclear cells promotes endothelial progenitor cell expansion and phenotype transition of anti-inflammatory macrophage and T lymphocyte to cells with regenerative potential.
abstract: BACKGROUND
  Cell-based therapies involving mononuclear cells (MNCs) have been developed for vascular regeneration to treat ischemic diseases; however, quality control of therapeutic MNCs has not been evaluated. We investigated the therapeutic potential of peripheral blood (PB) MNCs, operated by recently developed quality and quantity (QQ) culture of endothelial progenitor cells (EPCs).
  
  
  RESULTS
  PBs were collected from healthy volunteers; peripheral blood mononuclear cells (PBMNCs) isolated from these PBs were subjected to QQ culture for 7 days with medium containing stem cell factor, thrombopoietin, Flt-3 ligand, vascular endothelial growth factor, and interleukin-6. The resulting cells (QQMNCs) in EPC colony-forming assay generated significantly more definitive EPC colonies than PBMNCs. In flow cytometry, macrophages and helper T lymphocytes of QQMNCs became phenotypically polarized into angiogenic, anti-inflammatory, and regenerative subsets: classical M1 to alternative M2; T helper (Th)1 to Th2; angiogenic or regulatory T-cell expansion. Quantitative real-time polymerase chain reaction (qRT-PCR) assay revealed the predominant proangiogenic gene expressions in QQMNCs versus PBMNCs. Using murine ischemic hindlimb models, the efficacy of QQMNC intramuscular transplantation (Tx) was compared to that of PBMNCTx, cultured "early EPC" Tx (eEPCTx), and granulocyte colony-stimulating factor mobilized CD34(+) cell Tx (GmCD34Tx). Laser Doppler imaging revealed the blood perfusion recovery in ischemic hindlimbs after QQMNCTx superior to after PBMNCTx and eEPCTx, but also earlier than after GmCD34Tx. Histological evaluations and qRT-PCR assays in ischemic hindlimbs demonstrated that QQMNCTx, similarly to GmCD34Tx, enhanced angiovasculogenesis and myogenesis, whereas it preponderantly inhibited inflammation and fibrosis versus PBMNCTx and eEPCTx.
  
  
  CONCLUSIONS
  QQ culture potentiates the ability of PBMNCs to promote regeneration of injured tissue; considering the feasible cell preparation, QQ culture-treated PBMNCs may provide a promising therapeutic option for ischemic diseases.
  
  
  BACKGROUND
  irb.med.u-tokai.ac.jp/d/2/monthly/2010.html; IRB No.: 10R-020.URL: irb.med.u-tokai.ac.jp/d/2/monthly/201312.html; IRB No.: 13R228.
authors: Department of Regenerative Medicine Science, Tokai University School of Medicine, Isehara, Japan (H.M., T.S., A.S., T.A.).
authors: Haruchika
authors: H
authors: Masuda
authors: Department of Plastic and Reconstructive Surgery, Juntendo University School of Medicine, Tokyo, Japan (R.T., S.F.).
authors: Rica
authors: R
authors: Tanaka
authors: Department of Plastic and Reconstructive Surgery, Juntendo University School of Medicine, Tokyo, Japan (R.T., S.F.).
authors: Satoshi
authors: S
authors: Fujimura
authors: Department of Orthopedic Surgery, Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan (M.I.).
authors: Masakazu
authors: M
authors: Ishikawa
authors: Vascular Regeneration Research Group, Institute of Biomedical Research and Innovation IBRI, Kobe, Japan (H.A., A.K., T.A.).
authors: Hiroshi
authors: H
authors: Akimaru
authors: Department of Regenerative Medicine Science, Tokai University School of Medicine, Isehara, Japan (H.M., T.S., A.S., T.A.).
authors: Tomoko
authors: T
authors: Shizuno
authors: Department of Regenerative Medicine Science, Tokai University School of Medicine, Isehara, Japan (H.M., T.S., A.S., T.A.).
authors: Atsuko
authors: A
authors: Sato
authors: Teaching and Research Support Core Center, Tokai University School of Medicine, Isehara, Japan (Y.O., Y.I., J.I., Y.I., H.K.).
authors: Yoshinori
authors: Y
authors: Okada
authors: Teaching and Research Support Core Center, Tokai University School of Medicine, Isehara, Japan (Y.O., Y.I., J.I., Y.I., H.K.).
authors: Yumi
authors: Y
authors: Iida
authors: Teaching and Research Support Core Center, Tokai University School of Medicine, Isehara, Japan (Y.O., Y.I., J.I., Y.I., H.K.).
authors: Jobu
authors: J
authors: Itoh
authors: Teaching and Research Support Core Center, Tokai University School of Medicine, Isehara, Japan (Y.O., Y.I., J.I., Y.I., H.K.).
authors: Yoshiko
authors: Y
authors: Itoh
authors: Teaching and Research Support Core Center, Tokai University School of Medicine, Isehara, Japan (Y.O., Y.I., J.I., Y.I., H.K.).
authors: Hiroshi
authors: H
authors: Kamiguchi
authors: Vascular Regeneration Research Group, Institute of Biomedical Research and Innovation IBRI, Kobe, Japan (H.A., A.K., T.A.).
authors: Atsuhiko
authors: A
authors: Kawamoto
authors: Department of Regenerative Medicine Science, Tokai University School of Medicine, Isehara, Japan (H.M., T.S., A.S., T.A.) Vascular Regeneration Research Group, Institute of Biomedical Research and Innovation IBRI, Kobe, Japan (H.A., A.K., T.A.).
authors: Takayuki
authors: T
authors: Asahara
mesh_terms: D000818:Animals; D001808:Blood Vessels / Q000502:physiology; D002478:Cells, Cultured; D066026:Endothelial Progenitor Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D005434:Flow Cytometry; D006801:Humans; D015850:Interleukin-6 / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D008213:Lymphocyte Activation / Q000502:physiology; D008264:Macrophages / Q000166:cytology / Q000502:physiology*; D008297:Male; D008565:Membrane Proteins / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D018919:Neovascularization, Physiologic / Q000502:physiology; D012038:Regeneration / Q000502:physiology; D019089:Stem Cell Factor / Q000494:pharmacology; D013601:T-Lymphocytes / Q000166:cytology / Q000502:physiology*; D006377:T-Lymphocytes, Helper-Inducer / Q000166:cytology / Q000502:physiology; D013926:Thrombopoietin / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology
chemical_list: D015850:Interleukin-6; D008565:Membrane Proteins; D019089:Stem Cell Factor; D042461:Vascular Endothelial Growth Factor A; C084881:flt3 ligand protein; D013926:Thrombopoietin
keywords: anti‐inflammation; cell‐based therapy; peripheral blood mononuclear cells; serum‐free culture; vascular regeneration