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

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.

Selection of reference miRNAs for RT-qPCR assays in endometriosis menstrual blood-derived mesenchymal stem cells

“…After an extensive review of miRNAs with proven expression stability under experimental conditions similar to those in this study, we identified the most stable miRNAs: miR-101-3p, miR-23a-3p, miR-16-5p, miR-103a-3p, miR-24-3p, and miR-22-5p in MSCs [ 62 , 63 , 66 , 67 ]; miR-30e-5p in endometriosis [ 64 ]; and miR-191-5p, miR-17-5p, miR-25-3p, miR-103a-3p and miR-24-3p in both normal and tumor tissue conditions [ 60 , 61 ]. Considering the comprehensive ranking provided by RefFinder, cost-effectiveness, and our results suggesting the use of minimum three genes for optimal normalization, we recommend using miR-191-5p, miR-24-3p, and miR-103a-3p as internal controls for this study design.…”

Overexpression of lncRNA LINC00665 inhibits the proliferation and chondroblast differentiation of bone marrow mesenchymal stem cells by targeting miR-214-3p

“…The symptoms of patients with osteoarthritis are joint pain and limited joint movement, so the key to treatment is to relieve the pain of patients, restore joint function, and delay the progression of the disease [ 18 ]. Studies have noted that BMSCs may be involved in the treatment of osteoarthritis through their tissue repair function and anti-inflammatory properties [ 19 ]. Zheng et al [ 20 ] proposed that osteoarthritis is closely related to osteogenic differentiation of BMSCs, and ROCK1 signaling in BMSCs can assist in mediating the process of osteoarthritis.…”