Induction of stem-cell-derived functional neurons by NanoScript-based gene repression

Abstract

Even though gene repression is a powerful approach to exogenously regulate cellular behavior, developing a platform to effectively repress targeted genes, especially for stem-cell applications, remains elusive. Herein, we introduce a nanomaterial-based platform that is capable of mimicking the function of transcription repressor proteins to downregulate gene expression at the transcriptional level for enhancing stem-cell differentiation. We developed the "NanoScript" platform by integrating multiple gene repression molecules with a nanoparticle. First, we show a proof-of-concept demonstration using a GFP-specific NanoScript to knockdown GFP expression in neural stem cells (NSCs-GFP). Then, we show that a Sox9-specific NanoScript can repress Sox9 expression to initiate enhanced differentiation of NSCs into functional neurons. Overall, the tunable properties and gene-knockdown capabilities of NanoScript enables its utilization for gene-repression applications in stem cell biology.

Keywords: NanoScript; gene knockdown; nanoparticles; neuronal differentiation; transcription repressor proteins.

Figure 1. Schematic Representation of NanoScript-based Gene Repression

(a) When components of the transcriptional basal complex assemble on a target DNA sequence, such as the Sox9 promoter sequence, the corresponding gene is transcribed. (b) NanoScript-based gene expression is based on the synergistic effect of the DNA Binding Domain molecule for steric hindrance and the co-repressor molecule to disrupt the formation of the transcriptional basal complex on the target DNA sequence. (c) To demonstrate NanoScript-based repression in neural stem cells (NSCs), a GFP-specific NanoScript silences expression of GFP and a Sox9-specific NanoScript represses Sox9 to induce neuronal differentiation.

Figure 2. Construction and Characterization of NanoScript

(a) The magnetic core-shell nanoparticle (MCNP) was functionalized with PEG-terminated biomolecules, through thiol-gold interactions, to develop the NanoScript platform specific for either GFP or Sox9. (b) The hydrodynamic diameter and (c) transmission electron micrographs of NanoScript (scale bar = 20 nm). (d) A dye-labeled NanoScript (red) was transfected into rat NSCs and NanoScript was detected within the nucleus (blue) (scale bar = 20 μm).

Figure 3. NanoScript-GFP Effectively Silences GFP Expression

(a) Schematic representation of NanoScript-GFP downregulating GFP expression (i.e. reduction in green fluorescence from the cell) in GFP-labeled rat NSCs. (b) Fluorescence images of GFP-labeled rNSCs 4 days post transfection showing a maximal decrease in GFP expression (green) when NanoScript-GFP is transfected (scale bar = 20 μm). A 1 nM concentration of MCNPs was applied during the transfection. (c) Quantification of GFP expression in the fluorescence images corroborates the trend in the images and reveals that NanoScript-GFP has the highest GFP knockdown as compared to the controls. Quantification of GFP knockdown is an average from 6 images and standard error is from three independent trials.

Figure 4. NanoScript-Sox9 Represses Sox9 to Induce Functional Neuronal Differentiation

(a) Schematic representation of Sox9 repression in human NSCs by NanoScript-Sox9 induces enhanced neuronal differentiation. (b) Fluorescence images of hNSC stained with Tuj1 5 days post-transfection shows greater Tuj1 expression (red) when NanoScript-Sox9 is transfected. (Scale bar = 20 μm). (c) Gene expression analysis using qPCR in hNSCs reveals that repression of Sox9 correlates with an upregualtion of Tuj1. (Percent down-regulation of Sox9 and fold up-regulation of Tuj1 was calculated by normalizing to the housekeep gene, GAPDH, from the control) Standard error is from three independent trials (^* = P < 0.05). (d) Spontaneous calcium fluctuations via Fluo4 fluorescence (orange/yellow color) for an active neuron (white circle) during 18 seconds of imaging (scale bar = 20 μm). (e) Traces for the normalized fluorescence change (ΔF/F₀) representing spontaneous calcium ion influx for an active neuron (red line) and an inactive neuron (black line). Decreasing trend of the fluorescence is due to mild photobleaching.