| Background Blood transfusion is the first successful cell therapy,which is widely used for the clinical treatment of almost all diseases.Particularly,transfusion of RBCs can restore the oxygen transport capacity to improve the quality of life in patients with chronic anemia such as thalassemia and sickle cell disease,and can be even life-saving in several circumstances.However,red blood cell(RBC)transfusion is challenging due to several issues,including supply shortage,immune incompatibility,and blood-borne infections since donated blood from anonymous volunteers is the only source of RBCs.Therefore,the large-scale generation of cultured RBCs from stem cells in vitro could be a promising new source of consistent and safe alternatives to blood transfusion.Despite great progress has been made to induce erythrocytes from hematopoietic stem cells(HSCs)ex vivo,generating clinically applicable RBCs still remains an obstacle because of defects in limited expansion,the low proportion of enucleation and the low expression of adult globin during the terminal maturation.In addition,ineffective erythropoiesis,erythroblasts can be increased but fail to mature,is observed in diseases such as thalassemia,myelodysplastic syndrome(MDS)and chronic anemia,which further exacerbate the patient's anemia.Currently,the treatment of ineffective erythropoiesis has been hampered by the bottleneck of erythroid cell terminal maturation.Autophagy is currently considered as the main mechanism ofpromoting the eventual maturation of RBCs.Among the autophagy signaling pathways,mammalian target of rapamycin(mTOR)is the most important negative regulator.So far,the physiological function of mTOR signal in erythroid cell maturation is still unknown,and there is controversy whether mTOR inhibitors are used in anemia patients.Our previous animal studies found that in early erythropoiesis,mTORC1 signal is necessary for hematopoietic stem cell differentiation and proliferation,and inhibition of mTORC1 pathway in vivo and in vitro can partially correct the abnormal mature of erythrocytes differentiation and maturation.In this study,mTORC1 pathway inhibitor rapamycin was used to treat the cells at different time periods to evaluate the regulatory effect of mTORC1 on the targeted differentiation of human umbilical cord hematopoietic stem cells and to elucidate the relevant molecular mechanisms.Objective To study the effect of rapamycin on proliferation,differentiation and maturation during erythropoiesis from human umbilical cord blood HSCs and mechanism involved.To optimize the culture system for ex vivo generation of human erythrocytes from umbilical cord blood CD34+ cells,and improve the terminal maturation rate.Methods1)Human cord blood CD34+ cells were isolated.2)Human CD34+ cells were induced to differentiate into erythrocytes using the system without serum or feeding layer.3)At different time points of differentiation(Day8,Day11,Day18,Day22,Day44),50 n M rapamycin was added to the medium as the drug treatment group,and blank and DMSO as the control group.4)Using colony forming experiment to detect the proliferation of hematopoietic progenitor cell.5)Using flow cytometry to detect cell surface antigens,proliferation capacity,cell cycle,apoptosis,the proportion of enucleated cells and the levels reactive oxygen species(ROS).6)Wright-Giemsa and May-Grünwald-Giemsa(MGG)staining were used to observe the morphology of cells during differentiation and BFU-E/CFU-E colonies.7)Cells were stained using Mito Tracker Green,Lyso Tracker Red and CD235 a and observed under confocal microscope to detect the effect of mTOR inhibition on mitochondria clearance.8)Real-time quantitative PCR was used to detect the transcriptional level of globins,erythroid-specific genes,mTOR downstream genes and autophagy related genes.9)Western blot was used to detect the expression of mTOR downstream genes and autophagy related genes.Results1)CD34+ cells were successfully isolated from human umbilical cord blood and differentiated into erythrocytes.2)The number of BFU-E and CFU-E was significantly reduced after rapamycin treatment.3)At the early stage of differentiation,rapamycin reduced the number of erythroid progenitor cells by inducing cycle arrest and inhibiting proliferation but not apoptosis.The m RNA level and protein level of the cell cycle related gene P27 were increased.4)In the middle of differentiation,rapamycin treatment increased the rate of enucleated cells,promoted mitochondrial clearance and increased globin transcription.5)The phosphorylation level of downstream genes(p70S6 and S6)of mTOR pathway was decreased after rapamycin treatment,whereas the m RNA and protein expressions of most autophagy genes were increased.Conclusion1)We successfully established a serum-free and feeding-layer free culture system for generation of erythrocytes from human umbilical cord blood CD34+ cells,and rapamycin treatment from day 18 can significantly improve the efficiency of enucleation without reducing the number of cells.2)mTOR signaling pathway is necessary for the early differentiation of human erythrocytes.Inhibition of mTOR pathway leads to cell cycle arrest and cell proliferation inhibition,thus reducing the formation of erythoid colonies such as BFU-E and CFU-E,but the apoptosis rate and differentiation process have not been significantly changed.3)In the middle and late stage of human erythroid differentiation,the inhibition of mTOR pathway enhances the autophagy process,thereby improving the enucleation efficiency of red blood cells,promoting mitochondrial clearance,and ultimately promoting the end-stage maturation of red blood cells. |
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