Efficient siRNA Delivery by the Cationic LiposomeDOTAP in Human Hematopoietic Stem CellsDifferentiating into Dendritic Cells

[摘要] RNA interference technology is an ideal strategy to elucidate the mechanisms associated withhumanCD34+hematopoietic stem cell differentiation into dendritic cells. Simple manipulations invitro can unequivocally yield alloreactive or tolerogenic populations, suggesting key implications ofbiochemical players that might emerge as therapeutic targets for cancer or graft-versus-host disease.To knockdown proteins typically involved in the biology of dendritic cells, we employed ansiRNA delivery system based on the cationic liposome DOTAP as the carrier. Freshly-isolatedCD34+cells were transfected with siRNA for cathepsin S with negligible cytotoxicity and transfection rates(>60%) comparable to the efficiency shown by lentiviral vectors. Further, cathepsin S knockdown was performed during both cell commitment and through the entire 14-day differentiation process withrepeated transfection rounds that had no effect per se on cell development. Tested in parallel, other commercially-available chemical reagents failed to meet acceptable standards.In addition to safe and practical handling, a direct advantage of DOTAP over viral-mediatedtechniques is that transient silencing effects can be dynamically appraised through the recovery oftargeted proteins. Thus, our findings identify DOTAP as an excellent reagent for gene silencing inresting and differentiatingCD34+cells, suggesting a potential for applications in related preclinicalmodels.

[学科分类] 基础医学