Ex Vivo Large-scale Production And Preclinical Studies Of Megakaryocytes

Objectives:Platelet transfusion is the most important treatment to increase platelet counts and prevent excessive bleeding in patients.In recent years,the demand for platelets in the clinic has increased significantly,causing a severe shortage of donor-platelets.The ex vivo induced megakaryocyes from hematopoietic stem cells are expected to be a substitute for donor-platelets to alleviate the platelet resource shortage.This project aims to establish an efficient and large-scale platform to produce a large number of safe and effective megakaryocytes for clinical application.Methods:1.To develop a high-yield,high-purity megakaryocyte ex vivo culture system from CD34+ hematopoietic stem/progenitor cells.Umbilical cord blood CD34+ cells were isolated and various combinations of different cytokines,growth factors,and small molecule compounds were compared on stimulating CD34+ cell expansion and directed megakaryocytic differentiation to obtain the optimal combination,after which culture time were determined to achieve efficient production of megakaryocytes ex vivo.2.To achieve the large-scale production of megakaryocyte and identify their characteristics.The pilot-scale production platform for megakaryocytes was established with the 2L culture roller bottles and a rotary culture system.At the end of culture,the morphology and structure of megakaryocytes were identified by Wright Giemsa staining;the expression of cell-specific markers was analyzed by immunofluorescence staining;the DNA ploidy of megakaryocytes was detected by propidium iodide staining combined with flow cytometry;the expression levels of megakaryocyte differentiation-related transcription factors were detected by real-time quantitative polymerase chain reaction(RT-qPCR).3.To evaluate the safety and efficacy of megakaryocytes in mouse and non-human primate models.The obtained megakaryocyte progenitors and mature megakaryocytes were transplanted respectively into irradiated(2.5Gy)non-obese diabetic/severe combined immunodeficient(NOD/SCID)mice to analyze their in vivo metabolic kinetics and their platelet-release ability.CD34+cells from mobilized peripheral blood of cynomolgus monkeys were isolated and then megakaryocyte progenitors and mature megakaryocytes were produced by the developed culture system.A model of thrombocytopenia in cynomolgus monkeys was established with carboplatin,and transplantation of cynomolgus megakaryocyte progenitors and mature megakaryocytes was performed to evaluate their in vivo safety and efficacy to increase platelet counts.Results:1.After several rounds of screening and optimization,the optimal cytokine combinations to promote the expansion of CD34+ cells and directed differentiation of megakaryocytes were obtained,and an efficient two-stage culture system that was free of stroma,animal components,and genetic manipulations was developed to produce functional megakaryocytes with high yield and high purity.One human cord blood CD34+ cell could be induced by this system to produce up to 1.0×104 megakaryocytes with CD41a+ and CD42b+cells at 82.4%± 6.1%and 73.3%± 8.5%,yielding 30?650 fold higher cell numbers than reported previously.2.Standardization and large-scale production of megakaryocytes were achieved,which doubled the megakaryocyte yield in the pilot-scale production platform.Morphological studies revealed that the obtained mature megakaryocytes were large in size,with an average diameter of 29.1 ± 4.6 ?m and a pronounced pro-platelet structure.The DNA ploidy test showed that 4N or greater megakacyotes occupied 32.67%± 7.43%of the total cell population(4N,19.63%± 1.81%;8N,11.13%± 1.52%;8N?,4.00%±0.54%).RT-qPCR analysis illustrated that the expression levels of GATA-1,FOG-1,NF-E2 and ?-tubulin mRNA increased significantly at the end of culture period,demonstrating the differentiation and maturation of megakaryocytes at the molecular level.3.In the NOD/SCID mouse model,transplanted human megakaryocytic progenitors were capable of engrafting and producing functional platelets continuously;most of the transplanted mature megakarycoytes were entrapped in lung with the the strongest fluorescent signal in 0.5?3 hours(h),and meanwhile human platelets could be detected in mouse peripheral blood,peaking around 6?8h and declining gradually theirafter.In the cynomolgus thrombocytopenia model,autologous transplantation of megakaryocyte progenitors could shorten the platelet count nadir thus promoting the recovery of hematopoietic system;functional platelets were released in vivo 3h after transplantation with autologous or allogeneic mature megakaryocytes,which lasted for more than 48h.In addition,in the observation period of 12 months after transplantation.all animals were in apparent good health.Without any developmental abnormalities or tumor growth,supporting the term in vivo safety of megakaryocytes produced by this two stage culture system.Conclusion:The two stage culture system and the pilot-scale production platform established in this study can produce functional megakaryocytes from hematopoietic stem cells ex vivo with high yield and efficiency,providing new options for clinical treatment of thrombocytopenia due to various etiologies.