Study On Efficient Expansion Of Hematopoietic Stem Cells (CD34~+ Cells) In Vitro And Differentiation Into Red Blood Cells To Prepare General Blood

Background:Hematopoietic stem cells(HSCs)have been widely explored for both scientific research in the laboratory and treatment of blood disorders in the clinic.Previously,we optimized a HSC expansion medium consisting of various cytokines and nutrition supplements,which can effectively expand CD34+ cells derived from either human cord blood or mobilized peripheral blood.The expanded HSCs were demonstrated to retain long-term potential of repopulation and multi-lineage differentiation in non-obese diabetic/severe combined immunodeficiency(NOD/SCID)mice.Since HSCs have the capacity to self-renew and give rise to mature cells of all hematopoietic lineages,the ex vivo production of red blood cells(RBCs)on a large scale from HSCs has been considered as a promising approach to overcome the shortage of blood supply.Aims:The focus of the thesis project was on expansion and differentiation of HSCs,including in vivo studies of HEM-expanded CD34+ cells in the nonhuman primate model,optimization and development of a bottle turning device culture system for ex vivo generation of human erythrocytes on a large scale from HSCs.In addition,in vivo studies of induced erythrocytes in murine and nonhuman primate models were studied,aiming at providing preclinical evaluations for safety and functionality of ex vivo expanded CD34+cells and induced erythrocytes.Methods:1.CD34+ cells were isolated from the mobilized peripheral blood of nonhuman primates and expanded with HEM for 9 days.Expanded cells were transduced with the gene coding for green fluorescent protein(GFP).Nonhuman primates(n=11)were administered with cyclophosphamide,resulting in myelo-suppression before autotransplantation.The myelo-suppressed primates were randomly divided into three groups as follows:a control group treated with saline(n=3),a group with autologous CD34-cells(n=3),and a group treated with GFP-labeled,expanded autologous CD34+ cells(n=5),respectively.After autologous transplantation,routine blood tests and flow cytometry analysis were performed to determine the proportion of GFP+ cells in the peripheral blood and bone marrow.2.A procedure of four-stage ex vivo expansion and differentiation was developed in a modified IMDM basal medium supplemented with nutrients and selected cytokine combinations.Enriched CD34+ cells were cultured and expanded in the bottle-turning device system.During the differentiation process,erythroid markers(CD71 and CD235a)and enucleation efficiency(LDS-percentage)of cultured cells were evaluated by flow cytometry.Erythroid progenitor cells were confirmed by clonogenic capacity by colony-forming unit(CFU)assay.Hemoglobin content of the cells were determined quantitatively,and RT-qPCR analysis was performed to examine expression of erythroid-specific genes and the status of proto-oncogenes.Furthermore,xenotransplantation was performed in murine and nonhuman primate models to assess the safety and efficacy of ex vivo generated erythrocytes.Results:1.HEM could effectively expand nonhuman primate CD34+ cells to a sufficient number for autotransplantation.Expanded CD34+ cells retain multi-lineage differentiation as indicated by colony-forming unit assay.Results of autologous transplantation in nonhuman primates demonstrated that the expanded CD34+cells could improve the count nadir of white blood cell,platelet,and neutrophil,and could shorten the period of pancytopenia,resulting in enhanced hematological recovery following myelo-suppression.Transplanted CD34+ cells preserved both myeloid and lymphoid differentiation potentials in nonhuman primate bone marrow in one month after auto-transplantation.All primates transplanted with the expanded autologous CD34+ cells survived for over 18 months without any noticeable abnormalities.2.With the selected optimized culture conditions,we found that functional human erythrocytes could be efficiently produced from cord blood(CB)CD34+ cells using a bottle turning device culture system.One human CB CD34+ cell could be induced ex vivo to produce up to 200 million erythrocytes with a purity of 90.1 ±6.2%and 50 ± 5.7%for CD235a+ cells and enucleated cells,respectively.The yield of erythrocytes from one CB unit(5 million CD34+ cells)could be,in theory,equivalent to 500 blood transfusion units in the clinic.Moreover,induced human erythrocytes had a normal hemoglobin content and could continue to undergo terminal maturation in the murine xenotransplantation model.In nonhuman primate model,xenotransplantation of induced human erythrocytes enhanced hematological recovery and ameliorated the hypoxia situation in the primates with hemorrhagic anemia.Conclusions:1.Nonhuman primate CD34+ cells can be safely and efficiently expanded by HEM for autologous transplantation.Thus,our preclinical studies provide evidence of safety and efficacy for HEM-expanded CD34+ cells in clinical HSC transplantations.2.We have developed a large-scale culture system to produce functional human erythrocytes ex vivo.The results of our study suggest that the ex vivo-generated RBCs retain the normal features and functionality when they are compared with fresh RBCs and can be further explored as an alternative RBC source for clinical applications.