title: Hematopoietic stem cells expanded by fibroblast growth factor-1 are excellent targets for retrovirus-mediated gene delivery.
abstract: OBJECTIVE
  For the study of the function of genes of interest in hematopoietic stem cells (HSCs) and for successful gene therapy, it is fundamental to develop a method of efficient gene transfer into HSCs. In mice experiments, efforts have been made to raise the transduction efficiency by modifying the vectors, administrating 5-fluorouracil (5-FU) to donor mice, selecting cytokine cocktails to better sustain the long-term repopulating potential of the stem cells, and so on. The objective of this study is to examine whether the use of fibroblast growth factor-1 (FGF-1)-expanded bone marrow cells provide an improved source for retroviral gene delivery to HSCs.
  
  
  METHODS
  Unfractionated bone marrow cells from one mouse were cultured in serum-free medium containing FGF-1. Both floating and attached cells were transferred to retronectin precoated dishes and infected with virus supernatant from MP34 cells stably transduced with pMY/GFP retrovirus. After 3-day infection, the green fluorescence protein-positive fraction was sorted and the cells were transplanted to lethally irradiated mice.
  
  
  RESULTS
  The experiments illustrated that the number of bone marrow-derived competitive repopulation units (CRUs) was increased from 600 to 9300 per mouse after a 3-week culture period with FGF-1. Following retroviral transduction of the expanded cells, the absolute number of sorted retrovirus-transduced CRUs was 4200. Using these retrovirus-transduced cells in noncompetitive reconstitution assay, we achieved radiation protection and long-term bone marrow reconstitution in 100% of the recipients with average myeloid and lymphoid chimerisms of 70% and 50%, respectively, even if we transplanted 150 recipients with cells derived from a single donor mouse.
  
  
  CONCLUSIONS
  In conclusion, FGF-1-expanded bone marrow cells constitute an excellent source of stem cells that could be used in a range of gene delivery protocols.
authors.affiliation: Department of Hematology/Oncology, University of Tokyo, Japan. schiba-tky@umin.ac.jp
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Crcareva
authors.forename: Toshiki
authors.initials: T
authors.lastname: Saito
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kunisato
authors.forename: Keiki
authors.initials: K
authors.lastname: Kumano
authors.forename: Takahiro
authors.initials: T
authors.lastname: Suzuki
authors.forename: Mamiko
authors.initials: M
authors.lastname: Sakata-Yanagimoto
authors.forename: Masahito
authors.initials: M
authors.lastname: Kawazu
authors.forename: Aleksandar
authors.initials: A
authors.lastname: Stojanovic
authors.forename: Mineo
authors.initials: M
authors.lastname: Kurokawa
authors.forename: Seishi
authors.initials: S
authors.lastname: Ogawa
authors.forename: Hisamaru
authors.initials: H
authors.lastname: Hirai
authors.forename: Shigeru
authors.initials: S
authors.lastname: Chiba
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D018929:Cell Culture Techniques; D019070:Cell Lineage; D049109:Cell Proliferation / Q000187:drug effects; D016220:Fibroblast Growth Factor 1 / Q000494:pharmacology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D051379:Mice; D008810:Mice, Inbred C57BL; D012190:Retroviridae / Q000235:genetics*; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: D016220:Fibroblast Growth Factor 1; D049452:Green Fluorescent Proteins