Endogenous Controls for the Evaluation of Osteoarthritis-Related miRNAs in Extracellular Vesicles from Bone-Marrow-Derived Mesenchymal Stromal Cells and the Impact of Osteoarthritis Synovial Fluid.

Enrico Ragni,Marco Viganò,Carlotta Perucca Orfei,Federico Valli,Laura De Girolamo

doi:10.3390/biom12020316

Enrico Ragni, Marco Viganò + Show 3 more

Open Access

https://doi.org/10.3390/biom12020316

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Journal: Biomolecules	Publication Date: Feb 16, 2022
Citations: 1	License type: CC BY 4.0

Affiliation: Istituto Ortopedico Galeazzi

Abstract

Bone-marrow-derived stromal cells (BMSCs) have emerged as promising therapeutic option for the treatment of osteoarthritis (OA) due to their tissue regenerative and anti-inflammatory features. BMSCs’ clinical potential is mainly ascribed to their released factors and extracellular vesicles (EVs), whose therapeutic portfolio may be modulated by the environment in vivo or specific priming in vitro. Within the array of molecules shaping EVs’ power, miRNAs are considered privileged players. In this frame, a correct EV-miRNA detection and quantification is mandatory to understand and possibly boost BMSCs potential, either when envisioned as cell therapeutics or when proposed as producer of cell-free and clinical grade EVs. The aim of this study is to identify reliable reference genes (RGs) to study miRNAs in BMSC-EVs cultivated under standard or OA synovial fluid (OA-SF). miR-23a-3p and miR-221-3p emerged as the best candidates, respectively. Moreover, when both conditions were analyzed together, miR-24-3p resulted the most stable RGs, allowing for a sharper comparison of EVs content, further validated on the OA-related miRNA-193b-5p. The different RG stability ranking depending on the culturing conditions, as well as its divergence with respect to adipose (ASCs) and amniotic (hAMSCs) MSCs, confirm that miRNA RG selection in EVs is a mandatory step and that the identification of the most reliable candidate is greatly depending on the cell type and culturing/environmental conditions.

Highlights

The OA synovial fluid (OA-SF) was collected by puncture before arthroplasty
By Nanoparticle tracking analysis (NTA), Bone-marrow-derived stromal cells (BMSCs)-extracellular vesicles (EVs) were within the expected extracellular vesicle size range (Figure 1C), supporting what previously reported for this EV type [53]
An integrated method based on four well-known stability algorithms was proposed to identify reliable miRNA reference genes (RGs) in EVs released from BMSCs

Summary

Introduction

Laboratorio di Biotecnologie Applicate all’Ortopedia, IRCCS Istituto Ortopedico Galeazzi, Via R. Within the array of molecules shaping EVs’ power, miRNAs are considered privileged players. In this frame, a correct EV-miRNA detection and quantification is mandatory to understand and possibly boost BMSCs potential, either when envisioned as cell therapeutics or when proposed as producer of cell-free and clinical grade EVs. The aim of this study is to identify reliable reference genes (RGs) to study miRNAs in BMSC-EVs cultivated under standard or OA synovial fluid (OA-SF). MiR-23a-3p and miR-221-3p emerged as the best candidates, respectively When both conditions were analyzed together, miR-24-3p resulted the most stable RGs, allowing for a sharper comparison of EVs content, further validated on the OA-related miRNA-193b-5p. RG stability ranking depending on the culturing conditions, as well as its divergence with respect to adipose (ASCs) and amniotic (hAMSCs) MSCs, confirm that miRNA RG selection in EVs is a mandatory step and that the identification of the most reliable candidate is greatly depending on the cell type and culturing/environmental conditions

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