microRNA sequencing of MSC-derived Extracellular vesicles
Human bone marrow-derived MSCs were stimulated with Bronchoalveolar lavage fluid (BALF) from healthy individuals or patients with ARDS, Cystic Fibrosis, Cystic Fibrosis positive for Aspergillus or untreated untreated MSC. Extracellular vesicles were isolated from MSC conditioned medium following minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles (J Extracell Vesicles. 2014 Dec 22:3:26913. doi: 10.3402/jev.v3.26913. eCollection 2014). In Phase 1 of this study, differential expression as well as discriminant analysis was used to identify 14 microRNAs that were differentially expressed in MSC-derived EVs from MSCs exposed to BALF from ARDS patients compared to healthy controls. The effect of EVs on cellular physiology and was demonstrated in vitro by demonstration that wwo miRNAs involved in regulation of the cystic fibrosis transmembrane conductance regulator (CFTR), miRNA-145-5p and miRNA-138-5p, were also significantly increased in ARDS BALF-exposed hMSCs EVs. Functionally, EVs from hMSCs exposed to either ARDS or HV BALF had differential on CFTR Cl- secretion by cultured primary human bronchial epithelial cells, an effect predicted to reduce mucociliary clearance. Overall design: Translational study to demonstrate the acute respiratory distress syndrome (ARDS) inflammatory environment alters mesenchymal stromal cell (MSC) gene and protein expression as well as the microRNA (miRNA) content of MSC-extracellular vesicle (EVs). RNA was isolated from each EV preparation as described in the manuscript submitted and only samples that passed quality control with an A260/A280 above 1.80 were used for RNA sequencing (control=16; ARDS=12; HV=14). A total of 35 ul (35 ng/µl) of eRNA (EV preparation-derived RNA) was used for miRNA sequencing performed using the HTG EdgeSeq miRNA Whole Transcriptome Assay (miRNA WTA, as per manufacturer's instructions as published). The miRNA library was sequenced on a NextSeq (Illumina, Inc., San Diego, CA) using a V3 150-cycle kit with two index reads. PhiX (Roche, Mississauga, ON, CAN). Data were returned from the sequencer in the form of demultiplexed FASTQ files, with one file per original well of the assay. The HTG EdgeSeq Parser (v. 5.0.535.3181, HTG Molecular, Tucson, AZ, USA) was used to align the FASTQ files to the probe list to collate the data. Raw read counts for the 42 samples were imported into R to perform differential expression analyses with the R package DESeq2 28. Variance stabilization transformation was used to prepare data for analyses. Significance analyses of microarrays (SAM) was run using one class analysis approach to identify miRNAs over-represented in EVs derived from control hMSCs 29 based on the normalized data using variance stabilization transformation. The delta value was set to 7 (the best delta parameter value selected by the software with the lowest False Discovery Rate (FDR]) using 1000 permutations, FDR cut-off was 0.001 (%).