Abstract
Mesenchymal stem cells (MSCs) are promising tools for cell-based therapies due to their homing to injury sites, where they secrete bioactive factors such as cytokines, lipids, and nucleic acids, either free or conveyed within extracellular vesicles (EVs). Depending on the local environment, MSCs’ therapeutic value may be modulated, determining their fate and cell behavior. Inflammatory signals may induce critical changes on both the phenotype and secretory portfolio. Intriguingly, in animal models resembling joint diseases as osteoarthritis (OA), inflammatory priming enhanced the healing capacity of MSC-derived EVs. In this work, we selected miRNA reference genes (RGs) from the literature (let-7a-5p, miR-16-5p, miR-23a-3p, miR-26a-5p, miR-101-3p, miR-103a-3p, miR-221-3p, miR-423-5p, miR-425-5p, U6 snRNA), using EVs isolated from adipose-derived MSCs (ASCs) primed with IFNγ (iASCs). geNorm, NormFinder, BestKeeper, and ΔCt methods identified miR-26a-5p/16-5p as the most stable, while miR-103a-rp/425-5p performed poorly. Our results were validated on miRNAs involved in OA cartilage trophism. Only a proper normalization strategy reliably identified the differences between donors, a critical factor to empower the therapeutic value of future off-the-shelf MSC-EV isolates. In conclusion, the proposed pipeline increases the accuracy of MSC-EVs embedded miRNAs assessment, and help predicting donor variability for precision medicine approaches.
Highlights
Cell-based therapies are one of the most promising approaches in the medical arsenal to restore the lost function rather than producing new organs or tissue
After IFNγ stimulation, flow cytometry showed that IFNγ licensed-ASCs (iASCs)-extracellular vesicles (EVs) expressed both Mesenchymal stem cells (MSCs) (CD44, CD73, CD90, CD105) and vesicle markers (CD63, CD81), consistent with previously reported characteristics of MSC-EVs (Figure 1B) [22,32,43]
CD45 were not detectable. iASC-EVs were inspected by Nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM)
Summary
Cell-based therapies are one of the most promising approaches in the medical arsenal to restore the lost function rather than producing new organs or tissue. Several resources of cells can be used to renovate the damaged tissues, and human mesenchymal stromal cells (MSCs) have been identified as a leading candidate [2]. Cells”, since those molecules can orchestrate the trophism and regeneration of diseased tissues [4]. In this perspective, MSCs represent an innovation from current biopharmaceutical production, where the “medicine” of interest is not the cell itself but its released molecules [5]. MSCs represent an innovation from current biopharmaceutical production, where the “medicine” of interest is not the cell itself but its released molecules [5] This crucial difference greatly increases the grade of complexity in the field, taking into account both the manufacturing process and multiple donor variability
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