The Stemness Maintenance Of The Ex Vivo Expanded Umbilical Cord Blood CD133~+ Cells: Regulation Of The Overactivited Reactive Oxygen Species And P38 Mitogen-activated Protein Kinase

Object:To investigate the effect and mechanism of reactive oxygen species on the ex vivo expasion of umbilical cord blood CD133+cells.Methhod:A serum-and stroma-free culture system for umbilical cord blood CD133+cell ex vivo expansion was established. The levels of ROS, which reduced by antioxidant NAC in different concentration, were detected in several time points during the culture. HSC ability was investigated through the density of CD133+cell subgroup,CFU assay and 28day CAFC assay. The mechanism studies were performed, such as Carboxyfluorescein diacetate succinimidyl ester labeling,apoptosis assay,Senescence-associated (3 galactosidase (SA-(3-gal) staining and the expression of human p16INK4/p21WAF1Results:Accompanying with the impairment of HSC selfrenewal, the generation of intracellular ROS was increased during ex vivo expansion. The antioxidant NAC could eliminate ROS effectively according to the concentration. Otherwise, CD133+cells were increased in all groups, low-dose NAC led a higher increase than control group (p<0.05), but cell expansion was less in high-dose group (p<0.01). Furthermore, the numbers of CFU and CAFCs were hold in high levels in both of the NAC treated groups. Mechanism studies showed that NAC acted by suppressing proliferation, apoptosis and senescence.Conclusion:The ex vivo expasion of umbilical cord blood CD133+cells was regulated by the generation of intracellular ROS, which was acting through regulation of proliferation, apoptosis and senescence. HSC sternness could maintain in appropriate reduction of ROS, but over reduction could harm the proliferation of HSC. Object:To investigate the effect and mechanism of p38 MAPK on the ex vivo expasion of UCB CD 133+cells.Methhod:A serum-and stroma-free culture system for umbilical cord blood CD133+cell ex vivo expansion was established. During the culture, the activation of p38 MAPK was abrogated by SB203580, and the expression of P-P38 was detected in several time point. HSC ability was investigated through the density of CD133+cell subgroup,CFU assay,28day CAFC assay,migration rate and the expression of CXCR4. Repopulation ability was detected in NOD/SCID mice transplantation model. The mechanism studies were performed, such as Carboxyfluorescein diacetate succinimidyl ester labeling,apoptosis assay,Senescence-associated (3 galactosidase (SA-(3-gal) staining and the expression of humanp16INK4/p21WAFI.Results:Accompanying with the impairment of HSC selfrenewal, the activation of p38 MAPK was detected during the ex vivo expansion. SB203580 could abrogate the activation. Compared to control group, SB203580 enhanced expansion of early progenitors (CD133+ CD133+CD38-,CD133+CXCR4+, CFU-GEMM and CAFCs) after 7 d of culture (p<0.01). Furthermore, a robust human hematopoietic chimerism and rapid pace of engraftment were observed in non-obese diabetic severe combined immunodeficient mice, which were transplanted with SB203580-treated cells. Mechanism studies showed that SB203580 acted by suppressing apoptosis and senescence, but not by increasing proliferation and inhibiting differentiation.Conclusion:Inhibition of p38 MAPK promotes the ex vivo expansion and maintains the stemness of human HSCs during ex vivo expansion, which mainly via inhibiting of senescence and apoptosis. Object:To investigate the correlation and compare the effects of ROS and p38MAPK in the ex vivo expansion of UCB CD133+cells.Method:Besides the control group, UCB CD133+cells were cultured with NACn SB203580 or combination. The levels of ROS and expression of P-P38 were detected. The effects of expansion were evaluated by the density of CD133+cell subgroup,CFU assay and CAFC assay.Results:ROS was eliminated in all the three treated groups, a net 85%reduction of ROS was observed in combination group, while the ROS was reduced by 48%in SB203580 group and 37% in NAC group. Ferthermore, both SB203580 and NAC could abrogate the activation of p38 MAPK. Besides, the CD133+cells were expansion in SB group by 21.93±1.36 fold increase, in control group the number was 10.13±0.57; and NAC led a 14.50±1.19-fold increase. But the CD133+cells barely expansion in the combination group. In addition, the numbers of CFU and CAFCs were hold in higher levels in SB group compare to others.Conclusion:compare to elimination of ROS, Inhibition of p38MAPK producted a better effects on the ex vivo expansion of UCB CD133+cells; reactive oxygen species levels regulate hematopoiesis; ROS activate p38 MAPK, which further promotes ROS generation, forming a positive feedback loop to sustain ROS-p38 kinase signaling.