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, 14 (8), e0221679
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Human CAP Cells Represent a Novel Source for Functional, miRNA-loaded Exosome Production

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Human CAP Cells Represent a Novel Source for Functional, miRNA-loaded Exosome Production

Nikolas Zeh et al. PLoS One.

Abstract

Exosomes represent a promising delivery tool for nucleic acid-based pharmaceuticals. They are highly suitable for transporting therapeutic miRNAs to tumor cells, due to their natural membrane components. Further, exosomes are capable of effectively protecting nucleic acids against ribonucleases and enable the delivery of their content through cell membranes. However, no suitable production host for miRNA containing exosomes of non-tumorigenic origin has yet been identified. In this study we engineered an immortalised human amniocyte cell line (CAP® cells), whose exosomes were enriched and characterised. The cell line modifications not only enabled the production of GFP-labelled but also pro-apoptotic miRNA containing exosomes without negative influence on host cell growth. Furthermore, we demonstrated that pro-apoptotic miRNA containing CAP exosomes are taken up by ovarian cancer cells. Strikingly, delivery of functional exosomal miRNA led to downregulation of several reported target genes in the treated tumor cells. In summary, we revealed CAP cells of non-tumorigenic origin as a novel and efficient exosome production host with the potential to produce functional miRNA-loaded exosomes.

Conflict of interest statement

The authors have read the journal’s policy and the authors of this manuscript have the following competing interests: SS, SW, and NS are paid employees of CEVEC Pharmaceuticals GmbH. There are no patents, products in development or marketed products associated with this research to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Morphological and biochemical analysis of isolated CAP exosomes.
(A) Transmission electron microscopy pictures of parental (left) and modified (right) CAP exsomes. Scale bar represents 100 nm. (B) Western blot of 30 μg cellular and exosomal protein lysates per lane. Blotting performed on GRP78 (upper panel), turbo-GFP (middle panel) and CD9 protein (lower panel). Lane 1–4 represent cellular lysates, while lanes 5–8 depict exosomal lysate. (C) Quantification of Western blot Fig 1 B for CD9 signal. (D) Quantification of Western blot Fig 1 B for tGFP signal. (E) Quantification of Western blot Fig 1 B for GRP78 signal. (F) Flow cytometry analysis of GFP-positive exosomes isolated from parental and modified CAP cells.
Fig 2
Fig 2. Loading of pro-apoptotic miRNA-493 and miRNA-744 into exosomes via overexpression.
qPCR on overexpressed miRNAs of cellular and exosomal content. miRNA-493 shown in red bars, blue bars indicate miRNA-744 expression. miRNA overexpression in cells is given in blank bars, while checked bars represent overexpression in exosomes. CAP-CD63-GFP-Null cell line served as reference for miRNA overexpression cells, CAP-CD63-GFP-Null exosomes served as reference for exsosomes isolated from miRNA overexpression cells. U6 was used as housekeeping gene. Significance between cellular and exosomal overexpression was calculated via unpaired t-test [n = 3 replicates; mean ± SD; **p<0.01, ****p<0.0001].
Fig 3
Fig 3. Impact of CD63-GFP and miRNA overexpression on CAP cell growth.
(A) Viable cell density (VCD) of modified and of reference CAP cell lines during a 7 day cultivation, measured by trypan blue exclusion. (B) Viable cell density of modified and of reference CAP cell lines at day 6, measured via trypan blue exclusion. Significance was determined by using One-way ANOVA multiple comparison with Bonferroni correction [Each cell line cultured in 2 separate flasks; mean ± SD; n.s. = not significant, ****<0.0001]. (C) Viability of modified and of reference CAP cell lines during a 7 day cultivation, measured by trypan blue exclusion. (D) Viability of modified and of reference CAP cell llines at day 7, measured by trypan blue exclusion. Significance was determined by using One-way ANOVA multiple comparison with Bonferroni correction [Each cell line cultured in 2 separate flasks; mean ± SD; n.s. = not significant, ****<0.0001].
Fig 4
Fig 4. Uptake of exosomes from non-tumorigenic origin by ovarian cancer SKOV3 cells.
(A) Flow cytometric analysis of GFP-positive SKOV3 cells after co-culturing with CD63-GFP containing exosomes. Significance was calculated employing unpaired t-test [n = 3 replicates; mean ± SD; ****p<0.0001]. (B) qPCR on SKOV3 cells, co-cultured with miRNA-493 pre-loaded CAP exosomes. Fold change calculations referred to miRNA-Null exosomes treated SKOV3 cells. U6 served as loading control. (C) Confocal microscopy images of SKOV3 cells stained for organelle marker proteins after treatment with CD63-GFP containing exosomes. DAPI (blue) indicates nucleus staining, GFP (green) represents exosomes and Alexa647 (red) shows organelle staining of endosomes, endoplasmic reticulum, golgi and lysosomes. Scale bar represents 10 μm. (D) qPCR on miRNA-493 target genes STK38L, RAF1, FOXM1 and FRM1P in SKOV3 cells, after exosome co-culturing for 72 h. SKOV3 cells were treated with miRNA-Null and miRNA-493 loaded exosomes all 24 h with 250 μg exosomal protein. PPIA served as loading control. Significance was determined by unpaired t-test [n = 3 replicates; mean ± SD; n.s. = not significant; *p<0.05, **p<0.01].

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Grant support

This study was funded by CEVEC Pharmaceuticals GmbH in a cooperation-project with the University of Applied Sciences Biberach. CEVEC Pharmaceuticals funded the material for this study. CEVEC Pharmaceuticals GmbH also provided support for this study in the form of salaries for SS, SW, and NS. The funders had no additional role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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