|Budget Amount *help
¥5,400,000 (Direct Cost : ¥5,400,000)
Fiscal Year 2000 : ¥1,700,000 (Direct Cost : ¥1,700,000)
Fiscal Year 1999 : ¥1,500,000 (Direct Cost : ¥1,500,000)
Fiscal Year 1998 : ¥2,200,000 (Direct Cost : ¥2,200,000)
We demonstrated a significant ex vivo expansion of cord blood hematopoietic stem cells capable of repopulating in NOD/SCID mice, using a combination of stem cell factor (SCF), Flk2/Flt3 ligand (FL), thrombopoietin (TPO) and a complex of interleukin (IL)-6 and soluble IL-6 receptor (IL-6/sIL-6R). When 1 to 2x104 cord blood CD34+ cells and the cells cultured with SCF+FL, SCF+FL+TPO and SCF+FL+TPO+IL-6/sIL-6R for 7 days were transplanted into NOD/SCID mice, 6/25 (24%), 7/16 (44%), 7/14 (50%) and 13/16 (81%) recipients revealed successful engraftment, and the bone marrow cells of the engrafted recipients contained 1.4%, 2.3%, 6.7% and 11.5% of human CD45+ cells, respectively, 10 to 12 weeks after the transplantation. The CD45+ cells in the recipients engrafted with the cells cultured with SCF+FL+TPO+IL-6/sIL-6R consisted of various lineages and a large number of CD34+ cells, which formed human hematopoietic colonies in in vitro clonal culture. In comparion of the reconstitution between fresh CD34+ cells from 12 samples and their progenies cultured with SCF+FL+TPO+IL-6/sIL-6R, the proportion of CD45+ cells in recipient marrow was 10 times higher in the latter, indicating the significant expansion of the long-term repopulating hematopoietic stem cells. The expansion rate was estimated at 4.2-fold by a limiting dilution method. The addition of IL-3 to the cytokine combination abrogated the repopulating ability of the expanded cells. The present study may provide a novel culture method for the expansion of human transplantable hematopoietic stem cells aimed for clinical applications.