title: Therapeutic benefits in thalassemic mice transplanted with long-term-cultured bone marrow cells.
abstract: OBJECTIVE
  Autologous bone marrow (BM) cells with a faulty gene corrected by gene targeting could provide a powerful therapeutic option for patients with genetic blood diseases. Achieving this goal is hindered by the low abundance of therapeutically useful BM cells and the difficulty maintaining them in tissue culture long enough to complete gene targeting without differentiating. Our objective was to devise a simple long-term culture system, using unfractioned BM cells, that maintains and expands therapeutically useful cells for ≥4 weeks.
  
  
  METHODS
  From 2 to 60 million BM cells from wild-type (WT) mice or from mice carrying a truncated erythropoietin receptor transgene were plated with or without irradiated fetal-liver-derived AFT024 stromal cells in 25-cm(2) culture flasks. Four-week-cultured cells were analyzed and transplanted into sublethally irradiated thalassemic mice (1 million cells/mouse).
  
  
  RESULTS
  After 4 weeks, cultures with AFT024 cells had extensive "cobblestone" areas. Optimum expansion of Sca-1-positive cells was 5.5-fold with 20 × 10(6) WT cells/flask and 27-fold with 2 × 10(6) truncated erythropoietin receptor transgene cells. More than 85% of thalassemic mice transplanted with either type of cells had almost complete reversal of their thalassemic phenotype for at least 6 months, including blood smear dysmorphology, reticulocytosis, high ferritin plasma levels, and hepatic/renal hemosiderosis.
  
  
  CONCLUSIONS
  When plated at high cell densities on irradiated fetal-liver-derived stromal cells, BM cells from WT mice maintain their therapeutic potential for 4 weeks in culture, which is sufficient time for correction of a faulty gene by targeting.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, NC, USA. seigo.hatada@cshs.org
authors.forename: Seigo
authors.initials: S
authors.lastname: Hatada
authors.forename: William
authors.initials: W
authors.lastname: Walton
authors.forename: Tomoko
authors.initials: T
authors.lastname: Hatada
authors.forename: Anne
authors.initials: A
authors.lastname: Wofford
authors.forename: Raymond
authors.initials: R
authors.lastname: Fox
authors.forename: Naiyou
authors.initials: N
authors.lastname: Liu
authors.forename: Michael C
authors.initials: MC
authors.lastname: Lill
authors.forename: Jeffery H
authors.initials: JH
authors.lastname: Fair
authors.forename: Suzanne L
authors.initials: SL
authors.lastname: Kirby
authors.forename: Oliver
authors.initials: O
authors.lastname: Smithies
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000378:metabolism*; D016026:Bone Marrow Transplantation; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D018920:Coculture Techniques; D005260:Female; D005786:Gene Expression Regulation; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D013789:Thalassemia / Q000378:metabolism* / Q000628:therapy*; D013997:Time Factors; D014182:Transplantation, Autologous