Dataset Information

Evaluating Side Effects of Nanoparticle-mediated siRNA Delivery to Mesenchymal Stem Cells Using Next-generation Sequencing and Enrichment Analysis

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE87497 | GEO | 2016/09/30

SECONDARY ACCESSION(S): PRJNA344928

REPOSITORIES: GEO

ACCESS DATA

Similar Datasets

Project description:Multipotent stromal cells (MSCs) are currently in clinical trials for a number of inflammatory diseases. Recent studies have demonstrated the ability of MSCs to attenuate inflammation in rodent models of acute lung injury (ALI) suggesting that MSCs may also be beneficial in treating ALI. To better understand how human MSCs (hMSCs) may act in ALI, the lungs of immunocompetent mice were exposed to lipopolysaccharide (LPS) and 4 hr later bone marrow derived hMSCS were delivered by oropharyngeal aspiration (OA). Administration of hMSCs significantly reduced the expression of pro-inflammatory cytokines, neutrophil counts and total protein in bronchoalveolar lavage. There was a concomitant reduction in pulmonary edema as indicated by a decrease in lung wet/dry weight ratio. The anti-inflammatory effects of hMSCs were not dependent on localization to the lung, as intraperitoneal administration of hMSCs also attenuated LPS-induced inflammation in the lung. Microarray analysis revealed significant induction of TNF-α-induced protein 6 (TSG-6) expression by hMSCs 12 hr after OA delivery to LPS-exposed lungs. Knockdown of TSG-6 expression in hMSCs by RNA interference abrogated most of their anti-inflammatory effects. In addition, intra-pulmonary delivery of recombinant human TSG-6 reduced LPS-induced inflammation in the lung. These results show that hMSCs recapitulate the observed beneficial effects of rodent MSCs in animal models of ALI and suggest that the anti-inflammatory properties of hMSCs in the lung are explained, at least in part, by activation of hMSCs to secrete TSG-6. Eight- to 10-week-old female BALB/C mice were treated with either 1 mg/kg lipopolysaccharide (LPS) in 100 μl PBS or an equal volume of PBS, as vehicle control, by oropharyngeal aspiration (OA). Four hours after LPS exposure, 250,000 human multipotent stromal cells in 100 μl of PBS were given by OA and 30 min later a second dose of equal concentration was administered, for a total of 500,000 hMSC. As a control, 200 μl PBS was delivered as described above. After 12 h, total RNA was isolated from (A) lungs of LPS-exposed mice treated with either hMSCs (LPS+MSC) or PBS (LPS+PBS), and (B) lungs of PBS-exposed mice treated with hMSCs (PBS+MSC). To obtain additional controls, hMSCs were mixed in vitro with either LPS- (LPS+MSC in vitro mix) or PBS-exposed (PBS+MSC in vitro mix) mouse lungs just before RNA isolation. RNA samples containing mouse and human RNA were first analyzed for amount of human RNA based on human GAPDH signal from real-time RT PCR. Samples with similar human RNA content were used for both mouse and human microarrays

Project description:Nucleotides triphosphates are extracellular messengers binding to specific plasma membrane receptors (P2Rs) that modulate responses as different as proliferation, differentiation, migration or cell death on several cell types including hematopoietic stem cells. Little and controversial information is available on the role of extracellular nucleotides in human mesenchimal stem cells (hMSCs). In this study, we assessed whether P2Rs are expressed and functional in bone marrow-derived hMSCs. Our results demonstrated, at the mRNA and protein level, the expression of all P2X and P2Y receptor subtypes identified so far. P2R activation by their natural ligands adenosine triphosphate (ATP) and uridine triphosphate (UTP) induced in hMSCs, intracellular Ca2+ concentration changes, plasma membrane depolarization and permeabilization. hMSCs were resistant to the cytotoxic effects of high dose ATP despite the expression of permeabilizing P2Rs as demonstrated by the lack of morphological changes, significant release of intracellular markers of cell death or modification of the mitochondrial network. Gene expression profiling revealed the down-regulation of cell proliferation genes whereas genes involved in cell migration and cytokine production were strongly up-regulated by ATP. Functional studies confirmed the inhibitory activity of ATP on proliferation of hMSCs and clonogenic progenitors. Moreover, ATP exerted a chemotactic effect on hMSCs and increased their migration in response to the chemokine CXCL12. Finally, whereas ATP did not affect T-cell inhibitory activity of hMSCs, the nucleotide increased the production of pro-inflammatory cytokines by hMSCs. Thus, our data show that purinergic signaling modulates hMSC functions and point to a role for extracellular nucleotides on hMSCs biology. hMSCs from 6 healthy donors were seeded at a density of 2.5 x 103 cells/cm2 for 24 hours with or without 1mM ATP. We then assessed the transcriptome profile of ATPM-bM-^@M-^Streated and untreated cells using Affymetrix HG-U133 Plus 2 GeneChip array.

Project description:The methodology for the repair of critical-sized or non-union bone lesions has unpredictable efficacy due in part to our incomplete knowledge of bone repair and the biocompatibility of bone substitutes. Although human mesenchymal stem cells (hMSCs) differentiate into osteoblasts, which promote bone growth, their ability to repair bone has been unpredictable. We hypothesized that given the multi-stage process of osteogenesis, hMSC-mediated repair might be maximal at a specific time-point of healing. Utilizing a mouse model of calvarial healing, we demonstrate that the osteo-repair capacity of hMSCs can be substantially augmented by treatment with an inhibitor of peroxisome-proliferator-activated-receptor-γ, but efficacy is confined to the rapid osteogenic phase. Upon entry into the bone-remodeling phase, hMSC retention signals are lost, resulting in truncation of healing. To solve this limitation, we prepared a scaffold consisting of hMSC-derived extracellular matrix (ECM) containing the necessary biomolecules for extended site-specific hMSC retention. When inhibitor-treated hMSCs were co-administered with ECM, they remained at the injury well into the remodeling phase of healing, which resulted in reproducible and complete repair of critical-sized defects in 3 weeks. These data suggest that hMSC-derived ECM and inhibitor-treated hMSCs could be employed at optimal times to substantially and reproducibly improve bone repair. To gain insight into the superior healing potential of GW-hMSCs and also what might be accounting for their extended engraftment, microarray analyses on the RNA extracted from the calvarial tissue recovered after days 5 and 14 were performed. Equal amounts of total RNA from 4 animals per group and time point were pooled and animals receiving control (DMSO) or peroxisome proliferator-activated receptor-gamma inhibitor GW9662-treated hMSCs were compared with the assumption that murine cross-hybridization would be constant throughout the samples and thus be subtracted from the analysis.

Dataset Information

Evaluating Side Effects of Nanoparticle-mediated siRNA Delivery to Mesenchymal Stem Cells Using Next-generation Sequencing and Enrichment Analysis

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure