ABSTRACT
Objective To investigate the effect of bFGF on the grafting ability (grafing efficiency)of CD34 + cord blood cells seeded in 5 FU injured human bone marrow stromal cells.Methods Using combined methods of bone marrow stromal cell culture system,light microscopy and electron microscopy,flow cytometry,we investigated the toxic effects of 5 FU on human bone marrow stromal cells (hBMSC) and the benefit of bFGF on dynamics of hBMSC as well as the ability of the cord blood stem cell to bind hBMSC.Results After 3 hours incubation with 5 FU,significant changes in hBMSC cytosolic and nuclear morphology were observed.Nuclear splitting and abnormal shapes of the pseudopodia were found under the electron microscope.Flow cytometry revealed that apoptosis appeared before G 0/G 1,and that cells in G 0/G 1and G 2/M phases were remarkably lower,while the cells in S phase were remarkably higher than the controls.Two hours after adding the cord blood hematopoietic progenitor cells,the group treated with bFGF(injury with intervention of bFGF group,IBG)showed higher proprtion of the adherence layer compared to the group without adding bFGF(injury group,IG)(29% versus 12%) Conclusion 5 FU causes DNA damage in hBMSC.It also impairs the binding ability of cord blood stem cell to hBMSC.bFGF is able to repair hBMSC damage and improve the binding of CD34 + cord blood hematopoietic progenitor cells onto hBMSC.
ABSTRACT
Objective To elucidate the role of human bone marrow stromal cells (hBMSC) in combination with exogenous cytokines such as stem cell factor(SCF) and FLT 3 ligand (FL) in supporting the in vitro expansion of CD 34 + cells purified from cord blood.Methods CD 34 + cells were isolated from umbilical cord blood by using a high gradient magnetic cell sorting system(MACS),expanded in combination with SCF,IL3 (interleukin 3),IL 6 (interleukin 6),FL,EPO (erythropoietin),and were planted onto the pre established irradiated (20Gy) stroma layer(hBMSC plus combinations of cytokines) respectively.On day 10,total cells were counted, and hematopoietic progenitor cells were assessed by semisolid culture assay,CD 34 + cells were quantitated by FACS.Results After separation by MACS,the frequency of CD 34 + cells was 92%?0.04%.On day 2,almost all the inoculated cells adhered to the stroma layer,with only a small number in the supernatant.Then,cells in the supernatant increased gradually, but the percentage of CD 34 + cells decreased.Compared with control, expansion fold of CD 34 + cells, CFU GM, BFU E were significantly higher in hBMSC group (P 0.5).Conclusion ① CD 34 + cells from cord blood formed foci adherent to the monolayer and colony forming cells remained in the culture of 10days,indicating that hBMSC can support hematopoiesis in vitro;②using human bone marrow stromal cells in cooperation with exogenous cytokines may be a feasible way to expand hematopoietic stem/progenitor cells.