Effects Of Dendritic Cell-derived Exosomes On The Proliferation And Osteogenic Differentiation Of Mesenchymal Stem Cells

It is generally accepted that mesenchymal stem cells (MSCs) havepotent immuno-regulatory capacity mainly targeting dendritic cells (DC).Meanwhile, the questions about whether DC affects the biologicalproperties of MSCs and the molecules that mediate the reactions remainelusive. In this study, these possibilities have been investigated from aview-point of DC-derived exosomes (DCex). Firstly, the isolated andculture-expanded MSCs were identified in terms of cell morphology,surface phenotype analysis and their differentiation capacity. DC was alsoidentified by morphology and phenotype analysis. DCex were harvestedfrom the supernatants of DC culture medium by ultracentrifugation. Themorphology of DCex was observed under a transmission electronmicroscope and the surface phenotype of DCex was analyzed by flowcytometry (FCM). DCex protein concentration was measured by BCAmethod. Secondly, DCex labeled with DiI dye was co-cultured with DAPImarked MSCs, and the MSCs were then observed by confocal microscopyto define the cellular internalization of DCex. Finally, DCex was added intothe culture of MSCs, and the cell proliferation was tested by MTT test andfurther confirmed with a membrane dye dilution assay. Besides, thedifferentiation test was divided into3groups. MSCs were either exposed toDCex(10ug mL-1) or the standard osteogeneic induction condition. Thecells cultured in complete medium were served as the control. Theexpression levels of Runt related transcription factor2(Runx2) weredetected by real-time and traditional PCR. The cellular alkalinephosphatase (ALP) activity was also detected. The results showed thatMSCs and DC were successfully isolated and cultured. The morphology of MSCs was typical fusiform shape. Cells were induced to differentiate intoosteoblast and adipocyte The flow cytometry analysis results showed thatMSCs were positive for CD44and CD73, while negative for CD45, CD31and HLA-DR. Under the microscope, the dendritic cells were in irregularshape with slender synapses on their surface. DCex was either spherical oroval membrane vesicles with diameters of about40-100nm undertransmission electron microscope, the DCex expressed surface moleculesCD83, CD86, CD80and HLA-DR. DCex was incorporated by MSCs in atime-dependent manner. Compared with the blank control, the DCexexhibited potent promotion effect on the proliferation of MSCs when DCexwas added at concentrations or greater than5ug/ml (P<0.05). Thegrowth-promotion effect was obviously evidented when the MSCs wascultured for48h or longer. After cultured for7days, the MSCs treated withDCex highly expressed Runx2than the control group. After cultured for14days, ALP activity of the DCex treated MSC was markedly higher than thecontrol group (P<0.05).The results above show that DCex could be engulfed by MSCs, andthis internalization promotes MSCs growth and induces MSCs todifferentiate into osteoblast in vitro. It is assumed that DCex might be anew mediator in the interaction of DC on MSCs.