Date Published: March 23, 2017
Publisher: Public Library of Science
Author(s): Alfonso Eirin, Xiang-Yang Zhu, Amrutesh S. Puranik, John R. Woollard, Hui Tang, Surendra Dasari, Amir Lerman, Andre J. van Wijnen, Lilach O. Lerman, Benedetta Bussolati.
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.
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.
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. 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.
Mesenchymal stromal/stem cells (MSCs) are being tested in clinical trials to evaluate their therapeutic efficacy in a broad spectrum of diseases . The potential medical benefits of MSCs reside in their remarkable differentiation capabilities, as well as potent pro-angiogenic and immunomodulatory properties . Given that MSCs can be easily obtained from a variety of tissues, including adipose tissue, these cells offer an important advantage in clinical applications compared to other stem cell types.
The study was approved by the Mayo Clinic Animal Care and Use Committee. Fig 1 summarizes the experimental design and data analysis. Abdominal fat was collected from 3 female domestic pigs. Animals were anaesthetized with 0.25g of intramuscular tiletamine hydrochloride/zolazepam hydrochloride and 0.5g of xylazine, and anesthesia was maintained with intravenous ketamine (0.2mg/kg/min) and xylazine (0.03mg/kg/min). The abdominal region lateral to the umbilicus was draped and prepped under standard sterile technique using alcohol and local anesthesia (2mL 2% lidocaine). A small superficial 0.5cm skin incision was performed, and adipose tissue collected and stored in sterilized tubes. Analgesics (topical 1% lidocaine; Buprenorphine 0.005–0.02mg/kg IM, IV q6-12h) were administered for 24hrs days after fat biopsy.
The beneficial effects of MSCs have been attributed in part to the release of EVs, which participate in intercellular communication between MSCs and damaged cells. Coupled with their potential to alter the phenotype of recipient cells and exert tissue trophic and reparative effects , EVs have emerged as a novel and viable alternative to whole cell therapies. In this study, we have evaluated the molecular relatedness and putative functions of mRNAs, miRNAs, and proteins packed in MSC-derived EVs. Importantly, we found that the mRNA, miRNA, and protein content of EVs is distinct and mostly independent. Proteins present in EVs are distinct from those encoded by mRNAs included in EVs, and most of the miRNAs enriched in EVs target mRNAs distinct from those enriched in EVs. Hence, there is no direct regulatory correlation between mRNAs, miRNAs and proteins, in the sense that EVs do not incorporate mRNAs that encode for the same proteins that they contain. These findings are consistent with the premise that mRNAs are not translated in EVs and miRNAs do not actively silence translation of their cognate mRNAs.
In summary, our study shows that porcine MSC-derived EVs contain mRNAs, miRNAs, and proteins capable of modifying recipient cell phenotype and function, modulating multiple cellular pathways, and activating regenerative mechanisms. Moreover, differences in miRNA, mRNA, and protein composition between EVs and their parent MSCs suggest a complex mechanism of EV cargo sequestration and packaging. Notably, we identified a significant number of overlapping mRNAs and miRNAs TFs enriched in EVs, suggesting that interactions between mRNA and miRNA target TFs may be an important mechanism driving MSC-based repair. These observations support proposed shuttling mechanisms mediated by EV-dependent signaling between MSCs and recipient cells, and encourage development of EV-based regenerative strategies. Our present studies provide a platform for further studies to elucidate the molecular mechanisms by which proteins, transcriptional factors, and translational regulators transferred by MSC-derived EVs may activate tissue repair in recipient cells.