Objective: To investigate the effects of microvesicles(MV) isolated from human peripheral blood hematopoietic stem cells(PB-HSC) on immune regulation and hematopoiesis.
Methods: PB-HSCs were separated by density-gradient centrifugation and cultrued. The supernatants of PB-HSC at 48 h were harvested for isolation and purification of MV by using ultracentrifugation. The electron microscopy was used to observe the morphology of MV. The protein level in MV was quantified through bicinchoninic acid(BCA) protein assay. Flow cytometry was used to detect the immunophenotype of MV. Human peripheral blood mononuclear cells(PB-MNC) were isolated from healthy donor and treated with isolated MV. After being co-cultured for 12 h, confocal microscopy was used to observe the action mode of MV on PB-MNC. After being co-cultured for 48 h, the levels of IL-2, IL-6, IL-8, IL-10, IFN-γ and TNF-α were detected by ELISA. Flow cytometry was used to detect the changes of T cell subsets and the activation of T cell subsets as well as intracellular cytokine staining after co-culture for 48 h. The methylcellulose was used to assess the hematopoiesis-supportive function of MV as well as co-cultured supernatants.
Results: The eletron microscopy revealed that MV were elliptical membrane vesicles. The protein amount in MV ranges from 29 to 110 µg. Flow cytometry showed that MV expressed mix markers on the surface, especially highly expressed MV specific marker CD63(85.86%) and hematopoietic stem cell marker CD34(33.52%). After being co-cultured for 12 h, confocal microscopy showed that MV were merged with PB-MNC. After being co-cultured for 48 h, ELISA showed that the secretion of cytokines IL-6,IL-8, IL-10 as well as TNF-α was increased while the level of IL-2 and IFN-γ was not changed much. The results of flow cytometry showed that there was no significant change in T cell subsets and T cell activation. Staining of intracellular factor showed that IL-8 was increased significantly in CD11c+ cells. The colony-forming experiments revealed that MV and the co-cultured supernatants could facilitate the colony formation.
Conclusion: MV isolated from PB-HSC have immune-regulatery function and can prornote hematopoiesis.