Equine Adipose-Derived Mesenchymal Stromal Cells Release Extracellular Vesicles Enclosing Different Subsets of Small RNAs

Stefano Capomaccio; Katia Cappelli; Cinzia Bazzucchi; Mauro Coletti; Rodolfo Gialletti; Franco Moriconi; Fabrizio Passamonti; Marco Pepe; Stefano Petrini; Samanta Mecocci; Maurizio Silvestrelli; Luisa Pascucci

doi:10.1155/2019/4957806

Equine Adipose-Derived Mesenchymal Stromal Cells Release Extracellular Vesicles Enclosing Different Subsets of Small RNAs

Stem Cells Int. 2019 Mar 18:2019:4957806. doi: 10.1155/2019/4957806. eCollection 2019.

Authors

Stefano Capomaccio^{1

2}, Katia Cappelli^{1

2}, Cinzia Bazzucchi¹, Mauro Coletti^{1

2}, Rodolfo Gialletti^{1

2}, Franco Moriconi^{1

2}, Fabrizio Passamonti^{1

2}, Marco Pepe^{1

2}, Stefano Petrini³, Samanta Mecocci^{1

2}, Maurizio Silvestrelli^{1

2}, Luisa Pascucci^{1

2}

Affiliations

¹ Dipartimento di Medicina Veterinaria, Università degli Studi di Perugia, Via San Costanzo, 4, 06126 Perugia, Italy.
² Centro di Ricerca sul Cavallo Sportivo (CRCS), Università degli Studi di Perugia, Italy.
³ Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche, Italy.

Abstract

Background: Equine adipose-derived mesenchymal stromal cells (e-AdMSC) exhibit attractive proregenerative properties strongly related to the delivery of extracellular vesicles (EVs) that enclose different kinds of molecules including RNAs. In this study, we investigated small RNA content of EVs produced by e-AdMSC with the aim of speculating on their possible biological role.

Methods: EVs were obtained by ultracentrifugation of the conditioned medium of e-AdMSC of 4 subjects. Transmission electron microscopy and scanning electron microscopy were performed to assess their size and nanostructure. RNA was isolated, enriched for small RNAs (<200 nt), and sequenced by Illumina technology. After bioinformatic analysis with state-of-the-art pipelines for short sequences, mapped reads were used to describe EV RNA cargo, reporting classes, and abundances. Enrichment analyses were performed to infer involved pathways and functional categories.

Results: Electron microscopy showed the presence of vesicles ranging in size from 30 to 300 nm and expressing typical markers. RNA analysis revealed that ribosomal RNA was the most abundant fraction, followed by small nucleolar RNAs (snoRNAs, 13.67%). Miscellaneous RNA (misc_RNA) reached 4.57% of the total where Y RNA, RNaseP, and vault RNA represented the main categories. miRNAs were sequenced at a lower level (3.51%) as well as protein-coding genes (1.33%). Pathway analyses on the protein-coding fraction revealed a significant enrichment for the "ribosome" pathway followed by "oxidative phosphorylation." Gene Ontology analysis showed enrichment for terms like "extracellular exosome," "organelle envelope," "RNA binding," and "small molecule metabolic process." The miRNA target pathway analysis revealed the presence of "signaling pathways regulating pluripotency of stem cells" coherent with the source of the samples.

Conclusion: We herein demonstrated that e-AdMSC release EVs enclosing different subsets of small RNAs that potentially regulate a number of biological processes. These findings shed light on the role of EVs in the context of MSC biology.