Abstract
Exosomes are small (30–100 nm) membrane vesicles secreted by a variety of cell types and only recently have emerged as a new avenue for cell-to-cell communication. They are natural shuttles of RNA and protein cargo, making them attractive as potential therapeutic delivery vehicles. MicroRNAs (miRNAs) are short non-coding RNAs which regulate biological processes and can be found in exosomes. Here we characterized the miRNA contents of exosomes derived from human neural stem cells (hNSCs). Our investigated hNSC line is a clonal, conditionally immortalized cell line, compliant with good manufacturing practice (GMP), and in clinical trials for stroke and critical limb ischemia in the UK (clinicaltrials.gov: NCT01151124, NCT02117635, and NCT01916369). By using next generation sequencing (NGS) technology we identified the presence of a variety of miRNAs in both exosomal and cellular preparations. Many of these miRNAs were enriched in exosomes indicating that cells specifically sort them for extracellular release. Although exosomes have been proven to contain miRNAs, the copy number quantification per exosome of a given miRNA remains unclear. Herein we quantified by real-time PCR a highly shuttled exosomal miRNA subtype (hsa-miR-1246) in order to assess its stoichiometry per exosome. Furthermore, we utilized an in vitro system to confirm its functional transfer by measuring the reduction in luciferase expression using a 3’ untranslated region dual luciferase reporter assay. In summary, NGS analysis allowed the identification of a unique set of hNSC derived exosomal miRNAs. Stoichiometry and functional transfer analysis of one of the most abundant identified miRNA, hsa-miR-1246, were measured to support biological relevance of exosomal miRNA delivery.
Highlights
Exosomes are small membrane vesicles that originate from multi-vesicular bodies and are secreted by a variety of cell types [1, 2]
We further analyzed size distribution and number of particles per μl of exosome preparations using nanoparticle tracking analysis (NTA) and qNano. Based on these measurements and the shoulder associated with the major distribution peak (Fig 1A and 1B), our preparations have a modal size of 95 nm ± 2.3 standard error of the mean (SEM) (NTA) and 86 nm ± 2.2 SEM and confirmed populations with particle sizes consistent with exosomes
The identification of miRNA contents has a tendency to be biased by the choice of detection system analysis
Summary
Exosomes are small membrane vesicles that originate from multi-vesicular bodies and are secreted by a variety of cell types [1, 2]. Stoichiometry and Transfer Analysis of Exosomal miRNA. Their commercial affiliation with ReNeuron does not alter their adherence to PLOS ONE policies on sharing data and materials. MiRNA transfer via exosomes could bypass recipient cell transcriptional controls providing a relatively direct means of regulation. Studies, delivering purified exosomes to recipient cells, have reported transfer of miRNAs in experimental settings and support their use as a potential therapeutic strategy [8, 9]. The possibility of exosome-mediated miRNA transfer as a mode of intercellular communication is an attractive concept, the biological significance of miRNA transfer to target cells needs to be clarified [10]. Stoichiometric analysis of exosomal miRNA copy number contents might provide insight into the significance of miRNA-based intercellular communication
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