Efficient siRNA delivery by the cationic liposome DOTAP in human hematopoietic stem cells differentiating into dendritic cells.
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 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 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.
Martino
Sabata
S
Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Sezione di Biochimica e Biologia Molecolare, Università di Perugia, Via del Giochetto, 06100 Perugia, Italy.
di Girolamo
Ilaria
I
Tiribuzi
Roberto
R
D'Angelo
Francesco
F
Datti
Alessandro
A
Orlacchio
Aldo
A
D002403:Cathepsins / Q000235:genetics* / Q000378:metabolism; D002454:Cell Differentiation; D002470:Cell Survival; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000166:cytology* / Q000502:physiology; D005229:Fatty Acids, Monounsaturated; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000502:physiology; D006801:Humans; D008081:Liposomes / Q000737:chemistry; D000644:Quaternary Ammonium Compounds; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
D005229:Fatty Acids, Monounsaturated; D008081:Liposomes; D000644:Quaternary Ammonium Compounds; D034741:RNA, Small Interfering; D002403:Cathepsins; C028217:cathepsin S; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane