Therapeutic benefits in thalassemic mice transplanted with long-term-cultured bone marrow cells

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. Materials and 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-cm² 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 × 10⁶ WT cells/flask and 27-fold with 2 × 10⁶ 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.