miR-103a-3p and miR-22-5p Are Reliable Reference Genes in Extracellular Vesicles From Cartilage, Adipose Tissue, and Bone Marrow Cells

Abstract

Cartilage cells (CCs), adipose tissue (ASC)- and bone marrow (BMSC)-derived mesenchymal stromal cells (MSCs) have been shown as promising candidates for the treatment of osteoarthritis (OA). Despite their adaptive ability, exposure to chronic catabolic and inflammatory processes can limit their survival and healing potential. An attractive cell-free alternative or complementary strategy is represented by their secreted extracellular vesicles (EVs), having homeostatic properties on OA chondrocytes and synovial cells. In view of clinical translation, a thorough characterization of the shuttled therapeutic molecules, like miRNAs, is greatly needed to fingerprint and develop the most effective EV formulation for OA treatment. To date, a crucial pitfall is given by the lack of EV-miRNA-associated reference genes (RGs) for the reliable quantification and comparison among different therapeutic EV-based therapeutic products. In this study, the stability of 12 putative miRNA RGs (let-7a-5p, miR-16-5p, miR-22-5p, miR-23a-3p, miR-26a-5p, miR-29a-5p, miR-101-3p, miR-103a-3p, miR-221-3p, miR-423-5p, miR-425-5p and miR-660-5p), already proposed by literature in EV products from alternative sources, was assessed in EVs isolated from three donor-matched ASCs, BMSCs, and CCs through geNorm, NormFinder, BestKeeper, and ΔCt algorithms and the geometric mean of rankings. ASC-EVs and BMSC-EVs shared more similar molecular signatures than cartilage-derived EVs, although overall miR-103a-3p consistently ranked as the first and miR-22-5p as the second most stable EV-miRNA RG, whereas miR-221-3p behaved poorly. Further, to emphasize the impact of incorrect RG choice, the abundance of four OA-therapeutic miRNAs (miR-93-5p, miR-125b-5p, miR-455-3p, and miR-27b-3p) was compared. The use of miR-221-3p led to less accurate EV fingerprinting and, when applied to sift therapeutic potency prediction, to misleading indication of the most appropriate clinical product. In conclusion, miR-103a-3p and miR-22-5p will represent reliable RGs for the quantification of miRNAs embedded in MSC- and CC-EVs, a mandatory step for the molecular definition and comparison of the clinical potency of these innovative cell-free-based therapeutic products for OA in particular, as well as for a wider array of regenerative-medicine-based approaches.

Cartilage Protective and Immunomodulatory Features of Osteoarthritis Synovial Fluid-Treated Adipose-Derived Mesenchymal Stem Cells Secreted Factors and Extracellular Vesicles-Embedded miRNAs

“…Donors, ASCs passage, culturing conditions (except OA-SF treatment) and technical platforms for ELISA and qRT-PCR were identical to those used in this study. For miRNA comparison, miR-16-5p/23a-3p/103a-3p/22-5p/26a-5p/29a-5p were selected as reference genes according to previous publications from our group [ 34 , 35 , 36 , 37 ].…”

Identification of a suitable endogenous control miRNA in bone aging and senescence

“…Chen et al reported miR-25-3p as the most stable miRNA in serum samples from osteoporotic rats, monkeys and women, and with osteoblast and osteoclast differentiation ( Chen et al, 2016 ). Additionally, miRs-103a-3p and − 22-5p have been reported to be highly stable in extracellular vesicles released from cartilage cells, subcutaneous adipose tissue and BMSCs of osteoarthritic patients and miR-191-5p is shown to have high stability in human bone marrow stromal cell lines and primary cells ( Costé & Rouleux-Bonnin, 2020 ; Ragni et al, 2021 ). The results of each of these studies are unique and different from our results due to differences in methodologies, and the disease states and conditions being tested.…”

Identification of a suitable endogenous control miRNA in bone aging and senescence

“…Chen et al reported miR-25-3p as the most stable miRNA in serum samples from osteoporotic rats, monkeys and women, and with osteoblast and osteoclast differentiation (Chen et al, 2016). Additionally, miRs-103a-3p and -22-5p have been reported to be highly stable in extracellular vesicles released from cartilage cells, subcutaneous adipose tissue and BMSCs of osteoarthritic patients and miR-191-5p is shown to have high stability in human bone marrow stromal cell lines and primary cells (Costé & Rouleux-Bonnin, 2020; Ragni et al, 2021). The results of each of these studies are unique and different from our results due to differences in methodologies, and the disease states and conditions being tested.…”