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.

Keywords: adipose tissue mesenchymal stromal cells; bone marrow mesenchymal stromal cells; cartilage cells; extracellular vesicles; miRNAs; reference genes.

FIGURE 1

CC-, BMSC-, and ASC-EV phenotype characterization. (A) Size distribution of nanoparticles by NanoSight particle tracking analysis. Representative plots are shown. (B) TEMs of EVs showing particles with characteristic cup-shaped morphology and size consistent with the NanoSight profile. (C) The resolution of the reference bead mix indicates the flow cytometer performance in light scattering at default settings. The first cytogram depicts the SSC-H vs. 535/35 (green fluorescence triggering) channel. Four fluorescent populations (160, 200, 240, and 500 nm) were resolved (FITC+ gate) from the instrument noise (in black). The second plot shows EVs stained with CFSE to allow their identification and gating in the FITC channel (CFSE+ gate) vs. background noise, debris, and unstained particles. (D) After CFSE+ gating, with respect to antibody-unstained samples (in gray), antibody-treated EVs showed the presence of EV-defining molecules CD63 and CD81, while CD9 staining gave a weak signal. EVs were also strongly positive for stromal markers CD73 and CD90, with CD44 labeling allowing a complete shift of the population although without a sharp separation with respect to antibody-unstained (gray curve) samples. Representative cytograms are presented.

FIGURE 2

Expression of candidate RG miRNAs in CC-, BMSC-, and ASC-EVs. (A) C_RT values for miRNA RGs in different donors. Color from dark to light stands for donors 1–3, respectively. (B) The box plot graphs of the C_RT values for each RG, regardless of the donor difference, illustrate the interquartile range (box) and median. The whisker plot depicts the range of the values.

FIGURE 3

Unsupervised hierarchical clustering for the miRNA RG C_RT values. In the dendrogram, each row represents a miRNA, and each column represents a sample. The sample clustering tree is shown at the top. The color scale shown in the map illustrates the relative expression levels of miRNAs across all samples: red shades represent high expression levels (low C_RT), and blue shades represent low expression levels (high C_RT).

FIGURE 4

Influence of RG selection on cartilage and OA-related EV-miRNA profile. (A) PCA for the BMSC- and ASC-EV molecular signature, based on the amount ratios with CC-EVs set as 1, of cartilage-related miRNAs after stable miR-103a-3p and miR-22-5p or unreliable miR-221-3p RG normalization. (B) Effects of RG normalization on the differential expression of single-cartilage-related miRNAs in BMSC- and ASC-EVs with respect to CC-EVs set as 1, N = 3, ^§ p < 0.1, *p < 0.05, **p < 0.01, and ***p < 0.001.