Integrated transcriptomic and proteomic analysis of the molecular cargo of extracellular vesicles derived from porcine adipose tissue-derived mesenchymal stem cells

<div><p>Background</p><p>Mesenchymal stromal/stem cell (MSC) transplantation is a promising therapy for tissue regeneration. Extracellular vesicles (EVs) released by MSCs act as their paracrine effectors by delivering proteins and genetic material to recipient cells. To assess how their cargo mediates biological processes that drive their therapeutic effects, we integrated miRNA, mRNA, and protein expression data of EVs from porcine adipose tissue-derived MSCs.</p><p>Methods</p><p>Simultaneous expression profiles of miRNAs, mRNAs, and proteins were obtained by high-throughput sequencing and LC-MS/MS proteomic analysis in porcine MSCs and their daughter EVs (n = 3 each). TargetScan and ComiR were used to predict miRNA target genes. Functional annotation analysis was performed using DAVID 6.7 database to rank primary gene ontology categories for the enriched mRNAs, miRNA target genes, and proteins. STRING was used to predict associations between mRNA and miRNA target genes.</p><p>Results</p><p>Differential expression analysis revealed 4 miRNAs, 255 mRNAs, and 277 proteins enriched in EVs versus MSCs (fold change >2, p<0.05). EV-enriched miRNAs target transcription factors (TFs) and EV-enriched mRNAs encode TFs, but TF proteins are not enriched in EVs. Rather, EVs are enriched for proteins that support extracellular matrix remodeling, blood coagulation, inflammation, and angiogenesis.</p><p>Conclusions</p><p>Porcine MSC-derived EVs contain a genetic cargo of miRNAs and mRNAs that collectively control TF activity in EVs and recipient cells, as well as proteins capable of modulating cellular pathways linked to tissue repair. These properties provide the fundamental basis for considering therapeutic use of EVs in tissue regeneration.</p></div

miRNA enriched in EVs.

<p>A. Heat map, miRNA expression, and fold change showing that ssc-miR-183, ssc-miR-378, ssc-miR-140-3p, and ssc-miR-222 were upregulated in EVs compared to MSCs. B. Number (top) and distribution (bottom) of miRNA targets of EV-enriched miRNAs. C: Functional annotation clustering of miRNA targets.</p

Characterization of MSC-derived EVs.

<p>A: Transmission electron microscopy showing cultured MSCs releasing EVs. B: EVs express common EV (CD9, CD29, and CD63) and MSC (CD73 and CD105) markers. C: Size distribution of isolated EVs revealed a similar proportion of small microvesicles and exosomes.</p

Validation of RNAseq and proteomic analysis.

<p>Expression of the candidate miRNAs (miR-140-3p and miR-378), genes (SMAD2 and POU2F1), and proteins (C2 and TGFβ-1) was concordant with the RNAseq and proteomics findings. MSC-derived EVs (PKH26, red) were internalized by cultured of human umbilical vein endothelial cells (HUVECs) (CD31, green). DAPI DAPI = blue, nuclei. Incubation of HUVECs with EVs increased HUVEC expression of miR-140-3p, miR-378, SMAD2, POU2F1, C2, and TGFβ-1. *p<0.05 vs. MSCs, ‡p<0.05 vs. HUVECs.</p

Overview of experimental design and data analysis.

<p>Abdominal fat was collected from female domestic pigs, and mesenchymal stem cells (MSCs) and their daughter extracellular vesicles (EVs) isolated and characterized. mRNA and miRNA sequencing analysis and LC-MS/MS proteomic analysis were performed in both MSCs and EVs (n = 3 each). Differentially expression analysis was performed and EV-enriched miRNA, mRNA, and proteins identified. miRNA predicted targets were identified with TargetScan and ComiR. Functional annotation clustering analysis was performed using DAVID 6.7 database to obtain a ranking of primary gene ontology categories for the enriched mRNA, miRNA target genes, and proteins. Venn diagrams were used to visualize genes shared between each group and their interactions, and STRING to predict associations between mRNA and miRNA target genes.</p

Interactions between Transcription Factor (TF) miRNA targets and TF mRNAs enriched in EVs.

<p>TF miRNA targets and mRNA TF network derived from STRING. Nodes represent TF miRNA targets and mRNA TFs, and color lines their interactions according to the functional association networks from the STRING database. Red circles indicate common (TF) miRNA targets and TF mRNAs enriched in EVs.</p

Proteins enriched in EVs.

<p>A. Heat map of 277 proteins upregulated in EVs compared to MSCs. B. Panther analysis of molecular function and cellular component of proteins upregulated in EVs. C. Functional annotation clustering of EV-enriched proteins.</p

Interactions among miRNA, mRNA and proteins enriched in EVs.

<p>A. Venn diagram showing distribution of miRNA, mRNA, and proteins enriched in EVs, and their interactions. B. Functional annotation clustering of 41 common miRNA targets and mRNAs enriched in EVs. C. Distribution of transcription factor (TF) miRNA targets, TF mRNA, and TF proteins enriched in EVs.</p