Study on an efficient denucleation system for erythropoiesis from umbilical cord blood hematopoietic stem cells in vitro
Objective To study the effects of phosphatidylinositol trihydroxykinase(PI3K),histone deacetylase 2(HDAC2)and cytochalasin B on erythroid denucleation of umbilical cord blood hematopoietic progenitor cells in vitro and establish an efficient enucleation system.Methods Cord blood CD34+cells were enriched by magnetic sorting.Cytokine stem cell factor(SCF),interleukin 3(IL-3)and erythropoietin(EPO)were added on days 0 and 4 of culture,SCF and EPO were added on day 7 of culture,and only EPO was added on days 11,15 and 18 of culture.PI3K(0 ng/mL,25 ng/mL,50 ng/mL,and 100 ng/mL),HDAC2(0 ng/mL,60 ng/mL,150 ng/mL,and 300 ng/mL)and cytorelaxin B(0 ng/μL,25 ng/μL,50 ng/µL,and 75 ng/μL)were added on the 7th,11th,15th and 18th days,respectively.Orthogonal test was used to analyze the influence of four factors and four levels of the concentration,and addition time of three kinds of nucleating agents on the effect of nucleation,to obtain the best conditions for nucleation as optimal group,while the culture without nucleating agents as the control group for verification.On the 21st day of cell culture,CD235+and SYTO-64-cells were detected by flow cytometry,and the denucleation rate was calculated.Red blood cells were counted by hemocytometer,the content of 2,3-diphosphoglycerate(2,3-DPG)was detected by ELISA,and ATP was detected by chemiluminescence assay.The cell morphology was observed.Results Statistical analysis showed that the optimal enucleation condition was as follows:the PI3K with 100 ng/mL,HDAC2 with 150 ng/mL,and Cytochalasin B with 75 ng/uL were added on the 15th day.On the 21st day of culture,the rate of erythrocyte nucleation was(74.30±5.59)％in the optimal group and(28.30±14.10)％in the control group,with the optimal group higher than the control group(t=9.099,P=0.012).There was no significant difference in the morphology and function of red blood cells compared with the control group.The number of erythrocytes reached an average of 2 x 1010/L when the cells were cultured for 21 days.Conclusion The culture system established under the optimal conditions can promote the efficient denucleation of erythrocytes differentiated by hematopoietic stem cells in vitro.
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