| Aim:To identify the biological characteristic of microvesicles(MVs) derived from RPMI8226^U266^KM3myeloma cells and bone marrow mesenchymal stem cells(MSC), including their morphological observation, shedding frequency, size distribution, antigen expression and composition.Methods:Collect MVs from the culture condition of RPMI8226、U266、KM3myeloma cells and bone marrow mesenchymal stem cells by differential ultracentrifugation. Transmission electron microscope was used to check the morphological apperance of MVs. Bradford assay was utilized to quantitate their protein content. Flow cytometry analysis was carried out to investigate their shedding frequency, size distribution,antigen expression,and the miRNA expression of MVs derived from MSC was detected by miRNA arrays analysis.Results:MVs derived from myeloma cell lines and MSC show intact vesicles with diameter below1.0μm under transmission electron microscope observation. Protein quantification indicated that RPMI8226、U266、KM3myeloma cells and bone marrow mesenchymal stem cells(MSC) shed MVs in different protein content, they were (223±15) μg·24h-1·107cells-1,(182±11) ug·24h-·107cells-1,(143±9) ug·24h·107cells-1and (97±8) μg·24h-1·107cells-1,respectively. MVs derived from RPMI8226、U266、KM3myeloma cells express CD138, a classic antigen of human myeloma cells. MVs derived from MSC express CD29, CD44, CD73and CD90, which mimics the phenotype of their mother cells. Besides, we observed that MSC-MVs are rich of microRNAs which can positively regulate hematogenesis, such as miR-16,miR-21,miR-494.Conclusion:RPMI8226、U266、KM3myeloma cells and bone marrow mesenchymal stem cells(MSC) produce MVs in different frequency and varied size distribution. They carried similar antigen and composition to their parent cells. Role of myeloma cell-derived microvesicles in cell adaption to serum deprivationAim:To investigate the effects of serum deprivation (SD) on microvesicles (MVs) secreted from human myeloma cells and the implications for disease progression.Methods:RPMI8226, U266and KM3human myeloma cells were incubated in medium containing10%(non-SD) or1%fetal bovine serum (SD) and MVs were isolated. The levels and size distribution of MVs were analyzed with flow cytometry. The protein profiles of MVs were studied using2D SDS-PAGE, MALDI-TOF-MS and Western blotting. NF-κB activation was analyzed using EMSA. Angiogenesis was examined in Eahy926endothelial cells.Results:Exposure of RPMI8226cells to SD for24h did not alter the number of apoptotic cells. However, SD increased the number of MVs from RPMI8226, U266, and KM3cells to2.5-,4.3-and3.8-fold, respectively. The size distribution of SD MVs was also significantly different from that of non-SD MVs. Three proteins ZNF224, SARM and COBL in SD MVs were found to be up-regulated, which were involved in cell cycle regulation, signal transduction and metabolism, respectively. Co-culture of SD MVs and RPMI8226cells increased NF-κB activation in the target RPMI8226cells. Furthermore, SD MVs from RPMI8226cells significantly increased the microtubule formation capacity of Eahy926endothelial cells compared with non-SD MVs.Conclusion:SD elevates the levels of microvesicles with different size distribution and selectively enriched proteins in human myeloma cells in vitro. The selectively enriched proteins, especially ZNF224, may play key roles in regulation of myeloma cells, allowing better adaptation to SD. Effect of mesenchymal stem cell-derived microvesicles in ex vivo expansion of cord blood nucleated cells and CD34+stem/progenitor cellsAim:Several studies showed that mesenchymal stem cells derived microvesicles (MSC-MVs) rather than MSC transdifFerentiation particepate in tissue repair process in animal model of acute kidney injury, this indicated a potential significance of MSC-MVs in the biologic properties of their mother cell, we hypothesize that MSC-MVs may also mimic the beneficial effect of MSC in ex vivo expansion of hematopoietic stem/progenitor cells. This study was aimed to assess the biologic significance of MSC-MVs on human cord blood derived nucleated cells and CD34+stem/progenitor cells.Methods:The effects of MSC-MVs on the proliferation of cord blood derived nucleated cells and CD34+stem/progenitor cells were compared with the co-culture treatment of bone marrow derived MSC. Proportions of monocytes, granulocytes, T lymphocytes, B lymphocytes, natural killer (NK) cells and CD34+cells in cord blood derived nucleated cells were flow cytometrically detected. The maintenance of hematopoietic progenitor cells after ex vivo expansion was investigated by colony formation assay. Adhesion of CD34+progenitor cells to human umbilical vein endothelial cells were studied by MTT assay. Ex vivo chemotaxis function on CD34+stem/progenitor cells was measured by transwell system.Results:It was found that MSC-MVs significantly promotes ex vivo expansion of human cord blood derived nucleated cells and CD34+stem/progenitor cells, do not affect the clonogenicity, the adhesion of endothelium or the ex vivo chemotaxis function of human CD34+progenitor cells. The proliferative effect of10μug/ml MSC-MVs was not as potential as their mother cell, however, when MSC co-culture experiments were performed in combination with10μg/ml MSC-MVs, these small vesicles greatly enhanced the proliferative effect of MSC. Besides,7days of co-culture with10μ/ml MSC-MVs alone increased the proportions of monocytes and granulocytes in cord blood derived nucleated cells, which seemed to mimic the beneficial properties of MSC. Moreover,10μg/ml MSC-MVs showed more powful inhibition on the proportions of T lymphocytes and NK cells than their mother cells, indicating that MSC-MVs play an important role in the immunomodulatory function of MSC.Conclusion:Our findings indicate hematopoietic supportive and immunomodulative properties of MSC-MVs on human cord blood derived nucleated cells and CD34+progenitor cells. This suggest that MSC-MVs could be exploited as a potential new therapeutic approach in umbilical cord blood transplantation(UCBT). |
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