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 of maintaining them in tissue culture long enough for completing gene targeting without them 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 (tEpoR-tg), 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, the 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 tEpoR-tg 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.Conclusion-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.

Calcium Dependent CAMTA1 in Adult Stem Cell Commitment to a Myocardial Lineage

“…Human MSCs constitutively labeled with GFP in the cytoplasm or dsRed targeted to the mitochondria and mMSCs from C57BL/6 mice with the following genotypes: a beta Myosin Heavy Chain (βMHC)-YFP fusion transgenic mouse and αMHC-CFP/βMHC-GFP transgenic mouse [46] , [47] were prepared. Mouse MSCs were isolated from the bone marrow of the mice as previously reported [48] , [49] . The mMSCs demonstrated no fluorescence until after 8 days in co-culture, at which time few cells exhibited spontaneous faint fluorescence indicating the onset of expression of the MHC gene in these cells.…”

“…MSCs were isolated as previously described [48], [49]. Briefly, 8–12 week old male mice were sacrificed by cervical dislocation, and the femurs and tibiae were removed.…”

“…The mouse bone marrow mesenchymal stem cells (mMSCs) were prepared from C57BL/6 mice with the following genotypes: a βMHC-YFP fusion mouse and αMHC-CFP/βMHC-GFP transgenic mouse (both provided by Dr. Kumar Pandya [46] , [47] ). MSCs were isolated as previously described [48] , [49] . Briefly, 8–12 week old male mice were sacrificed by cervical dislocation, and the femurs and tibiae were removed.…”