Biological Characteristics Of Microvesicles Secreted By Human Peripheral Blood Hematopoietic Stem Cells

Hematopoietic stem cells are adult stem cells in the blood system and are a heterogeneous group.They have the potential of self-renewal and multilineage differentiation.According to its source,hematopoietic stem cells can be divided into bone marrow hematopoietic stem cells,peripheral blood hematopoietic stem cells,umbilical cord blood hematopoietic stem cells and fetal liver stem cells.For peripheral blood hematopoietic stem cells’ s collection is simple,and they can be collected in the short term,as well as the advantages of rapid reconstitution of hematopoietic and immune function,and high implantation rate after infusion,peripheral blood hematopoietic stem cells play an increasingly important role in transplantation and immunotherapy.At the same time,it was found that the colony forming cells of peripheral hematopoietic blood stem cells were more than the colony forming cells derived from other tissues.Microvesicles are sub micron double membrane structures secreted by cells,including exosomes secreted from endosomes,as well as microparticles secreted from the majority of cell membrane surfaces.It contains the components of original cell such as protein,lipid,mRNA as well as microRNA.After the fusion with the target cells,the content in microvesicles can be released into the target ones,which plays an important role in the process of cell information transmission.Microvesicles can be released by various types of cells,and are widely distributed in saliva,plasma,milk and other body fluids.Not only normal cells can secrete microvesicles,tumor cells can also secrete microvesicles:microvesilces can be detected in the body fluid in patients with cancer.Therefore,in the physiological and pathological state microvesicles secreted from different cells is a mixed group.The microvesicles vary with the source,quantity,size and composition of the cells.Peripheral blood hematopoietic stem cells have the function of immune regulation and hematopoietic recovery,and the microvesicles secreted by them may also have the ability to regulate the cytokines and promote colony formation.The results show thatmicrovesicles have little or no immunogenicity,and can be widely used if they have certain functions.However,there is little research on the microvesicles derived from the peripheral blood hematopoietic stem cells.The aim of this study was to investigate the biological characteristics of microvesicles derived from the peripheral blood hematopoietic stem cells,and to explore its immune regulation and hematopoietic function.Objective To extract and identify the microvesicles derived from the peripheral blood hematopoietic stem cells.To study it’s biological characteristics including the effects of microvesicles isolated from human peripheral blood hematopoietic stem cells on immune regulation and hematopoiesis.Content Isolate the healthy human peripheral blood hematopoietic stem cells and then cultured,extract and identify the microvesicles derived from the peripheral blood hematopoietic stem cells in the supernatant,and study its biological characteristics: to investigate that whether microvesicles derived from the peripheral blood hematopoietic stem cells have immune regulatory function on normal human peripheral blood mononuclear cells?Does the microvesicles derived from the peripheral blood hematopoietic stem cells affect the colony formation of hematopoietic cells?Methods(1)Mobilized peripheral blood hematopoietic stem cells were separated by density gradient centrifugation and cultrued.The supernatants of peripheral blood hematopoietic stem cells at 48 h were harvested for isolation and purification of microvesicles by using ultracentrfugation.(2)The electron microscopy was used to observe the morphology of microvesicles.The amount of protein in microvesicles was quantified through bicinchoninic acid(BCA)protein assay.Flow cytometry was used to detect the immunophenotype of microvesicles.(3)Human peripheral blood mononuclear cells(PB-MNC)were isolated from health donor and treated with isolated microvesicles.After co-cultured for 12 h,confocal microscopy was used to observe the mode of action of microvesicles to peripheral blood mononuclear cells.After co-cultured for 48 h,the production of IL-2,IL-6,IL-8,IL-10,IFN-γand TNF-α was detected by ELISA.The changes of T cell subsets and the activation of T cell subsets were detected by flow cytometry after co-cultured for 48 h.Flow cytometry was used to detect intracellular cytokine staining after co-culture for 48 h.(4)Methylcellulose wasused to assess the hematopoiesis-supportive function of microvesicles as well as co-cultured supernatants.Results Mobilized peripheral blood hematopoietic stem cells were extracted by density gradient centrifugation.Microvesicles secreted by human peripheral blood hematopoietic stem cells can be successfully extracted by ultracentrifugation.The eletron microscopy revealed that microvesicles were elliptical membrane vesicles.Flow cytometry showed that microvesicles express mix markers on the surface especially high expression of microvesicles specific marker CD63(85.86%)and the original cells’ marker such as hematopoietic stem cell marker CD34(33.52%),T cell marker CD3(43.98%)、CD45RA(23.54%)、CD45RO(68.3%),NK cell marker CD56(32.32%)and so on.After co-cultured for 12 h,confocal microscopy showed that microvesicles were merged with peripheral blood mononuclear cells.After co-cultured for 48 h,ELISA showed that the secretion of cytokines IL-6,IL-8,IL-10 as well as TNF-αwas increased while the amount of IL-2 and IFN-γ didn’t have much difference.The results of flow cytometry showed that there was no significant change in T cell subsets and T cell subsets.Intracellular factor staining showed that there was no significant change in cytokines in CD4+,CD8+,CD11c+ cells.The colony-forming experiments revealed that microvesicles and the co-cultured supernatants are likely to facilitate the colony formation.Conclusion Microvesicles isolated from peripheral blood mononuclear cells have immune-regulatery function and they are likely to support hematopoiesis.