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. 2007 Aug;13(8):1179-83.
doi: 10.1261/rna.527207. Epub 2007 Jun 7.

Adipocyte differentiation induced using nonspecific siRNA controls in cultured human mesenchymal stem cells

Yunhe Xu  1 , Sayed-Hadi Mirmalek-Sani, Feng Lin, Junlong Zhang, Richard O C Oreffo
Affiliations

Adipocyte differentiation induced using nonspecific siRNA controls in cultured human mesenchymal stem cells

Yunhe Xu et al. RNA. 2007 Aug.

Abstract

RNA interference (RNAi) is gene silencing induced by double-stranded RNA of 21-23 nucleotides in length, termed small interfering RNA, or siRNA. RNAi-based techniques have been widely applied to elucidate gene function, identify drug targets, and used in trials as a promising adjunct to silence disease-causing genes. However, emerging evidence suggests unexpected changes in expression of untargeted genes as a consequence of an off-target effect by RNAi in mammalian cells. To date, our understanding of such effects on stem cells is limited. We transfected human fetal femur-derived mesenchymal stem cells using commercially available nonspecific siRNA controls and examined adipocyte differentiation in the cells using morphology, histochemistry, and quantitative real-time PCR to examine the expression of key genes for adipogenic or osteogenic differentiation. We report here the induction of adipocyte differentiation in human mesenchymal stem cells using nonspecific siRNAs raising concerns as to the specificity of RNAi in stem cells and, critically, a need to understand and delineate the rules governing the specificity of RNAi.

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Figures

FIGURE 1.
FIGURE 1.
Adipocyte differentiation induced using nonspecific siRNA control in fetal human femur-derived mesenchymal stem cells (fetal MSCs) under adipogenic (A–G) or osteogenic (H–N) conditions. Cell controls: Cells were grown in adipogenic (A,D) or osteogenic (H,K) conditions. Mock transfection controls: Cells were transfected using Lipofectamine 2000 without siRNA followed by adipogenic (B,E) or osteogenic induction (I,L). NS-siRNA 1: Cells were transfected with the nonspecific siRNA control 1 followed by adipogenic (C,F) or osteogenic induction (J,M). Morphology (A–C,H–J) and Oil Red O staining (D–F,K–M) images were taken from cells following 9 d of adipogenic or osteogenic inductions. Images C, F, J, and M were taken from cells transfected with the nonspecific siRNA at 16 nM. Scale bars = 50 μm. (G,N) Quantification of adipocytes. Adipocytes were identified by accumulation of lipid droplets and staining for lipid with Oil Red O under phase microscopy (10× magnification) in cells following 7 d of adipogenic (G) or osteogenic (N) inductions. Adipocytes were counted from nine randomly selected fields in three wells (three fields a well) for each of treatment group, and data are presented as mean ± standard deviation (SD). The data were taken from a representative study of three experiments using different preparations of fetal MSCs.
FIGURE 2.
FIGURE 2.
Examining PPARγ-2 and Runx2 mRNA using reverse transcription and qRT-PCR in fetal MSCs. (A) Dose-dependent enhancement of nonspecific siRNA on PPARγ-2, but not Runx2 expression. RNA samples were extracted from cells grown in basal cell culture medium (basal) or cells transfected with the nonspecific siRNA control 1(NS-siRNA 1) at indicated concentrations or without siRNA (Mock), followed by adipogenic induction for 14 d. (B,C) PPARγ-2 expression in cells with mock or NS-siRNA 1 transfection and adipogenic induction for 0, 7, and 14 d, respectively. Each treatment was established in duplicate and each sample was examined in duplicate. PPARγ-2 or Runx2 mRNA levels in cells with mock transfection at day 14 of adipogenic induction (B,C) were taken as 1.0. Relative PPARγ-2 or Runx2 mRNA levels for each treatment are presented as mean ± SD.
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