Investigation of Content, Stoichiometry and Transfer of miRNA from Human Neural Stem Cell Line Derived Exosomes

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.

Fig 1. The characterization of exosomes derived from hNSCs.

Size distribution of exosomes analyzed with the NTA (A) and qNano (B), representative traces. In agreement with exosome sizes, isolated exosomes had a mode of approximately 100 nm. (C) Molecular characterization of exosomes and producer hNSCs by Western blotting. Protein extracts from hNSCs and exosomes were assessed using antibodies against exosomal protein markers (CD81 and CD61), and hNSC protein marker (MYC).

Fig 2. MiRNA next generation sequencing.

Cellular (A) and exosomal (B) total RNAs were processed by an Agilent 2100 Bioanalyzer. The corresponding virtual gel images generated by the software are depicted as electropherograms. (C) Representative diagram of differential miRNA distribution in exosomes compared to hNSC producers. MiRNA types preferentially released in exosomes are presented in red or retained within the hNSCs in blue, data expressed as log₂ ratio of exosomal/cellular miRNAs normalized read counts. Pie chart representation of the distribution of small RNA categories in hNSC (D) and exosome (E) samples.

Fig 3. Absolute quantification of selected miRNA.

(A) Example of standard curve obtained for miRNA quantification. MiRNA mimic was diluted to a concentration range of 3.01×10¹²–3.01×10⁷ copies per well. Each point plotted is an average of triplicate fluorescence values for each standard concentration measured. (B) Diagram showing the quantification of hsa-miR-1246 copy number per hNSC and per exosome (EXO). Exosome particle quantification was performed using two independent methods, NTA and qNano; cell number quantification was performed by hemocytometer analysis. The error bars represent ± SEM.

Fig 4. Functional transfer assessment of exosomal miRNA.

(A) In vitro assay validation of miR-1246/ 3’UTR-FAM53C mRNA binding was evaluated by measuring the relative luciferase reduction activity caused by the co-transfection of HeLa cells with the dual luciferase reporter plasmid and a range of dilution of hsa-miR-1246. Data were expressed as percent of control (scrambled miRNA) transfections, n = 6. (B) Biological functional transfer assessment of exosomal hsa-miR-1246 cargo. HeLa cells were pre-treated with purified exosomes and transfected with FAM53C 3’UTR dual luciferase plasmid. Relative reduction in luciferase activity in hsa-miR-1246 mimic and exosome treated samples was expressed as percent of control (scrambled miRNA) transfections, n = 6. The error bars represent ± SEM; *** p<0.001. (C) Western blot analysis of FAM53C in untreated (control), scrambled miRNA, hsa-miR-1246 mimic and exosome treated HeLa. FAM53C expression levels were normalised to α-tubulin following quantification by densitometry (ImageJ) and presented as relative ratio to the control sample.