RNA interference technology is an ideal strategy to elucidate the mechanisms associated with human CD34+ hematopoietic stem cell differentiation into dendritic cells. Simple manipulations in vitro can unequivocally yield alloreactive or tolerogenic populations, suggesting key implications of biochemical players thatmight emerge as therapeutic targets for cancer or graft-versushost disease. To knockdown proteins typically involved in the biology of dendritic cells, we employed an siRNA delivery system based on the cationic liposome DOTAP as the carrier. Freshly-isolated CD34+ 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 with repeated 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-mediated techniques is that transient silencing effects can be dynamically appraised through the recovery of targeted proteins. Thus, our findings identify DOTAP as an excellent reagent for gene silencing in resting and differentiating CD34+ cells, suggesting a potential for applications in related preclinical models.

Efficient siRNA Delivery by the Cationic Liposome DOTAP in Human Hematopoietic Stem Cells Differentiating into Dendritic Cells

MARTINO, Sabata
Writing – Review & Editing
;
DI GIROLAMO, ILARIA;TIRIBUZI, ROBERTO;DATTI, Alessandro
Writing – Review & Editing
;
ORLACCHIO, Aldo
2009

Abstract

RNA interference technology is an ideal strategy to elucidate the mechanisms associated with human CD34+ hematopoietic stem cell differentiation into dendritic cells. Simple manipulations in vitro can unequivocally yield alloreactive or tolerogenic populations, suggesting key implications of biochemical players thatmight emerge as therapeutic targets for cancer or graft-versushost disease. To knockdown proteins typically involved in the biology of dendritic cells, we employed an siRNA delivery system based on the cationic liposome DOTAP as the carrier. Freshly-isolated CD34+ 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 with repeated 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-mediated techniques is that transient silencing effects can be dynamically appraised through the recovery of targeted proteins. Thus, our findings identify DOTAP as an excellent reagent for gene silencing in resting and differentiating CD34+ cells, suggesting a potential for applications in related preclinical models.
2009
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11391/161774
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