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miR-22-5p and miR-29a-5p Are Reliable Reference Genes for Analyzing Extracellular Vesicle-Associated miRNAs in Adipose-Derived Mesenchymal Stem Cells and Are Stable under Inflammatory Priming Mimicking Osteoarthritis Condition

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Abstract

During the last two decades, mesenchymal stem cells (MSCs) gained a place of privilege in the field of regenerative medicine. Recently, extracellular vesicles (EVs) have been identified as major mediators of MSCs immunosuppressive as well as pro-regenerative activities in many disease models, including inflammatory/degenerative conditions as joint diseases and osteoarthritis. In order to shed light on EVs potential, a rigorous profiling of embedded proteins, lipids and nucleic acids (mRNA/miRNA) is mandatory. Nevertheless, reliable strategies to efficiently score miRNA cargo and modulation under diverse experimental conditions or treatments are missing. The aim of this work was to identify reliable reference genes (RGs) to analyze miRNA content in EVs secreted by adipose-derived MSCs (ASCs) and verify their consistency under inflammatory conditions that were proposed to enhance ASC-EVs immunomodulatory and regenerative potential. RefFinder algorithm, that integrates the currently available major computational programs (geNorm, NormFinder, BestKeeper, and Delta Ct method), allowed to identify miR-22-5p and miR-29a-5p as the most stable RGs. Notably, both miRNAs maintained the highest stability when EVs isolated from IFNg-treated ASCs were included in the analysis. In addition, considerable effects of suboptimal RGs choice on the reliable quantification of miRNAs involved at different levels (tissue homeostasis or macrophage polarization) in the osteoarthritis phenotype, and thus considered as promising therapeutic molecule, have clearly been demonstrated. In conclusion, a proper normalization method is not only needed for research purposes but also mandatory to characterize clinical products and predict their therapeutic potential, especially in the emerging field of MSCs derived-EVs as new tools for regenerative medicine.

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Acknowledgements

Authors thank Laura Dioni, Laura Cantone, Laura Perego and Mirjam Hoxha for their help and useful discussions. The present work was funded by Ministero della Salute, Ricerca Corrente.

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Authors and Affiliations

  1. Laboratorio di Biotecnologie Applicate all’Ortopedia, IRCCS Istituto Ortopedico Galeazzi, Via R. Galeazzi 4, 20161, Milan, Italy

    Enrico Ragni, Carlotta Perucca Orfei, Paola De Luca, Marco Viganò, Alessandra Colombini, Gaia Lugano & Laura de Girolamo

  2. EPIGET - Epidemiology, Epigenetics and Toxicology Lab, Department of Clinical Sciences and Community Health, University of Milan, Via San Barnaba 8, 20122, Milan, Italy

    Valentina Bollati

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  1. Enrico Ragni
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Contributions

Enrico Ragni: collection and/or assembly of data, data analysis and interpretation, conception and design, manuscript writing; Carlotta Perucca Orfei: collection and/or assembly of data, data analysis and interpretation; Paola De Luca: collection and/or assembly of data; Marco Viganò: data analysis and interpretation; Alessandra Colombini: data analysis and interpretation; Gaia Lugano: collection and/or assembly of data; Valentina Bollati: data analysis and interpretation; Laura de Girolamo: conception and design, manuscript writing, final approval of manuscript.

Corresponding author

Correspondence to Enrico Ragni.

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The authors have no conflict of interest.

Ethical Approval and Informed Consent

The study was carried out at IRCCS Istituto Ortopedico Galeazzi with Institutional Review Board approval and specimens were collected with patient informed consent (M-SPER-015 - Ver. 2–04.11.2016), and under the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

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Ragni, E., Perucca Orfei, C., De Luca, P. et al. miR-22-5p and miR-29a-5p Are Reliable Reference Genes for Analyzing Extracellular Vesicle-Associated miRNAs in Adipose-Derived Mesenchymal Stem Cells and Are Stable under Inflammatory Priming Mimicking Osteoarthritis Condition. Stem Cell Rev and Rep 15, 743–754 (2019). https://doi.org/10.1007/s12015-019-09899-y

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