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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.

SUBMITTER: Benoit DS

PROVIDER: S-EPMC3501203 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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

Benoit Danielle S W DS Boutin Molly E ME

Biomacromolecules 20121011 11

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 ar ...[more]

PMID: 23020123

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Project description:RNA interference has immense potential to modulate cell functions. However, effective delivery of small interfering RNA (siRNA) while avoiding deleterious side effects has proven challenging. This study investigates both intended and unintended effects of diblock copolymer nanoparticle (NP) delivery of siRNA delivery to human mesenchymal stem cells (hMSC). Specifically, siRNA delivery was investigated at a range of NP-siRNA:hMSC ratios with a focus on the effects of NP-siRNA treatment on hMSC functions. Additionally, next generation RNA sequencing (RNAseq) was used with enrichment analysis to observe side effects in hMSC gene expression. Results show NP-siRNA delivery is negatively correlated with hMSC density. However, higher NP-siRNA:hMSC ratios increased cytotoxicity and decreased metabolic activity. hMSC proliferation was largely unaffected by NP-siRNA treatment, except for a threefold reduction in hMSCs seeded at 4,000 cells/cm2. Flow cytometry reveals that apoptosis is a function of NP-siRNA treatment time and seeding density; ∼14% of the treated hMSCs seeded at 8,000 cells/cm2 were annexin V+-siRNA+ 24 hr after treatment, while 11% of the treated population was annexin V+-siRNA-. RNAseq shows that NP-siRNA treatment results in transcriptomic changes in hMSCs, while pathway analysis shows upregulation of apoptosis signaling and downregulation of metabolism, cell cycle, and DNA replication pathways, as corroborated by apoptosis, metabolism, and proliferation assays. Additionally, multiple innate immune signaling pathways such as toll-like receptor, RIG-I-like receptor, and nuclear factor-κB signaling pathways are upregulated. Furthermore, and consistent with traditional siRNA immune activation, cytokine-cytokine receptor signaling was also upregulated. Overall, this study provides insight into NP-siRNA:hMSC ratios that are favorable for siRNA delivery. Moreover, NP-siRNA delivery results in side effects across the hMSC transcriptome that suggest activation of the innate immunity that could alter MSC functions associated with their therapeutic potential.

Project description:RNA interference (RNAi) has immense potential to modulate cell functions. However, effective delivery of siRNA while avoiding deleterious side effects has proven challenging. This study investigates both intended and unintended effects of diblock copolymer nanoparticle (NP) delivery of siRNA delivery to human mesenchymal stem cells (hMSC). Specifically, siRNA delivery was investigated at a range of NP-siRNA:hMSC ratios with a focus on the effects of NP-siRNA treatment on hMSC functions. Additionally, next-generation RNA sequencing (RNA-seq) was used with enrichment analysis to observe side effects in hMSC gene expression. Results show NP-siRNA delivery is negatively correlated with hMSC density. However, higher NP-siRNA:hMSC ratios increased cytotoxicity and decreased metabolic activity. hMSC proliferation was largely unaffected by NP-siRNA treatment, except for a 3-fold reduction in hMSCs seeded at 4,000 cells/cm2. Flow cytometry reveals that apoptosis is a function of NP-siRNA treatment time and seeding density; ~14% of the treated hMSCs seeded at 8,000 cells/cm2 were annexin V+-siRNA+ 24 h after treatment, while 11% of the treated population was annexin V+-siRNA-. RNA-seq shows that NP-siRNA treatment results in transcriptomic changes in hMSCs, while pathway analysis shows upregulation of apoptosis signaling and downregulation of metabolism, cell cycle, and DNA replication pathways, as corroborated by apoptosis, metabolism, and proliferation assays. Additionally, multiple innate immune signaling pathways such as toll-like receptor (TLR), retinoic acid-inducible gene 1 (RIG-I)-like receptor, and nuclear factor-κB (NF-κB) signaling pathways are upregulated. Furthermore, and consistent with traditional siRNA immune activation, cytokine-cytokine receptor signaling was also upregulated. Overall, this study provides insight into NP-siRNA:hMSC ratios that are favorable for siRNA delivery. Moreover, NP-siRNA delivery results in side effects across the MSC transcriptome that suggest activation of the innate immunity that could alter MSC functions associated with their therapeutic potential.

Project description:The use of small molecular drugs with gene manipulation offers synergistic therapeutic efficacy by targeting multiple signaling pathways for combined treatment. Stimulation of mesenchymal stem cells (MSCs) with osteoinductive small molecule phenamil combined with suppression of noggin is a promising therapeutic strategy that increases bone morphogenetic protein (BMP) signaling and bone repair. Our cationic Sterosome formulated with stearylamine (SA) and cholesterol (Chol) is an attractive co-delivery system that not only forms stable complexes with small interfering RNA (siRNA) molecules but also solubilizes hydrophobic small molecules in a single vehicle, for directing stem cell differentiation. Herein, we demonstrate the ability of SA/Chol Sterosomes to simultaneously deliver hydrophobic small molecule phenamil and noggin-directed siRNA to enhance osteogenic differentiation of MSCs both in in vitro two- and three-dimensional settings as well as in a mouse calvarial defect model. These results suggest a novel liposomal platform to simultaneously deliver therapeutic genes and small molecules for combined therapy.Statement of significanceApplication of phenamil, a small molecular bone morphogenetic protein (BMP) stimulator, combined with suppression of natural BMP antagonists such as noggin is a promising therapeutic strategy to enhance bone regeneration. Here, we present a novel strategy to co-deliver hydrophobic small molecule phenamil and noggin-targeted siRNA via cationic Sterosomes formed with stearylamine (SA) and high content of cholesterol (Chol) to enhance osteogenesis and bone repair. SA/Chol Sterosomes demonstrated high phenamil encapsulation efficiency, supported sustained release of encapsulated drugs, and significantly reduced drug dose requirements to induce osteogenic differentiation of mesenchymal stem cells (MSCs). Simultaneous deliver of phenamil and noggin siRNA in a single vehicle synergistically enhanced MSC osteogenesis and calvarial bone repair. This study suggests a new non-phospholipid liposomal formulation to simultaneously deliver small molecules and therapeutic genes for combined treatment.

Dataset Information

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

Publications

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

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