title: Cell type and transfection reagent-dependent effects on viability, cell content, cell cycle and inflammation of RNAi in human primary mesenchymal cells.
abstract: The application of RNA interference (RNAi) has great therapeutic potential for degenerative diseases of cartilaginous tissues by means of fine tuning the phenotype of cells used for regeneration. However, possible non-specific effects of transfection per se might be relevant for future clinical application. In the current study, we selected two synthetic transfection reagents, a cationic lipid-based commercial reagent Lipofectamine RNAiMAX and polyethylenimine (PEI), and two naturally-derived transfection reagents, namely the polysaccharides chitosan (98% deacetylation) and hyaluronic acid (20% amidation), for siRNA delivery into primary mesenchymal cells including nucleus pulposus cells, articular chondrocytes and mesenchymal stem cells (MSCs). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous model gene to evaluate the extent of silencing by 20 nM or 200 nM siRNA at day 3 and day 6 post-transfection. In addition to silencing efficiency, non-specific effects such as cytotoxicity, change in DNA content and differentiation potential of cells were evaluated. Among the four transfection reagents, the commercial liposome-based agent was the most efficient reagent for siRNA delivery at 20 nM siRNA, followed by chitosan. Transfection using cationic liposomes, chitosan and PEI showed some decrease in viability and DNA content to varying degrees that was dependent on the siRNA dose and cell type evaluated, but independent of GAPDH knockdown. Some effects on DNA content were not accompanied by concomitant changes in viability. However, changes in expression of marker genes for cell cycle inhibition or progression, such as p21 and PCNA, could not explain the changes in DNA content. Interestingly, aspecific upregulation of GAPDH activity was found, which was limited to cartilaginous cells. In conclusion, non-specific effects should not be overlooked in the application of RNAi for mesenchymal cell transfection and may need to be overcome for its effective therapeutic application.
authors.affiliation: Department of Orthopaedics, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands.
authors.forename: Hsiao-yin
authors.initials: HY
authors.lastname: Yang
authors.affiliation: Department of Orthopaedics, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands.
authors.forename: Lucienne A
authors.initials: LA
authors.lastname: Vonk
authors.affiliation: Department of Orthopaedics, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands.
authors.forename: Ruud
authors.initials: R
authors.lastname: Licht
authors.affiliation: Department of Orthopaedics, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands.
authors.forename: Antonetta M G
authors.initials: AM
authors.lastname: van Boxtel
authors.affiliation: Department of Orthopaedics, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands.
authors.forename: Joris E J
authors.initials: JE
authors.lastname: Bekkers
authors.affiliation: Department of Orthopaedics, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands.
authors.forename: Angela H M
authors.initials: AH
authors.lastname: Kragten
authors.affiliation: Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark.
authors.forename: San
authors.initials: S
authors.lastname: Hein
authors.affiliation: Department of Material Chemistry, Uppsala University, Box 538, S-75121 Uppsala, Sweden.
authors.forename: Oommen P
authors.initials: OP
authors.lastname: Varghese
authors.affiliation: Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark.
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Howard
authors.affiliation: Department of Orthopaedics, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands.
authors.forename: F Cumhur
authors.initials: FC
authors.lastname: Öner
authors.affiliation: Department of Orthopaedics, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands; Faculty of Veterinary Medicine, University of Utrecht, The Netherlands.
authors.forename: Wouter J A
authors.initials: WJ
authors.lastname: Dhert
authors.affiliation: Department of Orthopaedics, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. Electronic address: L.B.Creemers@umcutrecht.nl.
authors.forename: Laura B
authors.initials: LB
authors.lastname: Creemers
mesh_terms: D053674:Aggrecans / Q000235:genetics; D002358:Cartilage, Articular / Q000166:cytology; D002453:Cell Cycle; D002470:Cell Survival; D002478:Cells, Cultured; D048271:Chitosan / Q000737:chemistry; D019902:Chondrocytes / Q000378:metabolism*; D024042:Collagen Type I / Q000235:genetics; D000090602:Collagen Type I, alpha 1 Chain; D024043:Collagen Type II / Q000235:genetics; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics; D051546:Cyclooxygenase 2 / Q000235:genetics; D015870:Gene Expression; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics; D006801:Humans; D006820:Hyaluronic Acid / Q000737:chemistry; D007249:Inflammation; D007403:Intervertebral Disc / Q000166:cytology; D007719:Knee Joint; D008055:Lipids / Q000737:chemistry; D008159:Lumbar Vertebrae; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D053495:Osteopontin / Q000235:genetics; D011094:Polyethyleneimine / Q000737:chemistry; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000235:genetics; D014162:Transfection
chemical_list: D053674:Aggrecans; C493956:CDKN1A protein, human; C499177:COL2A1 protein, human; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D024043:Collagen Type II; D050659:Core Binding Factor Alpha 1 Subunit; D050759:Cyclin-Dependent Kinase Inhibitor p21; D008055:Lipids; C086724:Lipofectamine; D018809:Proliferating Cell Nuclear Antigen; D034741:RNA, Small Interfering; C493709:RUNX2 protein, human; C506895:SPP1 protein, human; D053495:Osteopontin; D011094:Polyethyleneimine; D006820:Hyaluronic Acid; D048271:Chitosan; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)
keywords: Chondrocytes; MSCs; Non-specific effects; Nucleus pulposus; Transfection; siRNA