ABSTRACT
Objective:To investigate the influence of fetal liver AFT024 cells on the transfection efficiency of multidrug resistant gene 1(MDR1)and the in vitro expansion of CD34+ cells derived from umbilical cord blood.Methods:CD34+ cells were isolated from human umbilical cord blood by MACS CD34 Progenitor Cell Isolation Kit and co-cultured with AFT024 cells(AFT024 group)or cultured alone(control group)for 7 days.During the subsequent 14 days,retrovirus carrying MDR1 gene was supplemented twice a week to transfect CD34+ cells.On the 7th,14th and 21st day after culture,the number of total nucleated cells(TNC)was counted,the ratio of CD34+ cells was assayed by flow cytometry(FCM)and the number of CD34+ cells was calculated,and colony-forming cells(CFC)were counted by methylcellulose cultures.RT-PCR method was used to detect the level of MDR1 mRNA in the transfected cells.The expression and function of P-glycoprotein(P-gp)were evaluated by FCM assay and Rhodamine-123 efflux assay,respectively.The gene transfection efficiency was calculated by drug-resistant colony-forming cells assay.Results:(1)The MDR1 mRNA level in AFT024 group than that in control group.The gene transfection efficiency in AFT024 group was significantly higher than that in control group(46.0% vs 15.2%,P0.05).On the 14th day,the expansion fold of TNCs in control group was significantly higher than that in AFT024 group(P0.05).The expansion folds of CD34+ cells and CFCs in the AFT024 group were significantly higher than that of the control group(P
ABSTRACT
Objective To establish a method for ex vivo cord blood stem cell expansion,increase the number of the stem/progenitor,especially megakaryocyte progenitors so as to decrease the thrombocytopenia period after CBT.Methods Mononuclear cells (MNC) from 20 cord blood samples were cultured in presence of different cytokines:group 1:IL 3,IL 6,group 2:IL 3,IL 6,TPO,group 3:IL 3,IL 6,TPO and cord blood plasma.The final concentration of the cytokines was 50ng/ml.The control group had all medium except the cytokines and cord blood plasma.The cells were cultured for 21 days.At every 7 days,tests were done for nucleate cells number,CFU GM,CFU Mk,and CFU GEMM.Results At different times and in different groups,there were different changes.For NC,maximal increase were obtained in group 3 on day 21,which was 239.65?153.48.For CFU GM,the biggest expansion occurred on day 21 in group 3,the number being 184.17?140.40.For CFU Mk,the biggest expansion was obtained on day 14 in group 3,with the number of 135.34?105.20. The immature progenitor CFU GEMM achieved the max increase in group 3 on day 14,the number was 49.39?36.68. Conclusion The expansion of CFU Mk progenitor cells can be obtained in the presence of IL 3,IL 6,TPO.This combination also enhances the expansion of CFU GM and CFU GEMM.Addition of cord blood plasma can promote the expansion of mature and immature progenitor cells.