Ragni Enrico, Colombini Alessandra, De Luca Paola, Libonati Francesca, Viganò Marco, Perucca Orfei Carlotta, Zagra Luigi, de Girolamo Laura
IRCCS Istituto Ortopedico Galeazzi, Laboratorio di Biotecnologie Applicate all'Ortopedia, Milan, Italy.
IRCCS Istituto Ortopedico Galeazzi, Hip Department, Milan, Italy.
Front Bioeng Biotechnol. 2021 Feb 15;9:632440. doi: 10.3389/fbioe.2021.632440. eCollection 2021.
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.
软骨细胞（CCs）、脂肪组织（ASC）和骨髓（BMSC）来源的间充质基质细胞（MSCs）已被证明是治疗骨关节炎（OA）的有前景的候选细胞。尽管它们具有适应能力，但暴露于慢性分解代谢和炎症过程会限制其存活和愈合潜力。一种有吸引力的无细胞替代或补充策略是它们分泌的细胞外囊泡（EVs），其对OA软骨细胞和滑膜细胞具有稳态特性。鉴于临床转化，迫切需要对穿梭的治疗分子（如miRNAs）进行全面表征，以筛选和开发用于OA治疗的最有效的EV制剂。迄今为止，一个关键的陷阱是缺乏用于可靠定量和比较不同基于EV的治疗产品的EV-miRNA相关参考基因（RGs）。在本研究中，通过geNorm、NormFinder、BestKeeper和ΔCt算法以及排名的几何平均值，评估了文献中已提出的来自其他来源的EV产品中的12个假定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和miR-660-5p）在从三个供体匹配的ASC、BMSC和CCs中分离的EVs中的稳定性。ASC-EVs和BMSC-EVs共享比软骨来源的EVs更相似的分子特征，尽管总体上miR-103a-3p一直排名第一，miR-22-5p排名第二稳定的EV-miRNA RG，而miR-221-3p表现不佳。此外，为了强调错误选择RG的影响，比较了四种OA治疗性miRNAs（miR-93-5p、miR-125b-5p、miR-455-3p和miR-27b-3p）的丰度。使用miR-221-3p导致EV指纹识别不太准确，并且当应用于筛选治疗效力预测时，会对最合适的临床产品产生误导性指示。总之，miR-103a-3p和miR-22-5p将代表用于定量MSC-和CC-EVs中嵌入的miRNAs的可靠RGs，这是分子定义和比较这些创新的基于无细胞的OA治疗产品以及更广泛的基于再生医学的方法的临床效力的必要步骤。
