Insights into Inflammatory Priming of Adipose-Derived Mesenchymal Stem Cells: Validation of Extracellular Vesicles-Embedded miRNA Reference Genes as A Crucial Step for Donor Selection

Ragni, Enrico; Luca, Paola De; Orfei, Carlotta Perucca; Colombini, Alessandra; Viganò, Marco; Lugano, Gaia; Bollati, Valentina

doi:10.3390/cells8040369

Cited by 21 publications

(17 citation statements)

References 78 publications

(90 reference statements)

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“…Regarding EVs, the situation is even more difficult, and the establishment of reliable RGs is mandatory. Recently, our group proposed an in-silico pipeline to identify new miRNA RGs in EVs isolated from ASCs [37,38]. Several applets (geNorm, NormFinder, BestKeeper and ∆Ct method) [39][40][41][42] were used to score the stability of a selected panel of miRNAs to identify those resulting as more stable across either donors or divergent culturing conditions.…”

Section: Introductionmentioning

confidence: 99%

miRNA Reference Genes in Extracellular Vesicles Released from Amniotic Membrane-Derived Mesenchymal Stromal Cells

et al. 2020

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Human amniotic membrane and amniotic membrane-derived mesenchymal stromal cells (hAMSCs) have produced promising results in regenerative medicine, especially for the treatment of inflammatory-based diseases and for different injuries including those in the orthopedic field such as tendon disorders. hAMSCs have been proposed to exert their anti-inflammatory and healing potential via secreted factors, both free and conveyed within extracellular vesicles (EVs). In particular, EV miRNAs are considered privileged players due to their impact on target cells and tissues, and their future use as therapeutic molecules is being intensely investigated. In this view, EV-miRNA quantification in either research or future clinical products has emerged as a crucial paradigm, although, to date, largely unsolved due to lack of reliable reference genes (RGs). In this study, a panel of thirteen 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, miR-660-5p and U6 snRNA) that were identified in different EV types was assessed in hAMSC-EVs. A validated experimental pipeline was followed, sifting the output of four largely accepted algorithms for RG prediction (geNorm, NormFinder, BestKeeper and ΔCt method). Out of nine RGs constitutively expressed across all EV isolates, miR-101-3p and miR-22-5p resulted in the most stable RGs, whereas miR-423-5p and U6 snRNA performed poorly. miR-22-5p was also previously reported to be a reliable RG in adipose-derived MSC-EVs, suggesting its suitability across samples isolated from different MSC types. Further, to shed light on the impact of incorrect RG choice, the level of five tendon-related miRNAs (miR-29a-3p, miR-135a-5p, miR-146a-5p, miR-337-3p, let-7d-5p) was compared among hAMSC-EVs isolates. The use of miR-423-5p and U6 snRNA did not allow a correct quantification of miRNA incorporation in EVs, leading to less accurate fingerprinting and, if used for potency prediction, misleading indication of the most appropriate clinical batch. These results emphasize the crucial importance of RG choice for EV-miRNAs in hAMSCs studies and contribute to the identification of reliable RGs such as miR-101-3p and miR-22-5p to be validated in other MSC-EVs related fields.

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Section: Introductionmentioning

confidence: 99%

miRNA Reference Genes in Extracellular Vesicles Released from Amniotic Membrane-Derived Mesenchymal Stromal Cells

et al. 2020

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“…Nevertheless, in recent reports scoring RG stability for EV-miRNAs, U6 snRNA was shown to be an unreliable candidate. In fact, similar to EVs released by cardiosphere-derived-cells ( Gouin et al, 2017 ) and of particular importance for the cell types discussed in this manuscript, U6 snRNA performed poorly also in EVs released by MSCs isolated from adipose tissue ( Ragni et al, 2019a,b , d ) and amniotic membrane ( Ragni et al, 2020a ). This may be due to the mechanistical biogenesis of U6 snRNA that is processed by the Drosha complex in spite of the spliceosome ( Lee et al, 2003 ).…”

Section: Discussionmentioning

confidence: 61%

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

Ragni

Colombini

Luca

et al. 2021

Front. Bioeng. Biotechnol.

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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.

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“…Также известно, что хондроитина сульфат положительно влияет на функцию эндотелия, замедляет процессы атерогенеза [26,27]. Глюкозамин обладает регулирующим влиянием на аутофагию и запрограммированную клеточную смерть путем регуляции сигнального каскада m-TOR [28,29]. Применение фиксированной комбинации хондроитина и глюкозамина на животной модели спонтанного ОА у морских свинок линии Хартли способствовало нормализации клеточного фенотипа, ингибировало апоптоз [30].…”

Section: Discussionunclassified

Effectiveness of drugs with delayed-release structurally modified action depending on the phenotype of osteoarthritis

Кабалык

2020

Medicinskij sovet

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Aim. To evaluate the effectiveness of a fixed combination of honadroitin and glucosamine sulfate (Teraflex, Bayer) in the treatment of osteoarthritis (OA), depending on the molecular phenotype of the disease.Materials and methods. A 6-month prospective, open, randomized trial included 65 patients with OA of the knee joints who were prescribed therapy with Teraflex (Bayer) daily dose of 1500 mg + 1200 mg. Kinetic assessment of articular status was performed using a visual analogue pain scale and a WOMAC questionnaire, and serum concentrations of CRTAP (cartilage-associated protein), OSGIN-1 (oxidative stress-induced growth inhibitor 1), IL-1β (interleukin-1 beta) were determined in blood serum. Measurements of these parameters were made at the beginning of the study, after 3 and 6 months.Results. It was established that the rate of onset of the therapeutic effect and the effect on the molecular patterns of inflammation and oxidative stress depend on the phenotype of the disease. So, with oxidative and mixed phenotypes of the disease, clinical efficacy is observed in the treatment of teraflex after 3 months from the start of therapy. Indicators of oxidative stress during treatment decreased in the group of patients with the oxidative phenotype of the disease, while the level of interleukin-1 significantly decreased only in groups of patients with inflammatory and mixed OA phenotypes.Conclusions. The results indicate the effectiveness and safety of the drug Teraflex (Bayer) for the treatment of patients with OA. The results of the study indicate the targeted effect of a fixed combination of chondroitin + glucosamine on the molecular mechanisms of the disease.

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Insights into Inflammatory Priming of Adipose-Derived Mesenchymal Stem Cells: Validation of Extracellular Vesicles-Embedded miRNA Reference Genes as A Crucial Step for Donor Selection

Cited by 21 publications

References 78 publications

miRNA Reference Genes in Extracellular Vesicles Released from Amniotic Membrane-Derived Mesenchymal Stromal Cells

miRNA Reference Genes in Extracellular Vesicles Released from Amniotic Membrane-Derived Mesenchymal Stromal Cells

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

Effectiveness of drugs with delayed-release structurally modified action depending on the phenotype of osteoarthritis

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