Controlling mesenchymal stem cell gene expression using polymer-mediated delivery of siRNA

Abstract

siRNA treatment has great promise to specifically control gene expression and select cell behaviors but has delivery challenges limiting its use. Particularly for applications in regenerative medicine, uniform and consistent delivery of siRNA to control gene expression and subsequent stem cell functions, such as differentiation, is paramount. Therefore, a diblock copolymer was examined for its ability to effectively deliver siRNA to mesenchymal stem cells (MSCs). The diblock copolymers, which are composed of cationic blocks for siRNA complexation, protection, and uptake and pH-responsive blocks for endosomal escape, were shown to facilitate nearly 100% MSC uptake of siRNA. This is vastly superior to a commercially available control, DharmaFECT, which resulted in only ~60% siRNA positive MSCs. Moreover, the diblock copolymer, at conditions that result in excellent knockdown (down to ~10% of control gene expression), was cytocompatible, causing no negative effects on MSC survivability. In contrast, DharmaFECT/siRNA treatment resulted in only ~60% survivability of MSCs. Longitudinal knockdown after siRNA treatment was examined and protein knockdown persists for ~6 days regardless of delivery system (diblock copolymer or DharmaFECT). Finally, MSC phenotype and differentiation capacity was examined after treatment with control siRNA. There was no statistically significant differences on cell surface markers of diblock copolymer/siRNA or DharmaFECT/siRNA-treated or cells measured 2 weeks after siRNA delivery compared to untreated cells. Upon differentiation with typical media/culture conditions to adipogenic, chondrogenic, and osteogenic lineages and examination of histological staining markers, there was no discernible differences between treated and untreated cells, regardless of delivery mechanism. Thus, diblock copolymers examined herein facilitated uniform siRNA treatment of MSCs, inducing siRNA-specific gene and protein knockdown without adversely affecting MSC survival or differentiation capacity and therefore show great promise for use within regenerative medicine applications.

Figure 1

Poly(dimethylaminoethyl methacrylate)-b-poly(dimethylaminoethyl methacrylate-co-propylacrylic acid-co-butyl methacrylate) (pDMAEMA-b-p(DMAEMA-co-PAA-co-BMA)) polymer design for siRNA delivery to MSCs. Importantly, the first block, poly(dimethylaminoethyl methacrylate) (pDMAEMA, m) was designed to be partially protonated at physiological pH to allow for siRNA complexation and protection; the second block, n, was designed to be nearly charge neutral at physiologic pH (approximately 50% DMAEMA protonation and 50% PAA deprotonation) but to undergo a transition to more hydrophobic and membrane disruptive in lower pH environments of endo-lysosomal trafficking (m~60, n~70).

Figure 2

(A) Representative images demonstrating limited siRNA (green) uptake in untreated and siRNA only treated MSCs and punctate siRNA localization in DharmaFECT:siRNA treated MSCs while polymer:siRNA treated MSCs show robust, diffuse staining within the cytosol. Samples were treated for 24 hours with 37.5 nM siRNA with 4:1 charge ratio (polymer:siRNA) or standard treatment conditions (DharmaFECT:siRNA). Nuclei are stained with DAPI (blue fluorescence), bar = 100 µm. (B) Flow cytometry was used to quantify %-siRNA positive MSCs at 1, 4, and 24 hours after treatment (37.5 nM siRNA treatment with 4:1 charge ratio (polymer:siRNA) or standard treatment conditions (DharmaFECT:siRNA). Data are from three independent experiments conducted in triplicate with error bars representing standard deviation. Statistical significance was evaluated at a level of p<0.05 using 1-way analysis of variance to compare between groups at the same timepoints. * indicates significance compared to all other treatments.

Figure 3

Nonspecific cytotoxicity in MSCs at a variety of siRNA concentrations and charge ratios delivered via polymer diblock or DharmaFECT. (A) MSCs were treated with siRNA at different concentrations with charge ratio of 4:1 (diblock copolymer carrier) or by manufacturer’s recommendations (DharmaFECT) and after 24 hours analyzed for cell survivability using the alamarBlue metabolic assay. (B) MSCs were treated with siRNA at 37.5 nM with charge ratios of 1:1–8:1. Data are from three independent experiments conducted in triplicate relative to survivability of untreated cells (No Treatment) with error bars representing standard deviation. Statistical significance was evaluated at a level of p<0.05 use 1-way analysis of variance with * indicating significance from no treatment controls.

Figure 4

Specific siRNA-mediated knockdown of GAPDH in MSCs at a variety of siRNA concentrations and charge ratios delivered via polymer diblock or DharmaFECT carriers. (A) MSCs were treated with siRNA at different concentrations with charge ratio of 4:1 (diblock copolymer carrier) or by manufacturer’s recommendations (DharmaFECT) and after 48 hours analyzed for GAPDH gene expression using RT-PCR with β-actin as the housekeeping gene (see Table 1 for primer sequences used). (B) MSCs were treated with GAPDH siRNA at 37.5 nM with charge ratios of 1:1–8:1 using the diblock copolymer delivery system. Data are from three independent experiments conducted in triplicate relative to gene expression of untreated cells (No Treatment) with error bars representing standard deviation. Statistical significance was evaluated at a level of p<0.05 using one-way analysis of variance. All pairwise comparisons were found significant unless noted by NS (not significant).

Figure 4

Specific siRNA-mediated knockdown of GAPDH in MSCs at a variety of siRNA concentrations and charge ratios delivered via polymer diblock or DharmaFECT carriers. (A) MSCs were treated with siRNA at different concentrations with charge ratio of 4:1 (diblock copolymer carrier) or by manufacturer’s recommendations (DharmaFECT) and after 48 hours analyzed for GAPDH gene expression using RT-PCR with β-actin as the housekeeping gene (see Table 1 for primer sequences used). (B) MSCs were treated with GAPDH siRNA at 37.5 nM with charge ratios of 1:1–8:1 using the diblock copolymer delivery system. Data are from three independent experiments conducted in triplicate relative to gene expression of untreated cells (No Treatment) with error bars representing standard deviation. Statistical significance was evaluated at a level of p<0.05 using one-way analysis of variance. All pairwise comparisons were found significant unless noted by NS (not significant).

Figure 5

Longitudinal knockdown of GFP signal in GFP transgenic MSCs was assessed for the diblock copolymer and DharmaFECT delivery systems. GFP-MSCs were treated with siRNA at 37.5 nM with charge ratio of 4:1 (diblock copolymer carrier) or by manufacturer’s recommendations (DharmaFECT). At a variety of timepoints, GFP signal, which is normalized to DNA for each treatment to account for seeding fluctuations and nonspecific cytotoxicity of the control, DharmaFECT, was analyzed and is shown relative to untreated control cells at day 1, 2, 3, 4, 6, 8, 12 after siRNA treatment. Data are from three independent experiments conducted in triplicate relative to GFP expression of untreated cells (No Treatment) with error bars representing standard deviation. Statistical significance was evaluated at a level of p<0.05 using 1-way analysis of variance with & and * indicating significance from no treatment controls and polymer delivery, respectively.

Figure 6

Representative flow cytometry dot plots demonstrating no significant alterations in surface antigen expression by MSCs treated with siRNA. MSCs were treated with control siRNA at 37.5 nM with charge ratio of 4:1 (diblock copolymer carrier) or by manufacturer’s recommendations (DharmaFECT). Two weeks following treatment, MSCs were labeled with anti-CD90, anti-CD105, anti-CD45, and anti-CD44 antibodies and analyzed via flow cytometry. Fluorescence gating was established using both unstained cells and untreated cells. Data were analyzed from three independent experiments conducted in triplicate relative to untreated cells and statistical significance was evaluated at a level of p<0.05. The percentage of MSCs with the phenotype CD90+/CD105+/CD44+/CD45− for no treatment, polymer:siRNA, polymer alone, and DharmaFECT:siRNA treatments were 98.1 ± 0.4, 97.9 ± 0.2, 97.7 ± 0.8, and 93.6 ± 3.7, respectively, with no significant differences obtained upon pairwise comparison.

Figure 7

siRNA treated MSCs Retain Multilineage Potential. MSCs were treated with control siRNA at 37.5 nM with charge ratio of 4:1 (diblock copolymer carrier) or by manufacturer’s recommendations (DharmaFECT). 24 hrs after treatment, MSCs were differentiated using standard protocols for 3 weeks to induce osteogenesis (Von Kossa, mineralization), adipogenesis (oil red o, lipid droplets), or chondrogenesis (toluidine blue, glycosaminoglycans), bar = 100 µM.