Cell type and transfection reagent-dependent effects on viability, cell content, cell cycle and inflammation of RNAi in human primary mesenchymal cells

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

  • Hsiao Yin Yang, Department of Orthopaedics, University Medical Center Utrecht, Holland
  • Lucienne A. Vonk, Department of Orthopaedics, University Medical Center Utrecht, Holland
  • Ruud Licht, Department of Orthopaedics, University Medical Center Utrecht, Holland
  • Antonetta M G Van Boxtel, Department of Orthopaedics, University Medical Center Utrecht, Holland
  • Joris E J Bekkers, Department of Orthopaedics, University Medical Center Utrecht, Holland
  • Angela H M Kragten, Department of Orthopaedics, University Medical Center Utrecht, Holland
  • San Hein, Danmark
  • Oommen P. Varghese, Department of Materials Chemistry, Uppsala University, Uppsala Sweden, Sverige
  • Ken Howard
  • F. Cumhur Öner, Department of Orthopaedics, University Medical Center Utrecht, Holland
  • Wouter J A Dhert, Department of Orthopaedics, University Medical Center Utrecht, Holland
  • Laura B. Creemers, Department of Orthopaedics, University Medical Center Utrecht, Holland
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
OriginalsprogEngelsk
TidsskriftEuropean Journal of Pharmaceutical Sciences
Vol/bind53
Nummer1
Sider (fra-til)35-44
Antal sider10
ISSN0928-0987
DOI
StatusUdgivet - 12 mar. 2014

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