In Vitro Differentiation Of CD34 Positive Cells From Umbilical Cord Blood Into Megakaryocytes And Functional Platelets

Platelets,which range from 2-3 μm in diameter,are non-nucleated cell fragments released by multinucleated megakaryocytes in bone marrow.It has been documented that platelet play an important role in regulating hemostasis and blood coagulation in human body.However,due to the limited number of platelet and the fact that the storage period of platelets is usually between 5-7 days in vitro,it is necessary to continuously produce platelets in vitro to meet clinical requirements.In the present study,we isolated CD34 positive cells from umbilical cord blood,and established a differentiation approach in which those cells were differentiated into functional platelets in vitro.Objective: To establish a differentiation method in which CD34 positive cells from umbilical cord blood were differentiated into megakaryocytes and platelets in vitro.Moreover,we aimed to increase the quantity of megakaryocytes and platelets by using this method.Finally,we examined whether the differentiated platelets possessed the function in vitro and in vivo.Methods: The previously published method of differentiating CD34 positive cells to platelets was optimized.Various combinations of media and cytokines were employed at different stages of differentiation process.The differentiation process was divided into three stages: 1.CD34 positive cells differentiated into early megakaryocytes;2.Early megakaryocytes differentiated into mature megakaryocytes;3.Mature megakaryocytes produced platelets.Medium Ⅱ or SFM medium combined with cytokines SCF,TPO,IL3,IL6,IL11 were used to increase the proportion of platelet megakaryocytes and platelets differentiated from cord blood CD34 positive cells.RT-PCR was used to assess the expression of genes involved in the differentiation of CD34 positive cells into megakaryocytes and platelets.Moreover,transcriptome sequencing was performed to evaluate the gene expression profile of megakaryocytes at different differentiation stages.The protein level of CD62 P,which is an important molecule regulating the coagulation function of platelets in vivo,in megakaryocytes and platelets was examined by immunofluorescence staining.After platelets being activated with ADP,the ability of platelets to bind PAC1 was detected by flow cytometry.The established mouse model of thrombocytopenia was injected with the differentiated platelets and,the human platelet samples obtained from hospital,respectively.Then,the percentage of human platelet in the blood of the mice was detected by flow cytometry at different time points.Furthermore,the coagulation time and bleeding time of the mice were measured.Results:(1)By using our optimized method of CD34 positive cell differentiation,we were able to obtain approximately 90% of early megakaryocytes and more than 85% of mature megakaryocytes in vitro,as demonstrated by flow cytometry analysis.In addition,the purity of differentiated platelets could reach more than 75%.(2)RT-PCR and transcriptome analysis showed that genes related to megakaryocytes and platelets,such as CD41 a,CD42a,CD42 b,CD61 increased during differentiation process.RUNX1,FLI1,GATA1/2 and other genes that promote megakaryocyte formation increased with the differentiation to mature megakaryocytes.Genes associated with megakaryocyte differentiation were found to be gradually decreased,while the gene expression of FOG1 and EVI1,which are transcription factors promoting megakaryocyte differentiation to platelets,were gradually increased during differentiation.The expression level of CD62 P in differentiated platelets was similar to that of human platelets.(3)Immunofluorescence staining illustrated that CD62 P was expressed in megakaryocyte and platelet,suggesting that the differentiated platelets might have the ability of agglutination in vivo.Additionally,after platelets being activated by ADP,more than 72% platelets could bind with PAC1.(4)By using cyclophosphamide,the mouse model of thrombocytopenia was successfully established.As expected,the number of platelets in the blood of mice was significantly reduced,the weight of mice was declined,and the clotting time was prolonged.Interestingly,the differentiated platelets and human platelets,which were respectively injected into the tail vein of the mice,remarkably shortened the hemostatic time in the mice.It was found that the hemostatic and coagulation effects of differentiated platelets were close to human platelet samples.We observed that the quantity of human platelets and differentiated platelet in the blood of mice varied with time,which reached the peak level 4 hours after the injection and followed by a gradual reduction.Conclusion: We optimized the previously reported method of differentiating CD34 positive cells into megakaryocyte and platelets by dividing the process into three stages.By means of this approach,high purity of megakaryocytes and platelets could be obtained.We verified that the differentiated megakaryocytes had the ability to produce platelets,and the differentiated platelets were functional platelets that contribute to hemostasis and coagulation in vivo.