Directing neuronal differentiation of primary neural progenitor cells by gene knockdown approach
Low, Wei Ching
Yau, Winifred Wing Yiu
Stanton, Lawrence W.
Chew, Sing Yian
Date of Issue2012
School of Chemical and Biomedical Engineering
Directing differentiation of neural stem/progenitor cells (NPCs) to produce functional neurons is a promising remedy for neural pathological conditions. The major challenge, however, lies in the effective and efficient generation of a sizable population of neurons. A potential strategy is to incorporate RNA interference (RNAi) during directed stem cell differentiation to recapitulate the complex cell-signaling cascades that often occurs during the process. In this study, in vitro silencing of RE1-silencing transcription factor (REST) was carried out using small-interfering RNAs (siRNAs) to evaluate the efficacy of combining REST knockdown with conventional differentiation approaches to enhance neurogenesis. While earlier studies have demonstrated enhanced neuronal lineage commitment from embryonic stem cells and mesenchymal stem cells upon REST knockdown, the effects of REST silencing during other stages of neural development have not been extensively evaluated. We hypothesize that REST knockdown would enhance NPC development to mature neurons and that induced REST silencing can serve as a potential biochemical approach to direct cell fate. Under nonspecific induction conditions, REST knockdown induced eightfold higher Tuj1 mRNA expression at day 14 compared with untransfected cells and cells subjected to scrambled-siRNA treatment (controls). Immunostaining also revealed greater percentage of Tuj1 positive cells with REST knockdown. Combined with neuronal induction, REST silencing enhanced the kinetics of neuronal differentiation and the rate of maturation of committed neuronal cells. Specifically, upregulation of MAP2 occurred as early as 3 days after induction with REST silencing and the expression was comparable to the controls at day 14. Likewise, downregulation of REST generated more than twice the percentage of Tuj1 and MAP2 positive cells compared with controls at day 5 (p<0.05). Morphologically, REST-silencing enhanced the number and length of neurite extensions from Tuj1 positive cells (p<0.05), which was not evaluated in previous differentiation studies with REST knockdown. Taken together, these results demonstrate the efficacy of combining REST silencing during directed NPC differentiation to enhance the rate of differentiation and subsequent maturation of NPCs. This study also highlights the potential of RNAi as a biomedical strategy for guided stem cell differentiation.
DNA and cell biology
© 2012 Mary Ann Liebert, Inc. This paper was published in DNA and Cell Biology and is made available as an electronic reprint (preprint) with permission of Mary Ann Liebert, Inc. The paper can be found at the following official DOI: [http://dx.doi.org/10.1089/dna.2011.1557]. One print or electronic copy may be made for personal use only. Systematic or multiple reproduction, distribution to multiple locations via electronic or other means, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper is prohibited and is subject to penalties under law.