The Effect Of Antioxidant On The Ex Vivo Expansion Of Cord Blood CD34~+ Cells

The limited number of hematopoietic stem cells(HSCs)in cord blood remains a big harrier to their clinical applications in adult patients.Ex vivo expansion of HSCs is a feasible strategy which could overcome this problem.However,the level of intracellular reactive oxygen species(ROS)dramatically increases by the stimulation of cytokines and other stress when HSCs are cultured ex vivo.At physiological levels,ROS function as signal molecules which can regulate a variety of cellular functions,including proliferation,differentiation,senescence and apoptosis of HSCs.However,excessive production of ROS can inhibit HSC self-renewal and result in hematopoietic dysfunction.Regulation of intracellular ROS level is hence essential to maintain the physiological characteristic and functions of HSCs.In this research,freshly isolated cord blood CD34+cells were cultured in serum free medium with cytokines.Different antioxidants such as resveratrol,catechin,ginsenoside Rgl.notoginsenoside R1,puerarin,hyperoside and aloeemodin were studied for the effect on the level of intracellular ROS?colony-forming ability and proliferation of HSCs.Our results showed that the level of intracellular ROS was reduced and the growth of colony-forming cells was enhanced by the addition of antioxidant.Meanwhile,all the antioxidants but aloeemodin increased the proportion of CD34+cells and had no significant effect on the expansion of total cells.Further,CD34+cells were cultured in medium with or without resveratrol to analyze the effect of resveratrol on proliferation characteristic,physiological state and re-expansion ability of HSCs during ex vivo culture.The results revealed that the fold expansion of total cells was not significantly affected by the addition of 10 ?M resveratrol.However,after 14 days culture,the percentage of CD34+cells in resveratrol treated cultures retained 31.5±7.3%and increased significantly compared with those in control group which was 18.6±2.1%.And the proportion of apoptotic cells and aging cells in control cultures were 24.6±O.9%and 47.0±5.0%respectively,whereas those in resveratrol treated culture were both markedly decreased to 20.8±0.8%and 32.2±2.8%respectively.Moreover,after 14-day culture with resveratrol,the rate of apoptosis and aging CD34+cells were 31.5±5.7%and 2.5±0.4%respectively,which were both much lower than those of control group which were 42.2±2.6%and 5.7±1.4%respectively.Furthermore,the secondary culture of CD34+cells for 14 days revealed 44.9±11.6 folds expansion for resveratrol group and 23.2±7.8 folds expansion for control group.These results indicated that the addition of 10?M resveratrol not only promoted the ex vivo expansion of HSCs,but also ameliorated the physiological state and improved the re-expansion ability of CD34+cells.Our work provided new thinking for the optimization of ex vivo expansion of CD34+cells.